A method for purifying recombinant herpes zoster virus stock solution

Abstract

The present invention discloses a method for purifying a recombinant herpes zoster virus (HZV) stock solution, specifically relating to the field of biomedicine. The method comprises anion exchange chromatography, hydrophobic chromatography, and composite chromatography. The method can be used to prepare a high-purity recombinant HZV protein stock solution. The method has a high single-pass throughput for ultrafiltration concentrate, achieving a purity exceeding 98%. This method is suitable for large-scale production of recombinant HZV vaccine preparations requiring a purity exceeding 95%. The method can be scaled up or down according to production scale to produce purified recombinant HZV vaccines containing high-purity antigens.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a method for purifying a recombinant herpes zoster virus stock solution. Background Art

[0002] Herpes zoster (HZ) is caused by the varicella-zoster virus, which remains latent in the body. Infection with the herpes zoster virus can activate an acute infectious disease. The virus invades the human body through the upper respiratory tract or conjunctiva, causing systemic infection. The initial infection manifests as chickenpox in young children and can be latent in adults. After recovery from the initial infection, the virus can remain latent in the dorsal root ganglia of the spinal nerves for a long time. When the body's immunity decreases, it can grow and multiply again, causing herpes zoster, which usually manifests as unilateral skin blisters and pain distributed along the nerve segments. The most common complication of HZ is postherpetic neuralgia, the most common type of neuropathic pain. It can manifest as persistent pain or reappear after a period of relief, seriously affecting the patient's quality of life and placing a heavy disease burden on society. According to statistics, approximately 1 / 4 of the world's population is at risk of developing HZ, of which 2 / 3 of HZ patients are 50 years of age and above.

[0003] Although there are many methods to treat HZ, vaccine is still considered the most economical and effective measure to control HZ.

[0004] Recombinant shingles vaccine (CHO cells) is cultured using CHO cell lines. Problems such as host protein residues and nucleic acid residues in this process are key links in vaccine production quality control. At present, the domestic residual host cell DNA of CHO cells and other cultured cells is strictly limited to less than 10pg per dose, which is far less than the WHO and EU Pharmacopoeia standards of less than 10ng. Therefore, it is difficult to meet our national pharmacopoeia standards using traditional vaccine purification processes. Therefore, innovative research on the purification process of recombinant shingles vaccine (CHO cells) is needed.

[0005] Virus purification involves applying various physical and chemical methods to remove non-viral impurities, such as host cell components, without damaging or inactivating the virus, to produce a highly purified, concentrated viral sample. A well-designed viral antigen purification process effectively removes contaminants such as foreign proteins, host cell DNA, endotoxins, and other impurities introduced during the viral culture process, while maintaining excellent viral antigen recovery and purity.

[0006] Traditional viral vaccine purification uses a sucrose density gradient ultracentrifugation purification process, which requires large centrifuge equipment, is expensive, has a small sample load, takes a long time, and is not ideal for removing host cell DNA, making it unsuitable for the requirements of vaccine industrialization. Although the direct use of the complex chromatography method can separate proteins with high purity, it has strict separation conditions. Due to the excessively mixed materials, the relative processing capacity is low, the processing pressure is high, and the investment is high. In particular, it is easy to form a protein gel layer on the surface, which hinders the penetration of later samples and is not suitable for large-scale production needs. Conventional gel filtration chromatography, although its conditions are mild, reproducible, and easy to industrialize, is widely used in vaccine purification processes, but the effect of removing foreign proteins and DNA is not obvious, so it can only be used as an auxiliary means in vaccine preparation and purification processes. The following combined purification method has obvious advantages. Anion exchange chromatography can mainly remove most host cell proteins, impurity proteins, endotoxins and other impurities, and then the hydrophobic layer can be used to further remove host cell proteins and aggregates and other impurities. The final step is reconstitution chromatography, a multimodal chromatography medium that combines molecular sieve chromatography, ion exchange chromatography, and hydrophobic chromatography. It possesses both size exclusion and binding properties, enabling efficient capture. The unique design of this medium allows molecules smaller than 700 kD to enter the medium and bind to the octylamine ligand, further removing impurities such as host proteins, DNA fragments, endotoxins, and nucleases. Recombinant herpes zoster virus particles are further purified by flow-through, achieving further impurity removal and purification, further improving the purity of the resulting product.

