Preparation method of recombinant poliovirus

Abstract

The invention relates to a preparation method of a poliovirus solution, which comprises the following steps in sequence: a) providing cells which exist in a culture medium; b) regulating the cells to a proper cell density, and then contacting the poliovirus with the cells to obtain a mixed solution of the poliovirus and the cells; c) centrifuging the mixed solution, and collecting supernate; and d) purifying the supernate through a chromatographic step to obtain the poliovirus solution.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, and in particular to a method for preparing and purifying recombinant poliovirus. BACKGROUND

[0002] Poliovirus belongs to the Enterovirus genus of the Picornaviridae family, which can invade the central nervous system and damage the motor neuron cells of the anterior horn of the spinal cord, thereby causing poliomyelitis. Therefore, it is named as such. The virus has no envelope and appears as a spherical particle under an electron microscope, with a diameter of about 20-30 nm. According to the different antigenic immunotypes, poliovirus is divided into three serotypes, I, II and III. Researchers have screened natural attenuated mutant strains of the virus through long-term passage studies, and have developed inactivated and attenuated live vaccines using attenuated strains as the virus, which have been widely promoted for vaccination in the global range to achieve the purpose of preventing and ultimately eliminating poliomyelitis. In addition to being used for vaccine preparation, recombinant viruses of poliovirus after modification have been found to have the property of specifically killing tumor cells. The recombinant virus establishes a cell infection process through the recognition of CD155 molecules, and CD155 molecules are found to be overexpressed in most tumor cells, which endows the recombinant poliovirus with tumor cell targeting. After molecular modification, the recombinant poliovirus exhibits even better safety and tolerability. Therefore, the recombinant poliovirus has good potential to become a new type of oncolytic virus.

[0003] At present, the traditional upstream preparation process of poliovirus often adds an appropriate amount of serum to maintain the state of the cells, but the use of serum in the current production process has many defects and risks. First, the serum used in the cell culture stage often has some differences between different batches, which may affect the batch consistency of the product, increasing the instability of the production process and the difficulty of quality control. Second, in the production process of biological products, the use of serum can introduce the risk of potential blood-borne pathogenic microbial contamination, thereby affecting the production process and safety of the product. Third, the serum composition is complex, and the various impurities contained therein, such as proteins, hormones, polypeptides, and cytokines, can increase the difficulty of the subsequent purification process of the virus product. Finally, serum is expensive, increasing the cost of scaling up the production process.

[0004] Meanwhile, the column chromatography used in the traditional downstream preparation process of poliovirus has the following disadvantages: 1. The column needs to be packed, especially the packing of a gel column needs the experience of an operator, and there is a possibility of packing failure, and there is a problem of packing consistency, and a large chromatography column is often needed for commercial production, which increases the challenge of packing. 2. The current chromatography process has a low load of poliovirus, and multiple cycles are often needed for a single unit operation. In particular, the production efficiency of gel chromatography is low, and the cleaning process is complicated, time-consuming, and has a bottleneck in capacity after a certain scale, which greatly limits the improvement of the production efficiency of large-scale commercial production in the later stage. 3. There are many factors affecting the chromatography process, resulting in great uncertainty in the amplification process. 4. The virus prepared by the traditional column chromatography has low purity, and the virus prepared has high content of host nucleic acid and host protein, and the traditional column chromatography may cause contamination of exogenous microorganisms, which affects the efficiency of chromatography and the purity of the virus.

[0005] Therefore, it is necessary to develop a cost-effective, stable and safe poliovirus preparation process with higher purification efficiency, which can improve the purity, activity and safety of poliovirus while meeting the needs of large-scale production of poliovirus. SUMMARY

[0006] The poliovirus preparation and purification method provided by the present application overcomes the defects in the traditional production process, provides a method of using serum-free culture to reduce external contamination, and provides a method of microcarrier culture to facilitate the expansion of production scale. Both methods can achieve batch consistency of the product, increase the stability and quality control of the production process, ensure the safety of the production process, reduce the possibility of external contamination, and control the production cost to facilitate the expansion of the production scale.

[0007] On the other hand, the traditional column chromatography purification method is optimized in the present application. The improved chromatography purification method used in the present application has simple unit operation, fewer purification steps, shorter purification time, and is easy to scale up and commercialize. At the same time, the activity of the virus is well maintained, the batch stability of the virus is significantly improved, and the host cell DNA (HCD) and host cell protein (HCP) are greatly reduced.

[0008] The present application provides a method for preparing a poliovirus solution, which comprises the following steps in the order shown:

[0009] a) providing cells, the cells being present in a culture medium;

[0010] b) adjusting the cells to a suitable cell density, and then contacting the poliovirus with the cells to obtain a mixed solution of poliovirus and cells;

[0011] c) centrifuging the mixed solution and collecting the supernatant;

[0012] d) purifying the supernatant by a chromatography step to obtain the poliovirus solution.

[0013] In one embodiment, the culture medium in a) comprises a serum-containing medium.

[0014] In one embodiment, the cells in a) are cultured using microcarriers.

[0015] In one embodiment, the cells further comprise a fed-batch culture such that the glucose concentration in the cell culture system is not less than about 1 g / L.

[0016] In one embodiment, the feeding mode of the feed comprises a discontinuous feeding.

[0017] In one embodiment, the discontinuous feeding is a daily medium exchange from the second day of culture.

[0018] In one embodiment, the culture medium in a) further comprises a serum-free medium.

[0019] In one embodiment, the serum-free medium comprises a chemically defined serum-free medium.

[0020] In one embodiment, the serum-containing medium comprises at least a serum derived from an animal selected from the group consisting of bovine, equine, chicken, ovine, and human.

[0021] In one embodiment, the serum comprises fetal bovine serum, calf serum, adult bovine serum, equine serum, chicken serum, ovine serum, or human serum.

[0022] In one embodiment, the suitable cell density in b) is about 1 x 10 5 cells / ml to about 2 x 10 6 cells / ml.

[0023] In one embodiment, the multiplicity of infection (MOI) in b) at which the cells are contacted with the poliovirus is about 0.01 to about 0.5.

[0024] In one embodiment, the multiplicity of infection (MOI) at which the cells are contacted with the poliovirus is about 0.01 to about 0.1.

[0025] In one embodiment, the multiplicity of infection (MOI) at which the cells are contacted with the poliovirus is about 0.01 to about 0.1.

[0026] In one embodiment, wherein the contact time is about 5-7 days at the multiplicity of infection (MOI) conditions described.

[0027] In one embodiment, wherein the multiplicity of infection (MOI) of the poliovirus is about 0.05 to about 0.2.

[0028] In one embodiment, wherein the mixed solution in b) comprises poliovirus, cells, cell debris, and / or media.

[0029] In one embodiment, wherein the viral titer in the supernatant in c) is at least 1.00E+06 CCID 50 / ml,

[0030] 2.00E+06 CCID 50 / ml, 3.00E+06 CCID 50 / ml, 4.00E+06 CCID 50 / ml, 5.00E+06 CCID 50 / ml,

[0031] 6.00E+06 CCID 50 / ml, 7.00E+06 CCID 50 / ml, 8.00E+06 CCID 50 / ml, 9.00E+06 CCID 50 / ml,

[0032] 1.00E+07 CCID 50 / ml, 2.00E+07 CCID 50 / ml, 3.00E+07 CCID 50 / ml, 4.00E+06 CCID 50 / ml,

[0033] 5.00E+07 CCID 50 / ml, 6.00E+07 CCID 50 / ml, 7.00E+07 CCID 50 / ml, 8.00E+07 CCID 50 / ml,

[0034] 9.00E+07 CCID 50 / ml, 1.00E+08 CCID 50 / ml, 2.00E+08 CCID 50 / ml, 3.00E+08 CCID 50 / ml,

[0035] 4.00E+08CCID 50 / ml, 5.00E+08CCID 50 / ml, 6.00E+08CCID 50 / ml, 7.00E+08CCID 50 / ml

[0036] 8.00E+08CCID 50 / ml, 9.00E+08CCID 50 / ml, 1.00E+09CCID 50 / ml, 2.00E+09CCID 50 / ml

[0037] 3.00E+09CCID 50 / ml, 4.00E+09CCID 50 / ml or 5.00E+09CCID 50 / ml.

[0038] In one embodiment, the viral titer in the supernatant described in c) is approximately 1.00E+06CCID. 50 / ml to 5.00E+09CCID 50 / ml.

[0039] In one embodiment, the chromatographic step in d) includes the use of ion exchange chromatography, affinity chromatography, and / or size exclusion chromatography.

[0040] In one embodiment, the chromatographic step in d) includes the use of gel column chromatography, monolithic column chromatography, and / or anion exchange chromatography.

[0041] In one embodiment, the purification in d) further includes a pretreatment performed prior to the chromatographic step in d).

[0042] In one embodiment, the pretreatment includes adding nucleases and / or filtration for sterilization.

[0043] In one embodiment, the pretreatment further includes ultrafiltration.

[0044] In one embodiment, the purification in d) is carried out when the pH of the supernatant in c) is approximately 6.0-10.0.

[0045] In one embodiment, the chromatographic step in d) further includes elution with an eluent.

[0046] In one embodiment, the elution includes using linear gradient elution and / or one-step elution.

[0047] In one embodiment, the pH of the eluent is approximately 5.0-8.0.

[0048] In one embodiment, the eluent includes an eluent salt, which includes NaCl, KCl, Na2SO4 and / or Na3PO4, K2SO4 and / or K3PO4.

[0049] In one embodiment, the elution salt is NaCl.

[0050] In one embodiment, the concentration of the elution salt is approximately 10-500 mM.

[0051] In one embodiment, the anion exchange chromatography is used after the gel column chromatography and / or the monolithic column chromatography.

[0052] In one embodiment, the cells are African green monkey kidney (Vero) cells.

[0053] In one embodiment, the poliovirus comprises wild-type poliovirus and / or its mutants, and the mutants comprise substitutions, mutations, insertions, and / or deletions of one or more genes.

[0054] This application also provides a poliovirus solution prepared by the above method, wherein the poliovirus solution has a virus titer of at least 1.00E+06CCID. 50 / ml, 2.00E+06CCID 50 / ml, 3.00E+06CCID 50 / ml, 4.00E+06CCID 50 / ml, 5.00E+06CCID 50 / ml, 6.00E+06CCID 50 / ml, 7.00E+06CCID 50 / ml

[0055] 8.00E+06CCID 50 / ml, 9.00E+06CCID 50 / ml, 1.00E+07CCID 50 / ml, 2.00E+07CCID 50 / ml

[0056] 3.00E+07CCID 50 / ml, 4.00E+06CCID 507.00E+07 CCID / ml 50 8.00E+07 CCID / ml 50 9.00E+07 CCID / ml

[0057] 7.00E+07 CCID / ml 50 8.00E+07 CCID / ml 50 9.00E+07 CCID / ml 50 1.00E+08 CCID / ml 50 2.00E+08 CCID / ml

[0058] 1.00E+08 CCID / ml 50 2.00E+08 CCID / ml 50 3.00E+08 CCID / ml 50 4.00E+08 CCID / ml 50 5.00E+08 CCID / ml

[0059] 1.00E+08 CCID / ml 50 2.00E+08 CCID / ml 50 3.00E+08 CCID / ml 50 4.00E+08 CCID / ml 50 5.00E+08 CCID / ml

[0060] 1.00E+08 CCID / ml 50 2.00E+08 CCID / ml 50 3.00E+08 CCID / ml 50 4.00E+08 CCID / ml 50 5.00E+08 CCID / ml 50 / ml.

[0061] The present application also provides a poliovirus solution prepared by the above method, wherein the virus titer of the poliovirus solution is about 1.00E+06 CCID 50 / ml to 5.00E+09 CCID 50 / ml.

[0062] The present application provides a method for preparing a poliovirus harvest, comprising the following steps in the order shown:

[0063] a) providing cells, wherein the cells are present in a culture medium;

[0064] b) adjusting the cells to a suitable cell density, and then contacting the poliovirus with the cells to obtain a mixed solution of poliovirus and cells;

[0065] c) obtaining the poliovirus harvest from the mixed solution.

[0066] In one embodiment, wherein the poliovirus harvest comprises poliovirus, cells and / or cell debris.

[0067] In one embodiment, wherein the medium in a) is a serum-containing medium.

[0068] In one embodiment, wherein the cells in a) further comprise culturing with microcarriers.

[0069] In one embodiment, wherein the cells further comprise fed-batch culturing such that the glucose concentration in the cell culture system is not less than about 1 g / L.

[0070] In one embodiment, wherein the feeding mode of the feed comprises discontinuous feeding.

[0071] In one embodiment, wherein the discontinuous feeding is daily medium exchange from the second day of culturing.

[0072] In one embodiment, wherein the medium in a) is a serum-free medium.

[0073] In one embodiment, the serum-free medium comprises a chemically defined serum-free medium.

[0074] In one embodiment, wherein the serum-containing medium comprises serum from at least one animal source selected from the group consisting of bovine, equine, chicken, ovine and human.

[0075] In one embodiment, the serum comprises fetal bovine serum, calf serum, young bovine serum, equine serum, chicken serum, ovine serum or human serum.

[0076] In one embodiment, wherein the suitable cell density in b) is about 1 x 10 5 cells / ml to about 2 x 10 6 cells / ml.

[0077] In one embodiment, wherein the multiplicity of infection (MOI) of poliovirus in b) is about 0.01 to about 0.5.

[0078] In one embodiment, wherein the contact time under the multiplicity of infection (MOI) of poliovirus in b) is about 3 to 6 days.

[0079] In one embodiment, wherein the multiplicity of infection (MOI) of poliovirus in b) is about 0.01 to about 0.1.

[0080] In one embodiment, wherein the contact time is about 5-7 days at the multiplicity of infection (MOI) conditions described.

[0081] In one embodiment, wherein the multiplicity of infection (MOI) of the poliovirus is about 0.05 to about 0.2.

[0082] In one embodiment, wherein the mixed solution in b) comprises poliovirus, cells, cell debris, and / or culture medium.