[0007] In contrast, multi-step purification processes offer significant advantages. Chinese patent CN118702780A discloses a method for purifying recombinant herpes zoster protein, comprising a four-step purification process using Diamond MIX-A, Diamond Butyl Mustang, Diamond Q, and Diamond MIX-AMustang as chromatographic media, in sequence. The multimodal strong anion exchange media, Diamond MIX-A and Diamond MIX-A Mustang, effectively remove HCPs, impurities, and a small amount of pigment. The hydrophobic interaction media, Diamond Butyl Mustang, provide consistency in the purification scheme, removing most pigments and further impurities and HCPs. The strong anion exchange media, Diamond Q, is highly effective in removing HCPs. However, the four-step purification method employed in this patent is a small-scale laboratory experiment, resulting in a low sample load and impurity scalability for large-scale industrial production. Furthermore, the method requires frequent changes of eluent, resulting in cumbersome steps and low yields. Summary of the Invention

[0008] To this end, the present invention provides a method for purifying a recombinant herpes zoster virus stock solution to solve the above-mentioned problems in the prior art.

[0009] First, the multi-step purification process is less dependent on large-scale equipment, significantly reducing equipment purchase and maintenance costs and reducing production site requirements. Second, by combining different chromatographic techniques, such as anion exchange chromatography and hydrophobic chromatography, and finally using a composite chromatographic filler, the multi-step purification process can more efficiently remove impurities and improve virus purity. Anion exchange chromatography provides initial virus purification, while hydrophobic chromatography further removes impurities such as host cell DNA. Finally, composite chromatography, as the final polishing step, combines the advantages of anion chromatography, hydrophobic chromatography, and molecular sieves, further enhancing purification efficiency and ultimately achieving a higher purity of the resulting viral product. Furthermore, the multi-step purification process can significantly shorten purification time. Compared to traditional purification processes, because each chromatographic step removes a portion of impurities, the multi-step purification process can achieve the desired purification effect more quickly, shortening the production cycle. In summary, the multi-step purification process exhibits significant advantages in viral vaccine purification, including reduced equipment investment, high purification efficiency, and time savings. These advantages make the multi-step purification process highly suitable for the needs of vaccine industrialization.

[0010] In order to achieve the above object, the present invention provides the following technical solutions:

[0011] According to one aspect of the present invention, a method for purifying a recombinant herpes zoster protein stock solution is provided, comprising the following steps:

[0012] Step 1, anion exchange chromatography

[0013] The clarified protein filtrate obtained by the primary filtration is eluted with an anion exchange chromatography eluate equilibrated with an equilibration solution and collected to obtain an initial protein solution;

[0014] Step 2: Hydrophobic chromatography

[0015] The inactivated solution of the initial protein solution after inactivation by the inactivator is eluted by hydrophobic chromatography, and the obtained eluate is concentrated to obtain a concentrated protein solution;

[0016] Step 3: Composite chromatography

[0017] The concentrated protein solution is eluted using a composite chromatography with a multi-mode chromatography filler, and the obtained eluate is sterile filtered to obtain a purified recombinant herpes zoster protein stock solution.

[0018] Furthermore, in step 1, the primary filtration is first deep filtration through a composite glass fiber, and then filtered through a polyethersulfone membrane to obtain a clarified protein primary filtrate; wherein the pore size of the composite glass fiber filter element is 0.45~0.8μm, and the pore size of the polyethersulfone membrane filtration is 0.2~0.45μm.

[0019] Furthermore, in the step 1, the anion exchange chromatography column model is a Cytiva BPG450 chromatography column, and the filler is Samc Q40.

[0020] Furthermore, in step 1, the balancing solution is a mixture of phosphate buffer and sodium chloride solution. As an example, the sodium chloride concentration is preferably 0.2-1.0 M, more preferably 0.55 M; the pH of the phosphate buffer is 7.0-8.0, preferably pH 7.4.