[0083] In one embodiment, wherein the virus titer of the poliovirus harvest in c) is at least 1.00E+06 CCID 50 / ml, 2.00E+06 CCID 50 / ml, 3.00E+06 CCID 50 / ml, 4.00E+06 CCID 50 / ml, 5.00E+06 CCID 50 / ml, 6.00E+06 CCID 50 / ml, 7.00E+06 CCID 50 / ml, 8.00E+06 CCID 50 / ml, 9.00E+06 CCID 50 / ml, 1.00E+07 CCID 50 / ml, 2.00E+07 CCID 50 / ml, 3.00E+07 CCID 50 / ml, 4.00E+06 CCID 50 / ml, 5.00E+07 CCID 50 / ml, 6.00E+07 CCID 50 / ml, 7.00E+07 CCID 50 / ml, 8.00E+07 CCID 50 / ml, 9.00E+07 CCID 50 / ml, 1.00E+08 CCID 50 / ml, 2.00E+08 CCID 50 / ml, 3.00E+08 CCID 50 / ml, 4.00E+08 CCID 50 / ml, 5.00E+08 CCID 50 / ml, 6.00E+08 CCID 50 / ml, 7.00E+08 CCID 50 / ml, 8.00E+08 CCID 50 / ml, 9.00E+08 CCID50 50 / ml, 1.00E+09 CCID50 50 / ml, 2.00E+09 CCID50 50 / ml, 3.00E+09 CCID50 50 / ml, 4.00E+09 CCID50 50 / ml, or 5.00E+09 CCID50 50 / ml.

[0084] In one embodiment, wherein the virus titer of the poliovirus harvest of c) is from about 1.00E+06 CCID50 50 / ml to 5.00E+09 CCID50 50 / ml.

[0085] In one embodiment, wherein the cell is a Vero cell.

[0086] In one embodiment, the poliovirus comprises a wild-type poliovirus and / or a mutant thereof, and the mutant comprises one or more substitutions, mutations, insertions, and / or deletions of a gene.

[0087] The present application also provides a method of purifying a poliovirus, comprising performing a chromatography step on a mixed solution of poliovirus and cells.

[0088] In one embodiment, wherein the virus titer of the mixed solution is at least 1.00E+06 CCID50 50 / ml,

[0089] 2.00E+06 CCID50 50 / ml, 3.00E+06 CCID50 50 / ml, 4.00E+06 CCID50 50 / ml, 5.00E+06 CCID50 50 / ml,

[0090] 6.00E+06 CCID50 50 / ml, 7.00E+06 CCID50 50 / ml, 8.00E+06 CCID50 50 / ml, 9.00E+06 CCID50 50 / ml,

[0091] 1.00E+07 CCID50 50 / ml, 2.00E+07 CCID50 50 / ml, 3.00E+07 CCID50 50 / ml, 4.00E+06 CCID 50 / ml,

[0092] 5.00E+07 CCID 50 / ml, 6.00E+07 CCID 50 / ml, 7.00E+07 CCID 50 / ml, 8.00E+07 CCID 50 / ml,

[0093] 9.00E+07 CCID 50 / ml, 1.00E+08 CCID 50 / ml, 2.00E+08 CCID 50 / ml, 3.00E+08 CCID 50 / ml,

[0094] 4.00E+08 CCID 50 / ml, 5.00E+08 CCID 50 / ml, 6.00E+08 CCID 50 / ml, 7.00E+08 CCID 50 / ml,

[0095] 8.00E+08 CCID 50 / ml, 9.00E+08 CCID 50 / ml, 1.00E+09 CCID 50 / ml, 2.00E+09 CCID 50 / ml,

[0096] 3.00E+09 CCID 50 / ml, 4.00E+09 CCID 50 / ml, or 5.00E+09 CCID 50 / ml of poliovirus.

[0097] In one embodiment, wherein the mixed solution has a viral titer of about 1.00E+06 CCID 50 / ml to 9.00E+08 CCID 50 / ml.

[0098] In one embodiment, wherein the mixed solution comprises poliovirus, cells, cell debris, and / or culture medium.

[0099] In one embodiment, wherein the cells comprise cells obtained using serum culture, serum-free culture, and / or microcarrier culture.

[0100] In one embodiment, it further comprises centrifuging the poliovirus mixed solution to obtain a supernatant.

[0101] In one embodiment, it further comprises that the supernatant pH is about 6.0-10.0 when performing the chromatography step.

[0102] In one embodiment, wherein the chromatography step comprises using ion exchange chromatography, affinity chromatography and / or size exclusion chromatography.

[0103] In one embodiment, wherein the purification further comprises pre-treatment.

[0104] In one embodiment, wherein the pre-treatment comprises adding nuclease and / or filter sterilization.

[0105] In one embodiment, wherein the pre-treatment further comprises ultrafiltration.

[0106] In one embodiment, wherein the chromatography step comprises using gel column chromatography, monolithic column chromatography and / or anion exchange chromatography.

[0107] In one embodiment, the chromatography step further comprises using eluent for elution.

[0108] In one embodiment, wherein the elution mode of the eluent is linear gradient elution and / or one-step elution.

[0109] In one embodiment, wherein the pH of the eluent is about 5.0-8.0.

[0110] In one embodiment, wherein the eluent comprises elution salt, and the elution salt comprises NaCl, KCl, Na2SO4 and / or Na3PO4, K2SO4 and / or K3PO4.

[0111] In one embodiment, wherein the elution salt is NaCl.

[0112] In one embodiment, wherein the concentration of the elution salt is about 10-500 mM.

[0113] In one embodiment, the anion exchange chromatography is used after the gel column chromatography and / or the monolithic column chromatography.

[0114] In one embodiment, wherein the cell is Vero cell.

[0115] In one embodiment, the poliovirus comprises wild-type poliovirus and / or its mutant, and the mutant comprises substitution, mutation, insertion and / or deletion of one or more genes.

[0116] In one embodiment, the purified poliovirus has a viral titer of at least 1.00E+06 CCID 50 / ml, 2.00E+06 CCID 50 / ml, 3.00E+06 CCID 50 / ml, 4.00E+06 CCID 50 / ml, 5.00E+06 CCID 50 / ml, 6.00E+06 CCID 50 / ml, 7.00E+06 CCID 50 / ml, 8.00E+06 CCID 50 / ml, 9.00E+06 CCID 50 / ml, 1.00E+07 CCID 50 / ml, 2.00E+07 CCID 50 / ml, 3.00E+07 CCID 50 / ml, 4.00E+06 CCID 50 / ml, 5.00E+07 CCID 50 / ml, 6.00E+07 CCID 50 / ml, 7.00E+07 CCID 50 / ml, 8.00E+07 CCID 50 / ml, 9.00E+07 CCID 50 / ml, 1.00E+08 CCID 50 / ml, 2.00E+08 CCID 50 / ml, 3.00E+08 CCID 50 / ml, 4.00E+08 CCID 50 / ml, 5.00E+08 CCID 50 / ml, 6.00E+08 CCID 50 / ml, 7.00E+08 CCID 50 / ml, 8.00E+08 CCID 50 / ml, 9.00E+08 CCID 50 / ml, 1.00E+09 CCID 50 / ml, 2.00E+09 CCID 50 / ml, 3.00E+09 CCID 50 / ml, 4.00E+09 CCID 50 / ml, or 5.00E+09 CCID 50 / ml.

[0117] In one embodiment, the purified poliovirus has a viral titer of about 1.00E+06 CCID 50 / ml to 5.00E+09 CCID 50 / ml.

[0118] The present application also provides a pharmaceutical composition comprising a poliovirus solution prepared using the method of claim as previously described, and / or a purified poliovirus prepared by the method as previously described, and optionally a pharmaceutically acceptable adjuvant.

[0119] The present application also provides the use of a poliovirus solution prepared according to the method as previously described, a purified poliovirus prepared by the method as previously described, and / or a pharmaceutical composition as previously described, for the manufacture of a medicament for the prevention, alleviation and / or treatment of a disease and / or a condition.

[0120] The present application also provides a method for the treatment, alleviation and / or prevention of a disease and / or a condition, the method comprising administering to a patient in need a therapeutic amount of a poliovirus solution prepared by the method as previously described, a purified poliovirus prepared by the method as previously described, and / or a pharmaceutical composition as previously described.

[0121] The present application also provides a poliovirus solution prepared according to the method as previously described, a purified poliovirus prepared by the method as previously described, and / or a pharmaceutical composition as previously described, for use in the treatment, alleviation and / or prevention of a disease and / or a condition.

[0122] Other aspects and advantages of the present application will become readily apparent to those skilled in the art from the following detailed description, wherein only the preferred embodiment of the application is shown and described. As will be realized, the application is capable of modifications in various obvious aspects, all without departing from the spirit and scope of the application as described. Accordingly, the drawings and descriptions should be regarded as illustrative in nature and not restrictive. BRIEF DESCRIPTION OF DRAWINGS

[0123] The specific features of the application involved are shown in the appended claims. The characteristics and advantages of the application involved can be better understood by referring to the detailed description of the exemplary embodiments and to the attached drawings. The drawings are briefly described as follows:

[0124] Figure 1 The figure shows the cell growth curves for each group of microcarrier cell culture.

[0125] Figure 2 The figure shows the titer summary for each group of microcarrier culture process at different days post infection. Detailed Implementation

[0126] The following specific embodiments illustrate the implementation of the invention. Those skilled in the art can easily understand other advantages and effects of the invention from the content disclosed in this specification.

[0127] Terminology Definition

[0128] As used herein, the term "poliovirus" refers to a virus belonging to the genus Enterovirus of the family Picornaviridae. The poliovirus includes wild-type polioviruses of type I, II, or III. The poliovirus also includes recombinant polioviruses, such as attenuated variants of poliovirus, for example, the variant of poliovirus constructed by Ming Te Yeh et al. (Geneticstabilization of attenuated oral vaccines against poliovirus types 1 and 3, Nature 2023 Jul; 619(7968):135-142.). In one embodiment, the poliovirus is an attenuated poliovirus (with lower neurotoxicity), such as the Sabin strain (or possibly type I, II, or III Sabin strains). In one embodiment, the recombinant poliovirus is a variant of an attenuated poliovirus, such as a poliovirus modified with Sabin I as its backbone. Its preparation method can be any recombinant poliovirus vaccine preparation process known to those skilled in the art. In poliovirus preparation processes, viral titer (or potency, i.e., the median infectious dose in cell culture, CCID) is typically measured. 50 Poliovirus activity was assessed using viral titer ( / ml), viral recovery rate, and viral RNA copy number. Viral titer refers to the amount of virus per unit volume of liquid that infects half of the cultured cells within a given time period, and is used to measure viral activity. Viral RNA copy number typically refers to the number of viral RNA molecules in a specific volume of sample, and is used to assess viral abundance.

[0129] As used herein, the term "suitable cell density" refers to a cell density at which cells are in a healthy growth state at the time of viral inoculation and have sufficient space for viral replication after inoculation, i.e., at which viral inoculation is performed such that the degree of cytopathic effect is at least 70%, 75%, 80%, 85%, 90%, or 95%. In this application, the suitable cell density is approximately 1 × 10⁻⁶ cells / year. 5 cells / ml - approximately 2 × 10 6 cells / ml.

[0130] As used herein, the term "contacting" means bringing a molecule within the physical proximity of a second molecule, either directly or indirectly. The molecule can be in contact with the second molecule through a buffer, oxygen, a solution or a medium, etc. In the present application, the contacting includes, for example, placing a virus in a culture vessel containing a cell. The contacting also includes, for example, placing an antibody in a beaker, microtiter plate, cell culture flask, or microarray, etc. containing a polypeptide. The contacting described in the present application can occur in vivo, ex vivo, or in vitro. The day of the contacting is day 0.

[0131] The term "medium" refers to a source of nutrients for cell growth or maintenance. As understood by one of skill in the art, the source of nutrients can contain components required for cell growth and / or survival, or can contain components that aid in cell growth and / or survival. Vitamins, essential or non-essential amino acids (e.g., cysteine and cystine), and trace elements (e.g., copper) are examples of medium components. Any of the media provided herein can also be supplemented with any one or more of insulin, plant hydrolysate, and animal hydrolysate.

[0132] As used herein, the term "serum-containing medium" refers to a cell culture medium in which there is animal serum, such as fetal bovine serum, calf serum, adult bovine serum, horse serum, chicken serum, sheep serum, or human serum, in the medium. The serum-containing medium can be serum added to any basal medium commonly known to one of skill in the art, such as DMEM, Ham's F12, Medium 199, McCoy's, or RPMI. The basal medium can comprise a number of components, including amino acids, vitamins, organic and inorganic salts, and a carbohydrate source, each present in an amount to support cell culture, which is commonly known to one of skill in the art. In one embodiment, the cell is a cell used for culturing poliovirus. In one embodiment, the cell is a vero cell.

[0133] As used herein, the term "microcarrier" refers to a micro-particle used for cell culture, composed of natural dextran or synthetic polymers, which enables cells to attach to its surface and grow as a monolayer when in suspension, thereby increasing the area for cell attachment growth. The microcarriers described in the present application have a diameter between 60-250 μm and exist in a suspended state during the culture process. The microcarriers described in the present application include, but are not limited to, commercial microcarriers such as Cytodex series, Cytopore, Cytoline, etc. In one embodiment, the microcarrier is a spherical microcarrier Cytodex-1.

[0134] As used herein, the term "serum-free medium" refers to a cell culture medium that does not contain animal serum such as fetal bovine serum, bovine serum albumin, or human serum albumin. In one embodiment, the serum-free medium is a chemically defined, serum-free and animal protein-free medium. The animal protein-free medium refers to a cell culture medium that does not contain proteins and / or protein components such as albumin, transferrin, insulin, or growth factors from multicellular, non-plant, higher eukaryotes. Animal proteins and protein components are distinguished from non-animal proteins, small peptides, and oligopeptides that can be included in the animal protein-free cell culture medium according to the present application, which are obtainable from plants (typically 10-30 amino acids in length) or from lower eukaryotes such as yeast. Serum-free media suitable for use in the present application can be well known to those skilled in the art and are generally readily available from commercial sources or prepared under standard methods from conventional ingredients. Serum-free media that can be used in the present application include, but are not limited to, custom serum-free media for vero cells. In one embodiment, the present application uses a chemically defined serum-free medium, for example, NutriVero FlexlO, CD Vero-S. In one embodiment, the present application can also use the Pesche series of media, VERO SFM01, VERO SFM01 pro serum-free medium, OPM-VERO SFM6C3. In one embodiment, the present application uses an ultra-low protein serum-free medium, for example, VP-SFM AGT. In one embodiment, the present application uses a serum-free medium containing HEPES and sodium bicarbonate buffer, for example, ProVero-1 NAO, and the like.