[0021] Furthermore, in step 2, the inactivator is Tween 80 or Tween 80 and tributyl phosphate. As an example, the preferred concentration is 0.1-1.0%, more preferably 0.5%.

[0022] Furthermore, in the step 2, the hydrophobic chromatography column uses a Cytiva BPG200 chromatography column, and the filler is PhentyI Chromstar HP.

[0023] Furthermore, in step 2, the inactivation solution is first mixed with an ammonium sulfate solution before being loaded for elution, and the eluent is an ammonium sulfate solution. As an example, the concentration of the ammonium sulfate solution is 1-2M, and the preferred final concentration is 2M.

[0024] Among the inactivation conditions, the inactivation temperature is 2-8°C, preferably 4°C.

[0025] Furthermore, in step 2, the concentration method is ultrafiltration concentration. As an example, the pore size of the ultrafiltration membrane is 10-50 kDa.

[0026] Furthermore, in step 3, Capto Core 700 (Cytiva) was selected as the multimodal chromatography filler.

[0027] The method further includes storing the subpackaged product at a temperature below -65°C in the dark.

[0028] According to another aspect of the present invention, a recombinant herpes zoster virus vaccine preparation is provided, which comprises the recombinant herpes zoster protein stock solution purified by the above purification method, a protective agent, and pharmaceutically common excipients and carriers.

[0029] As examples, stock solution protectants include, but are not limited to, human serum albumin, gelatin, or polyvinyl pyrrolidone.

[0030] The present invention has the following advantages:

[0031] The present invention adopts a multi-step continuous purification method, using a deep filtration method combined with a polyethersulfone membrane filtration to remove some impurity protein components in the recombinant herpes zoster virus, and then applying an ion exchange chromatography method based on the difference in charge on the surface of the recombinant herpes zoster virus antigen (the ion exchange chromatography method achieves separation based on the difference in the type and quantity of charge on the surface of the recombinant herpes zoster virus antigen and the host cell protein), and utilizing the difference in the hydrophobic area on the surface of the recombinant herpes zoster virus antigen and the impurity protein to apply a hydrophobic chromatography method (the hydrophobic chromatography method achieves separation and purification based on the difference in the hydrophobic area exposed on the surface of the recombinant herpes zoster virus antigen and the hydrophobic area on the surface of the cell protein) to perform multi-step chromatographic purification, thereby achieving continuous multi-step purification of the recombinant herpes zoster protein stock solution.

[0032] The present invention can be used to prepare high-purity recombinant herpes zoster protein stock solution, and the single processing volume of ultrafiltration concentrate is high The purity can reach more than 98%, which is suitable for large-scale production of recombinant herpes zoster vaccine preparations that require a purity greater than 95%. It can be expanded or contracted according to the production scale and used to produce refined recombinant herpes zoster vaccines with high-purity antigens.

[0033] The recombinant herpes zoster protein stock solution purified by the process of the present invention can effectively remove host cell impurity proteins and DNA to obtain a high-purity virus stock solution sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the embodiments or the description of the prior art. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other implementation drawings based on the provided drawings without inventive effort.

[0035] The structures, proportions, sizes, etc. illustrated in this specification are intended only to complement the contents disclosed herein and to facilitate understanding and reading by persons familiar with the art. They are not intended to limit the conditions under which the present invention may be implemented and therefore have no substantive technical significance. Any structural modifications, changes in proportions, or adjustments in sizes, without affecting the efficacy and objectives of the present invention, shall still fall within the scope of the technical contents disclosed herein.

[0036] Figure 1 HPLC chromatogram showing the purity of the purified recombinant herpes zoster virus stock solution protein provided in Example 1 of the present invention;

[0037] Figure 2 This is an HPLC chromatogram showing the purity of the purified recombinant herpes zoster virus stock solution protein provided in Example 2 of the present invention. DETAILED DESCRIPTION

[0038] The following describes the implementation of the present invention using specific embodiments. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. Obviously, the embodiments described are only a portion of the present invention, not all of it. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention.

[0039] The present invention uses a multi-step purification process of anion exchange chromatography, hydrophobic chromatography, and complex chromatography to purify the recombinant virus vaccine stock solution, comprising the following steps:

[0040] 1) Primary filtration: Use composite glass fiber for deep filtration, and then filter through a polyethersulfone membrane to obtain a clarified protein primary filtrate.

[0041] 2) Anion exchange chromatography: The clarified protein filtrate obtained in the above step was equilibrated in an equilibration solution (a mixture of phosphate buffer with a pH of 7.0-8.0 and NaCl with a concentration of 0.2-1.0 M) and then loaded onto an anion exchange column (Cytiva BPG450 column, with Samc Q40 as the filler) for elution and collection.

[0042] 3) Inactivation: The initial protein solution is inactivated using an inactivator (0.1-1.0% Tween 80) at 2-8°C.

[0043] 4) Hydrophobic chromatography: The inactivated solution was subjected to hydrophobic column chromatography (the chromatography column used was Cytiva BPG200 chromatography column, and the filler was PhentyI Chromstar HP). After adding 10-40% ammonium sulfate solution to the inactivated solution, the sample was loaded, and then rinsed with 10-40% ammonium sulfate solution to the baseline, and the elution peak was collected.

[0044] 5) Ultrafiltration concentration: The purified solution of the elution peak collected above was concentrated using an ultrafiltration membrane (0.22 μm), and the solution was resuspended and replaced with the equilibrium solution of the reconstituted chromatography (filler model: Capto Core 700 from Cytiva).

[0045] Recombinant herpes zoster virus stock solution: Combibo, VZV-gE.

[0046] Example 1

[0047] This example uses a multi-step purification process to purify the recombinant herpes zoster virus stock solution:

[0048] 1) Primary Filtration: Prepare 500-1000 L of recombinant herpes zoster virus stock culture medium and perform deep filtration using a composite glass fiber filter element with a pore size of 0.8 + 0.45 μm at a flow rate of 0.5 L / min and a pressure of less than 15 psi. Then, filter through a 0.45 μm polyethersulfone membrane at a flow rate of 0.5 L / min and a pressure of less than 15 psi to obtain a clarified protein primary filtrate.

[0049] 2) Anion Exchange Chromatography: Using a Cytiva BPG450 column and Samc Q40 filler, the clarified protein filtrate was equilibrated with a phosphate buffer (pH 7.4) at 0.55 M NaCl. The sample load was no more than 250 g at a flow rate of 238 L / h to 477 L / h. The eluted protein was collected using an ion exchange eluent to obtain the initial protein solution.

[0050] 3) Inactivation: The initial protein solution was inactivated using 0.5% Tween-80 as an inactivator at 4°C for 16 hours.

[0051] 4) Hydrophobic chromatography: Hydrophobic column chromatography was performed using a Cytiva BPG200 column and PhentyI Chromstar HP packing. After adding anion purification inactivation solution to a final concentration of 20% ammonium sulfate, the column was equilibrated with 20% ammonium sulfate, 0.25 M PBS buffer for 4 column volumes before loading the sample. The sample volume was 200 g and the loading flow rate was 47 L / h-90 L / h. The flow rate was terminated when the flowthrough peak reached a certain height. The column was then flushed to the baseline with 20% ammonium sulfate, 0.25 M PBS buffer, and the elution peak was collected with 0.25 M PBS buffer.

[0052] 5) Ultrafiltration concentration: Use an ultrafiltration membrane with a pore size of 50 kDa to concentrate the purified solution, and resuspend and replace the solution with the reconstitution chromatography equilibrium solution five times.

[0053] 6) Composite chromatography: Use multimodal chromatography filler for composite chromatography, with a sample loading flow rate of 238 L / h-477 L / h, elute 5CV after loading, and collect the flow-through.

[0054] 7) The protein purity of the vaccine stock solution after the above purification process was tested by HPLC chromatography, such as Figure 1 As shown, the protein purity was 98.51%; the HCP content was 75.66 ppm; and the yield was 51%.