[0135] As used herein, the term "feed" refers to the addition of extra nutrients to promote the growth of cells during cell culture. In one embodiment, the feed used in the present application includes carbon source nutrients. For example, the carbon source nutrient is glucose. The feed used in the present application can also include amino acids, vitamins, antibiotics, pH buffers, trace elements, inorganic salts, and / or nitrogen source nutrients. In one embodiment, the feed used in the present application is a complete medium. The addition of feed can be by methods known in the art, for example, continuous feed, intermittent feed addition, semi-continuous exchange feed, or discontinuous exchange feed. Continuous feed refers to the constant addition of feed to the culture system at a certain rate. Intermittent feed addition refers to the periodic addition of a certain amount of nutrients rather than continuous addition. Semi-continuous exchange feed refers to the periodic removal of part of the culture broth containing the product, followed by the addition of the same volume of feed. Discontinuous exchange feed refers to the addition of feed to the culture system as needed for cell growth or metabolism, to extend the growth phase of the cells.

[0136] As used herein, the term "purification" or "purifying" or "isolation" or "isolating" refers to the separation of viral particles from a mixture of viruses and removal of other cellular components, proteins, nucleic acids, lipids, and the like impurities to obtain a high purity of the virus. In one embodiment, the purification of the present application refers to the separation and purification of viral particles by chromatography steps, such as separation of viruses by chromatographic methods such as ion exchange chromatography, affinity chromatography, molecular exclusion chromatography, and the like. In one embodiment, the purification separates and purifies viral particles by using one type of chromatographic method alone. For example, purification of viral particles by using molecular exclusion chromatography. For example, purification of viral particles by using more than one type of molecular exclusion chromatography. For example, purification of viral particles by using ion exchange chromatography. For example, purification of viral particles by using more than one type of ion exchange chromatography. In one embodiment, the purification separates and purifies viral particles by a combination of chromatographic methods. For example, ion exchange chromatography can be combined with molecular exclusion chromatography to purify viral particles.

[0137] As used herein, the term "ion exchange chromatography" refers to a separation technique based on the reversible exchange of ions between the sample and the opposite charge on the ion exchanger, such as ion exchange chromatography can be cation exchange chromatography, can be anion exchange chromatography. In one embodiment, the ion exchange chromatography includes ion exchange resin chromatography. In one embodiment, the ion exchange chromatography includes monolithic column chromatography. In one embodiment, the monolithic column chromatography comprises a porous structure of a continuous monolith. For example, the porous structure of the monolithic column chromatography can be composed of porous polymethyl methacrylate. In one embodiment, the ion exchange chromatography includes strong anion exchange chromatography, such as Super Q 650M anion exchange chromatography. The packing of the ion exchange chromatography described in the present application can also include, but is not limited to, SP (sulfopropyl), CM (carboxymethyl), Q (quaternary ammonium group), or DEAE (diethylaminoethyl). The use of QA is basically the same as Q, and is also commonly used in strong anion exchange chromatography. In some cases, QA and Q can be used interchangeably to refer to the same stationary phase group (quaternary ammonium group).

[0138] As used herein, the term “monolithic column” refers to a chromatographic column with a continuous porous monolithic structure, which can be used without the need for packing the chromatographic column step. The monolithic column is divided into organic monolithic column and inorganic monolithic column. In one embodiment, the present application uses organic monolithic column. For example, the present application uses polymethyl methacrylate monolithic column chromatography. In one embodiment, the monolithic column used by the present application can also carry functional groups. For example, with QA functional groups. For example, with DEAE functional groups. DEAE is a weak anion exchange group, and QA (quaternary ammonium group) is a strong anion exchange group. For example, the monolithic column can be a form of chromatography for separation using anion exchange, in which case the monolithic column can be referred to as anion exchange monolithic column chromatography, which can be a special form of anion exchange chromatography, which uses monolithic column as carrier, so that the separation efficiency is higher, the flow resistance is smaller, and it is particularly suitable for separating macromolecules such as proteins and nucleic acids. The core of both is to separate negatively charged molecules by positively charged groups, but the introduction of monolithic column enhances the separation performance and speed of anion exchange chromatography.

[0139] As used herein, “BAKERBOND poly QUAT” is a commercially available strong anion exchange chromatography packing;

[0140] “NW Rose Viral M” and “Cytiva Core 400” are two fillers of anion exchange chromatography commercially available; “Super Q 650M” represents a high-performance anion exchange chromatography packing suitable for the purification of biological macromolecules such as viruses, proteins, etc. The anion exchange chromatography packing used in the present application includes but is not limited to other anion exchange resins or membranes functionally equivalent to the above-mentioned fillers.

[0141] As used herein, the term “molecular exclusion chromatography” refers to a chromatographic technique that separates molecules according to their size in solution. In one embodiment, the packing of molecular exclusion chromatography is gel. For example, the molecular exclusion chromatography packing can be dextran gel, can be polyacrylamide gel, can be agarose gel, can be hydrophilic gel.

[0142] As used herein, the term "pre-treatment" refers to pre-treating the sample before purification, preliminary purification of cell debris, unlysed tissue components, and other insoluble impurities in the sample to avoid the effect of these impurities on the purification result or clogging the chromatography column in the subsequent steps. In one embodiment, the pre-treatment can be addition of a nuclease. The nuclease can be a DNA-removing nuclease, can be an RNA-removing nuclease. For example, the nuclease is Benzonase®. In one embodiment, the pre-treatment can be ultrafiltration concentration of the virus particles. For example, the ultrafiltration concentration can be hollow fiber ultrafiltration concentration of polyvinylidene fluoride (PVDF), polyethersulfone (PES), or polysulfone (PSF). The hollow fiber membrane used herein has a larger membrane area than a conventional hollow fiber membrane, for example, the membrane area is at least 150%-200% larger than the original membrane area. In one embodiment, the pre-treatment can be filter sterilization, wherein the filtration can be removal of large particles through a 0.22 μιη, 0.45 μιη, or 0.8 μιη filter.

[0143] As used herein, the term "cell" refers to its meaning as generally accepted in the art. For exemplary nucleic acid molecules of the application, the term is used in its usual biological sense and does not refer to an intact multicellular organism, e.g., specifically not to a human. Cells can be present within an organism, e.g., a bird, a plant, and a mammal, e.g., a human, a cow, a sheep, an ape, a monkey, a pig, a dog, and a cat. Cells can be prokaryotic (e.g., bacterial cells) or eukaryotic (e.g., mammalian or plant cells). Cells can be of somatic or germ line origin, totipotent or pluripotent, dividing or non-dividing. Cells can also be derived from or can comprise a gamete or an embryo, a stem cell, or a fully differentiated cell.