[0055] Example 2

[0056] This example uses a multi-step purification process to purify the recombinant herpes zoster protein stock solution:

[0057] 1) Primary Filtration: Prepare 500-1000 L of recombinant herpes zoster virus stock culture medium and perform deep filtration using a composite glass fiber filter element with a pore size of 0.8 + 0.45 μm at a flow rate of 0.5 L / min and a pressure of less than 15 psi. Then, filter through a 0.45 μm polyethersulfone membrane at a flow rate of 0.5 L / min and a pressure of less than 15 psi to obtain a clarified protein primary filtrate.

[0058] 2) Anion Exchange Chromatography: Use a Cytiva BPG450 column and Samc Q40 packing material. Equilibrate the clarified protein filtrate with 1.0 M NaCl in a phosphate buffer (pH 7.0) at this concentration before loading the sample. The sample volume should not exceed 250 g. The sample flow rate should be 6 ml / min. Elute with an ion exchange buffer and collect the eluent.

[0059] 3) Inactivation: The initial protein solution was inactivated at 2°C using 0.1% Tween 80 and tributyl phosphate (1:1) as an inactivator for 16 hours.

[0060] 4) Hydrophobic Chromatography: Hydrophobic column chromatography was performed using a Cytiva BPG200 column and PhentyI Chromstar HP packing. After adding anion purification inactivation solution to a final concentration of 10% ammonium sulfate, the column was equilibrated with 10% ammonium sulfate, 0.25 M PBS buffer for 4 column volumes before loading the sample. The sample volume was 200 g and the loading flow rate was 3 ml / min. The flow rate was terminated when the flowthrough peak reached a certain height. The column was then flushed to the baseline with 10% ammonium sulfate, 0.25 M PBS buffer, and the elution peak was collected with 0.25 M PBS buffer.

[0061] 5) Ultrafiltration concentration: Use an ultrafiltration membrane with a pore size of 50 kDa to concentrate the purified solution, and resuspend and replace the solution with the reconstitution chromatography equilibrium solution five times.

[0062] 6) Composite chromatography: Multimodal chromatography medium was used for composite chromatography. The sample loading flow rate was 10 ml / min. After loading, the sample was eluted for 5 CV and the flow-through was collected.

[0063] 7) The protein purity of the vaccine stock solution after the above purification process was tested by HPLC chromatography, such as Figure 2 As shown, the protein purity was 98.12%, the HCP content was 85.26 ppm, and the yield was 50%.

[0064] 8) Add the stock solution protective agent to the flow-through solution, filter it through 0.22 μm sterilization, and store it at -70°C after packaging.

[0065] Example 3

[0066] This example uses a multi-step purification process to purify the recombinant herpes zoster virus stock solution:

[0067] 1) Primary Filtration: Prepare 500-1000 L of recombinant herpes zoster virus stock culture medium and perform deep filtration using a composite glass fiber filter element with a pore size of 0.8 + 0.45 μm at a flow rate of 0.5 L / min and a pressure of less than 15 psi. Then, filter through a 0.45 μm polyethersulfone membrane at a flow rate of 0.5 L / min and a pressure of less than 15 psi to obtain a clarified protein primary filtrate.

[0068] 2) Anion Exchange Chromatography: Use a Cytiva BPG450 column and Samc Q40 packing material. Equilibrate the clarified protein filtrate with a phosphate buffer (pH 8.0) at a salinity of 0.05-0.1 M NaCl. Load no more than 250 g of sample at a flow rate of 6 ml / min. Elute with an ion exchange buffer and collect.

[0069] 3) Inactivation: The initial protein solution was inactivated using 1.0% Tween-80 as an inactivator at 2-8°C for 16 hours.

[0070] 4) Hydrophobic Chromatography: Hydrophobic column chromatography was performed using a Cytiva BPG200 column and PhentyI Chromstar HP packing. After adding anion purification inactivation solution to a final concentration of 40% ammonium sulfate, the column was equilibrated with 40% ammonium sulfate, 0.25 M PBS buffer for 4 column volumes before loading the sample. The sample volume was 200 g and the loading flow rate was 3 ml / min. The flow rate was terminated when the flowthrough peak reached a certain height. The column was then flushed to the baseline with 40% ammonium sulfate, 0.25 M PBS buffer, and the elution peak was collected with 0.25 M PBS buffer.