[0144] As used herein, the term "cell debris" refers to non-viral components released from cells upon death or injury. In one embodiment, the cell debris includes, but is not limited to, organelle fragments, cell membrane fragments, proteins, and inclusion bodies, etc. In one embodiment, the cell debris mainly includes host nucleic acids and host proteins.

[0145] As used herein, the term "virus harvest" refers to a solution containing virus particles collected from the cultured cells during the virus culture process, which typically contains viruses, cell debris, culture medium residues, and other possible impurities. The term "mixed solution" refers to a solution containing cells and virus particles, which can contain viruses, cell debris, culture medium residues, stabilizers, and other possible virus preservation additives. The term "supernatant" refers to a clarified solution obtained by centrifugation or other pre-treatment of a solution containing virus particles, which can be directly used for chromatography purification. The term "virus stock" or "DS" refers to a high-activity virus solution after a purification step, which is further concentrated, filter sterilized, etc., to finally obtain a virus stock, which can be used for formulation.

[0146] As used herein, the term "about" means ± 50% of the recited value. DETAILED DESCRIPTION

[0148] The present application is directed to the development of a method for purifying any poliovirus or derivative thereof in useful quantities, particularly poliovirus or variants thereof for use as vaccines or viral vectors for laboratory or industrial scale needs. The present application improves upon the deficiencies of existing purification methods that use serum culture, low efficiency column chromatography purification, making the scale-up of poliovirus production purification process simpler and more efficient, and effectively reducing the purification time, significantly improving the production efficiency.

[0149] In one aspect, the present application provides a method for purifying poliovirus or variants thereof, comprising the steps in the order shown:

[0150] a) providing cells, said cells being present in a culture medium;

[0151] b) adjusting the cells to a suitable cell density, and then contacting the poliovirus with the cells to obtain a mixed solution of poliovirus and cells;

[0152] c) centrifuging the mixed solution to collect the supernatant;

[0153] d) purifying the supernatant by a chromatography step to obtain a solution of said poliovirus.

[0154] In certain embodiments, the poliovirus is a wild-type virulent poliovirus, such as a type I poliovirus, a type II poliovirus, or a type III poliovirus. In certain embodiments, the poliovirus is a type I poliovirus Mahoney or Brunenders, a type II poliovirus type MEF (or MEF-1), or a type III poliovirus Saukett. In certain embodiments, the poliovirus is a naturally attenuated poliovirus, such as a live attenuated poliovirus (less neurovirulent), such as a Sabin strain (possibly a type I, type II, or type III Sabin strain). In one embodiment, the recombinant poliovirus is a variant of a live attenuated poliovirus, such as a recombinant poliovirus engineered on the backbone of Sabin I.

[0155] Virus culture

[0156] To overcome the batch-to-batch variability caused by traditional virus culture processes, reduce the contamination of exogenous factors, increase the safety of the product, reduce the instability of the production process and the cost of scale-up, and achieve larger-scale production of poliovirus, the present application improves the virus culture method.

[0157] The cells used in the present application are African green monkey kidney cells (vero cells) and the methods of resuscitating and passaging the cells are any methods known to those skilled in the art.

[0158] For example, the frozen vero cells are resuscitated in complete medium, which includes 90% DMEM and 10% FBS, and when the cells grow to 80-90% confluency, they are passaged using 0.25% trypsin for digestion, followed by the addition of complete medium to stop the digestion, and then passaged by centrifugation, discarding the supernatant, resuspension, etc.

[0159] In certain embodiments, the present application includes culturing cells using serum-free culture methods for the production of poliovirus.

[0160] The serum-free culture methods used in the present application mean that the serum is absent from the culture medium used for cell growth and infection. In one embodiment, the serum-free culture methods are performed without any components of direct animal origin (e.g., serum or serum components, etc.). In one embodiment, the serum-free culture methods are performed in chemically defined media. In one embodiment, any additives supplemented to the culture medium during the infection of the virus also do not contain components of animal origin. The serum-free culture can be performed in a batch, fed-batch, continuous culture system, etc. in a culture dish, a spinner flask, or a bioreactor. The serum-free culture media suitable for use in the present application can be well known to those skilled in the art and are generally available in large quantities from commercial channels or prepared according to standard methods under conventional conditions. The serum-free culture media that can be used in the present application include, but are not limited to, custom serum-free media for vero cells. In one embodiment, the present application uses a chemically defined serum-free culture medium, such as NutriVero Flex10, CD Vero-S. In one embodiment, the present application can also use VEROSFM01, VERO SFM01 pro serum-free medium, Pesche series culture, OPM-VERO SFM6C3. In one embodiment, the present application uses a serum-free culture medium with ultra-low protein, such as VP-SFM AGT. In one embodiment, the present application uses a serum-free culture medium containing HEPES and sodium bicarbonate buffer, such as ProVero-1 NAO, etc.

[0161] In the methods of the present application, lipids and / or hydrolysates and / or other supplements can be supplemented to the serum-free culture medium to further improve the yield.

[0162] In other embodiments, the present application includes culturing cells using microcarrier culture methods for the production of poliovirus.

[0163] Microcarriers suitable for use in the present application are known to those skilled in the art and are generally available commercially in large quantities or can be prepared under standard conditions according to standard methods. Microcarriers described herein include, but are not limited to, Cytodex series, Cytopore, Cytoline, and the like. In one embodiment, the microcarriers are spherical microcarriers Cytodex-1.

[0164] The amount of microcarriers used in the present application balances the cost and viral infection titer, i.e., the viral harvest titer is at least 4-fold higher than that of conventional methods. In certain embodiments, the amount of microcarriers used in the present application is controlled to be about 2 g / L to about 6 g / L. In one embodiment, the amount of microcarriers used in the present application is controlled to be about 2 g / L to about 4 g / L.

[0165] The present application also includes a method for improving the viability of cells grown in microcarrier conditions, comprising adding a feed to the culture medium containing microcarriers, the feed resulting in a sugar content in the culture system of no less than about 0.5 g / L, about 1 g / L, about 1.5 g / L, about 2 g / L, or more.

[0166] In one embodiment, the feed used in the present application includes a carbon source nutrient. For example, the carbon source nutrient is glucose.

[0167] In one embodiment, the feed used in the present application can also include amino acids, vitamins, antibiotics, pH buffers, trace elements, inorganic salts, and / or nitrogen source nutrients.

[0168] In one embodiment, the feed used in the present application is fresh complete medium.

[0169] The method of feed addition in the present application can be any method known in the art, such as continuous feed, intermittent feed addition, semi-continuous exchange feed, or discontinuous exchange feed.

[0170] In one embodiment, the feed used in the present application is added by discontinuous exchange feed.

[0171] The microcarrier culture method used in the present application provides an increase of nearly 30% in the total amount of cells before infection, a 4-fold increase in viral harvest titer, and a significant reduction in the volume of harvest liquid compared to conventional cell factory processes, which is more conducive to subsequent purification operations.

[0172] In certain embodiments, the present application also improves the viral infection parameters in small-scale processes and scaled-up processes, respectively.