[0071] 5) Ultrafiltration concentration: Use an ultrafiltration membrane with a pore size of 50 kDa to concentrate the purified solution, and resuspend and replace the solution with the reconstitution chromatography equilibrium solution five times.

[0072] 6) Reconstitution chromatography: Multimodal chromatography medium was used for reconstitution chromatography. The sample was loaded at a flow rate of 10 ml / min. After loading, the sample was eluted for 5 CV and the flow-through was collected.

[0073] 7) The protein purity of the vaccine stock solution after purification by the above purification process was tested by HPLC chromatography, and the protein purity result was 98.03%; the HCP content was 85.65 ppm; and the yield was 52%.

[0074] 8) Add the stock solution protective agent to the flow-through solution, filter it through 0.22 μm sterilization, and store it at -70°C after packaging.

[0075] Example 4

[0076] This example uses a multi-step purification process to purify the recombinant herpes zoster virus stock solution:

[0077] 5 L of recombinant herpes zoster virus stock culture medium was prepared, and the other conditions were exactly the same as in Example 1. The obtained vaccine stock solution was tested for protein purity, and the protein purity result was 98.05%; the HCP content was 89.65 ppm; and the yield was 45%.

[0078] Example 5

[0079] This example uses a multi-step purification process to purify the recombinant herpes zoster virus stock solution:

[0080] 10 L of recombinant herpes zoster virus stock culture medium was prepared, and the other conditions were exactly the same as in Example 1. The obtained vaccine stock solution was tested for protein purity, and the protein purity result was 97.65%; the HCP content was 88.25 ppm; and the yield was 47%.

[0081] Comparative Example 1

[0082] Using the method disclosed in patent CN118702780A, a primary filtration sample of recombinant herpes zoster virus (primary filtration: 500 mL of recombinant herpes zoster virus stock culture medium was prepared and deep filtered using a composite glass fiber filter element with a pore size of 0.8 + 0.45 μm, a flow rate controlled at 0.5 L / min, and a pressure less than 15 psi. The sample was then filtered through a 0.45 μm polyethersulfone membrane at a flow rate controlled at 0.5 L / min and a pressure less than 15 psi to obtain a clarified protein primary filtrate) was sequentially purified using Diamond MIX-A, Diamond Butyl Mustang, Diamond Q, and Diamond MIX-AMustang as chromatographic media.

[0083] The protein yield obtained by purification was 13.25%, the protein purity was 97.02%; the HCP content was 180 ppm.

[0084] Comparative Example 2

[0085] Preliminary filtration of this comparative example: 500 mL of recombinant herpes zoster virus stock culture solution was prepared and filtered using a 0.5 μm microporous membrane. Other procedures were consistent with those in Example 1. The protein purity results were 60% in yield and 86.25% in protein purity; the HCP content was 380 ppm.

[0086] Comparative Example 3

[0087] This comparative example did not use anion exchange chromatography, and the other aspects were completely consistent with Example 1. The yield of the purified protein was 62%, the protein purity was 85.23%, and the HCP content was 280 ppm.

[0088] Comparative Example 4

[0089] In this comparative example, the equilibrium solution and eluent of anion exchange chromatography were both phosphate buffer with a pH of 7.4. Other aspects were consistent with those of Example 1. The yield of the purified protein was 35%, the protein purity was 85.23%, and the HCP content was 500 ppm.

[0090] Comparative Example 5

[0091] In this comparative example, the composite chromatography was replaced with Diamond Q chromatography and Diamond MIX-A Mustang chromatography. The elution conditions in this part were based on the method of patent CN118702780A. The purified protein had a yield of 37% and a protein purity of 75.23%; the HCP content was 350 ppm.

[0092] Comparative Example 6

[0093] This comparative example did not use hydrophobic chromatography, and the other procedures were identical to those in Example 1. The yield of the purified protein was 61%, the protein purity was 80.27%, and the HCP content was 260 ppm.