[0173] The methods described herein for infecting cells with poliovirus and / or for propagating virus are suitably performed at a temperature, e.g., about 33°C to 38°C. When the cells have a density of about 1 x 105 about 2 x 10 5 about 3 x 10 5 about 4 x 10 5 about 5 x 10 5 about 6 x 10 5 about 7 x 10 5 about 8 x 10 5 about 9 x 10 5 about 1 x 10 6 about 2 x 10 6 about 3 x 10

[0174] The cells are contacted with the poliovirus so that the poliovirus infects the cells and propagates. For example, a batch of poliovirus seed is added to pre-warmed virus maintenance medium, which is then added to the cell culture and allowed to adsorb to the cells, with constant agitation, and the virus culture is started.

[0175] In the methods described herein, the poliovirus can infect the cells at a multiplicity of infection (MOI) of from about 0.001 to about 10. In certain embodiments, the infection can be at an MOI of from about 0.01 to about 0.5. In one embodiment, the infection is at an MOI of from about 0.01 to about 0.1. In one embodiment, the infection is at an MOI of from about 0.05 to about 0.2.

[0176] In the methods described herein, the poliovirus is harvested within about 0.5 days to about 10 days after infection. In one embodiment, the poliovirus is harvested within about 3 days to about 6 days after infection. In one embodiment, the poliovirus is harvested within about 5 days to about 7 days after infection.

[0177] After the poliovirus has propagated in the cells, the virus or components thereof are harvested from the cell culture. This can be accomplished by conventional methods known to those of ordinary skill in the art. Virus produced and released in the cell culture medium can be separated from the biological tissue of the cells by conventional methods such as centrifugation or filtration, and harvested in the supernatant. In this case, the centrifugation or filtration is the harvesting step. The virus can be harvested using conventional methods, for example, at the end of the culture by collecting the medium containing the virus suspension, which can be filtered using, for example, a 0.22 μm, 0.45 μm, or 0.8 μm filter and optionally stored at 4°C.

[0178] In the methods described herein, viral infection of cells can be performed at the scale of one cell factory, ten cell factories, or ten ten cell factories. The scale of the cell factories can also be adjusted as needed for poliovirus, for example, to scale up to pilot or factory scale.

[0179] In the viral culture methods used herein, the viral titer in the harvest fluid can be at least about 1.00E+06 CCID 50 / ml, about 2.00E+06 CCID 50 / ml, about 3.00E+06 CCID 50 / ml, about 4.00E+06 CCID 50 / ml, about 5.00E+06 CCID 50 / ml, about 6.00E+06 CCID 50 / ml, about 7.00E+06 CCID 50 / ml, about 8.00E+06 CCID 50 / ml, about 9.00E+06 CCID 50 / ml, 1.00E+07 CCID 50 / ml, about 2.00E+07 CCID 50 / ml, about 3.00E+07 CCID 50 / ml, about 4.00E+06 CCID 50 / ml, about 5.00E+07 CCID 50 / ml, 6.00E+07 CCID 50 / ml, 7.00E+07 CCID 50 / ml, 8.00E+07 CCID 50 / ml, 9.00E+07 CCID 50 / ml, 1.00E+08 CCID 50 / ml, 2.00E+08 CCID 50 / ml, 3.00E+08 CCID 50 / ml, 4.00E+08 CCID 50 / ml, 5.00E+08 CCID 50 / ml, 6.00E+08 CCID 50 / ml, 7.00E+08 CCID 50 / ml, 8.00E+08 CCID 50 / ml, 9.00E+08 CCID 50 / ml, 1.00E+09 CCID 50 / ml, 2.00E+09 CCID 501.00E+06 CCID / ml, 3.00E+09 CCID 50 1.00E+06 CCID / ml, 4.00E+09 CCID 50 1.00E+06 CCID / ml, or 5.00E+09 CCID 50 1.00E+06 CCID / ml, indicating that the virus culture method of the present application can obtain a better virus yield.

[0180] In one embodiment, the virus titer in the harvest fluid can be about 1.00E+06 CCID 50 / ml to 9.00E+08 CCID 50 / ml.

[0181] In one embodiment, the virus titer in the harvest fluid can be about 1.00E+06 CCID 50 / ml to 5.00E+08 CCID 50 / ml.

[0182] In one embodiment, the virus titer in the harvest fluid can be about 1.00E+06 CCID 50 / ml to 1.00E+08 CCID 50 / ml.

[0183] Chromatography purification

[0184] In the present application, the chromatography step can include, but is not limited to, one or more chromatography methods using ion exchange chromatography, affinity chromatography and / or molecular exclusion chromatography.

[0185] In one embodiment, the supernatant of the virus mixed solution is pretreated before or after the chromatography step. Generally, in the pretreatment step, cell debris and other insoluble impurities are removed from the supernatant, and it is adjusted to conditions suitable for chromatography purification.

[0186] In one embodiment, the pretreatment step includes adding a nuclease and filtering sterilization; optionally, further including ultrafiltration.

[0187] The nuclease can be a DNA-removing nuclease or an RNA-removing nuclease.

[0188] For example, the nuclease is Benzonase All-Purpose Nuclease.

[0189] In certain embodiments, the chromatography step includes using ion exchange chromatography.

[0190] The improved ion exchange chromatography process used in the present application has simple unit operations, fewer purification steps, shorter purification times, is easy to scale up and commercialize, the activity of the virus is well maintained, and the residual amount of HCP is also low. The following advantages are obtained when applied to large-scale production: 1: high resolution capability combined with low shear flow characteristics to achieve high quality yields; 2. Improved productivity, benefiting from high loading and ready-to-use format, increased yield and reduced run time with higher throughput; 3. Scalable plug-and-play format, cGMP-compliant ready-to-use format, scalability from 1 mL to 40 L has been validated; 4. Ideal choice for macromolecular applications: powerful tool for nanoscale biomolecule purification.

[0191] Ion exchange chromatography allows the use of a variety of commercially available chromatography materials known for the separation and purification of biological materials. Chromatography supports of different base materials (natural or synthetic polymers) can be used in the present application. These base materials can have different shapes, including particulate supports, membranes, and monoliths. The ion exchange chromatography described in the present application includes the use of cation exchange chromatography and anion exchange chromatography. Anion exchange chromatography can include one or more functional groups including, but not limited to, DEAE, EDA (ethylenediamine), and Q. Cation exchange chromatography can likewise include one or more functional groups including, but not limited to, SO3 (sulfonyl) and CM (carboxymethyl). These functional groups can be attached to any resin suitable for use in the present application.

[0192] For example, the present application uses anion exchange chromatography including methacrylic acid.

[0193] For example, the present application uses anion exchange chromatography with quaternary ammonium groups.

[0194] For example, the present application uses monolith ion exchange chromatography including a polymethacrylic acid methyl ester matrix.

[0195] For example, the present application uses monolith ion exchange chromatography with quaternary ammonium groups.

[0196] For example, the present application uses monolith ion exchange chromatography with DEAE functional groups.

[0197] The ion exchange chromatography used in the present application can also include a combination of the various ion exchange chromatographies described above, wherein the functional groups and matrices of the ion exchange chromatographies can each independently be selected from the functional groups and matrix materials known to those skilled in the art.