[0094] Comparative Example 7

[0095] This comparative example did not use composite chromatography, and other aspects were completely consistent with Example 1. The yield of the purified protein was 58%, the protein purity was 78.21%, and the HCP content was 350 ppm.

[0096] The present invention can be used to prepare a high-purity recombinant herpes zoster virus stock solution. The single processing amount of the ultrafiltration concentrate can reach 50 g, the recovery rate is high, the purity can reach more than 97%, and the HCP content is not higher than 100 ppm. It is suitable for the large-scale production of recombinant herpes zoster vaccine preparations requiring a purity greater than 98%.

[0097] The multi-step purification process of the present invention has a low degree of dependence on large-scale equipment, greatly reducing the purchase and maintenance costs of equipment, and also reducing the requirements for production sites.

[0098] The multi-step purification process of the present invention combines different chromatographic techniques, such as anion exchange chromatography and hydrophobic chromatography, and finally uses a composite chromatographic filler to more efficiently remove impurities and improve virus purity. Anion exchange chromatography provides initial virus purification, while hydrophobic chromatography further removes impurities such as host cell DNA.

[0099] The complex chromatography of the present invention is used as the last step of purification, combining the advantages of anion chromatography, hydrophobic chromatography and molecular sieve, further improving the purification effect, so that the final viral product purity is higher. In addition, the multi-step purification process can also greatly shorten the purification time. Compared with the traditional purification process, since the chromatography technology can remove a part of the impurities in each step, the multi-step purification process can achieve the expected purification effect faster and shorten the production cycle. In summary, the multi-step purification process has shown obvious advantages in the purification of viral vaccines, including low equipment investment, high purification efficiency, time saving, etc. These advantages make the multi-step purification process more suitable for the needs of vaccine industrialization.

[0100] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made thereto. Therefore, such modifications and improvements, without departing from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A method for purifying a recombinant herpes zoster virus stock solution, characterized in that: The following steps are involved: Step 1, anion exchange chromatography The clarified protein filtrate obtained by the primary filtration is eluted with an anion exchange chromatography eluent equilibrated with an equilibration solution and collected to obtain an initial protein solution; wherein the anion exchange chromatography column model is a Cytiva BPG450 column, and the filler is Samc Q40; The primary filtration is performed by deep filtration through a composite glass fiber, followed by filtration through a polyethersulfone membrane to obtain a clarified protein primary filtrate. The pore size of the composite glass fiber filter element is 0.8 ± 0.45 μm, and the pore size of the polyethersulfone membrane is 0.45 μm. The flow rate is controlled at 0.5 L / min and the pressure is less than 15 psi. The equilibrium solution is a mixture of phosphate buffer and sodium chloride solution; the sodium chloride concentration is 0.2-1.0 M; and the pH of the phosphate buffer is 7.0-8.

0. Step 2: Hydrophobic chromatography The inactivated solution of the initial protein solution after inactivation by an inactivator is eluted by hydrophobic chromatography, and the obtained eluate is concentrated to obtain a concentrated protein solution; wherein, the hydrophobic chromatography column uses a Cytiva BPG200 chromatography column filled with PhentyIChromstar HP; the anion purification inactivation solution is added to a final concentration of 10-40% ammonium sulfate, and the chromatography column is equilibrated with 0.25M PBS buffer for 4 column volumes before loading the sample, and the column is rinsed to the baseline with 10-40% ammonium sulfate and 0.25M PBS buffer, and the elution peak is collected with 0.25M PBS buffer; the purified solution is concentrated using a 50kDa pore size ultrafiltration membrane and resuspended with the reconstitution chromatography equilibration solution and the solution is exchanged 5 times; Step 3: Composite chromatography The concentrated protein solution was eluted using a multiplex chromatography using a multimodal chromatography filler. After loading the sample, elution was performed for 5CV, and the flow-through was collected. The obtained eluate was sterile filtered to obtain a purified recombinant herpes zoster protein stock solution. Among them, Capto Core 700 was selected as the multimodal chromatography filler.

2. The method for purifying a recombinant herpes zoster virus stock solution according to claim 1, wherein: In the step 2, the inactivator is Tween 80 or Tween 80 and tributyl phosphate.

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