[0198] The viruses described herein can be further purified from the supernatant or pre-treated supernatant based on their surface charge. The supernatant or pre-treated supernatant can be loaded directly onto the chromatography column or the supernatant or pre-treated supernatant can be adjusted to the appropriate conditions before loading. The appropriate conditions are conditions under which the supernatant or pre-treated supernatant binds to the positive (negative) charge functional groups of the ion exchange chromatography.

[0199] In one embodiment, the appropriate conditions are conditions under which the pH of the supernatant or pre-treated supernatant is adjusted to bind to the ion exchange chromatography functional groups.

[0200] For example, the pH of the supernatant or pre-treated supernatant can be adjusted to between about 6.0 and about 10.0.

[0201] For example, the pH of the supernatant or pre-treated supernatant can be adjusted to between about 7.0 and about 10.0.

[0202] For example, the pH of the supernatant or pre-treated supernatant can be adjusted to between about 8.0 and about 10.0.

[0203] For example, the pH of the supernatant or pre-treated supernatant can be adjusted to between about 7.0 and about 9.0.

[0204] For example, the pH of the supernatant or pre-treated supernatant can be adjusted to between about 7.0 and about 8.0.

[0205] For example, the pH of the supernatant or pre-treated supernatant can be adjusted to between about 6.0 and about 6.5.

[0206] For example, the pH of the supernatant or pre-treated supernatant can be adjusted to between about 6.5 and about 7.0.

[0207] For example, the pH of the supernatant or pre-treated supernatant can be adjusted to between about 7.5 and about 8.0.

[0208] For example, the pH of the supernatant or pre-treated supernatant can be adjusted to between about 8.5 and about 9.0.

[0209] For example, the pH of the supernatant or pre-treated supernatant can be adjusted to between about 9.5 and about 10.0.

[0210] In one embodiment, the appropriate conditions are conditions under which the conductivity of the supernatant or pre-treated supernatant is adjusted to bind to the ion exchange chromatography.

[0211] For example, the conductivity of the supernatant or pre-treated supernatant can be adjusted to at most 15 ms / cm.

[0212] For example, the conductivity of the supernatant or pre-treated supernatant can be adjusted to at most 10 ms / cm.

[0213] For example, the conductivity of the supernatant or pre-treated supernatant can be adjusted to about 2.0 to about 8.0 ms / cm.

[0214] For example, the conductivity of the supernatant or pre-treated supernatant can be adjusted to about 3.0 to about 7.0 ms / cm.

[0215] For example, the conductivity of the supernatant or pre-treated supernatant can be adjusted to about 4.5 to about 6.5 ms / cm.

[0216] For example, the conductivity of the supernatant or pre-treated supernatant can be adjusted to about 5.0 to about 6.0 ms / cm.

[0217] For example, the conductivity of the supernatant or pre-treated supernatant can be adjusted to about 5.0 to about 5.5 ms / cm.

[0218] For example, the conductivity of the supernatant or pre-treated supernatant can be adjusted to about 5.5 to about 6.0 ms / cm.

[0219] In one embodiment, under the above conditions, the negatively charged viral particles bind to the positively charged functional groups on the ion exchange chromatography surface.

[0220] The loading amount described herein is calculated according to the upper limit of the purification capacity of the chromatography column and the viral titer in the supernatant, which is known to those skilled in the art.

[0221] The viral particles and other impurities are separated and eluted by an eluent when the ion exchange chromatography is used for purification according to the present application. The eluent can be a salt solution with strong ionic strength. The eluent includes, but is not limited to, NaCl, KCl, Na2SO4 and / or Na3PO4, K2SO4 and / or K3PO4.

[0222] The ion exchange chromatography elution used in the present application is performed under suitable conditions. The suitable conditions refer to conditions under which the viral yield is ensured while the impurity proteins are removed to the maximum extent.

[0223] In one embodiment, the suitable conditions for elution are adjusting the elution pH.

[0224] For example, the elution pH adjustment is about 5.0 to about 8.0. For example, the elution pH adjustment is about 5.0 to about 7.0. For example, the elution pH adjustment is about 5.0 to about 6.0. For example, the elution pH adjustment is about 5.0 to about 5.5. For example, the elution pH adjustment is about 5.5 to about 6.0. For example, the elution pH adjustment is about 6.0 to about 6.5. For example, the elution pH adjustment is about 6.5 to about 7.0. For example, the elution pH adjustment is about 7.0 to about 7.5. For example, the elution pH adjustment is about 7.5 to about 8.0.

[0225] In one embodiment, the suitable condition during elution is adjusting the elution salt concentration.

[0226] For example, the elution salt concentration adjustment is about 10-500 mM. For example, the elution salt concentration adjustment is about 50-500 mM. For example, the elution salt concentration adjustment is about 50-300 mM. For example, the elution salt concentration adjustment is about 50-150 mM. For example, the elution salt concentration adjustment is about 150-250 mM. For example, the elution salt concentration adjustment is about 250-500 mM.

[0227] In one embodiment, the suitable condition during elution is adjusting the elution mode.

[0228] For example, the elution mode is one-step elution. One-step elution refers to the elution process is performed in the same elution solution. Under the above elution salt concentration and elution pH conditions, one-step elution can be completed in a shorter time, reducing the possible pollution during the elution process, and the operation is simple, which can realize rapid separation.

[0229] For example, the elution mode is linear gradient elution. Linear gradient elution refers to gradually changing the composition of the elution solution during the elution process, and different components are separated according to different protein and column binding strengths. Linear gradient elution can provide more refined virus separation capacity, which can be used to separate complex samples with different binding properties.

[0230] In the ion exchange chromatography method used in the present application, after selecting suitable sample loading conditions and suitable elution conditions, the virus titer after purification can be at least about 1.00E+07 CCID 50 / ml, about 2.00E+07 CCID 50 / ml, about 3.00E+07 CCID 50 / ml, about 4.00E+06 CCID 50 / ml, about 5.00E+07 CCID 50 / ml, about 6.00E+07 CCID 50 / ml, approximately 7.00E+07CCID 50 / ml, approximately 8.00E+07CCID 50 / ml, approximately 9.00E+07CCID 50 / ml, approximately 1.00E+08CCID 50 / ml, 2.00E+08CCID 50 / ml, 3.00E+08CCID 50 / ml, 4.00E+08CCID 50 / ml, 5.00E+08CCID 50 / ml, 6.00E+08CCID 50 / ml, 7.00E+08CCID 50 / ml, 8.00E+08CCID 50 / ml, 9.00E+08CCID 50 / ml, 1.00E+09CCID 50 / ml, 2.00E+09CCID 50 / ml, 3.00E+09CCID 50 / ml, 4.00E+09CCID 50 / ml or 5.00E+09CCID 50 / ml indicates that the process method described in this application can significantly increase the purity of poliovirus.

[0231] In one embodiment, the purified virus titer can be approximately 1.00E+07CCID. 50 / ml to 5.00E+09CCID 50 / ml.

[0232] In one embodiment, the purified virus titer can be approximately 5.00E+07CCID. 50 / ml to 5.00E+09CCID 50 / ml.

[0233] In one embodiment, the purified virus titer can be approximately 5.00E+07CCID. 50 / ml to 1.00E+09CCID 50 / ml.

[0234] In the ion exchange chromatography method used in the present application, the recovery of the purified virus can be at least about 40% to about 99%, about 40% to about 90%, about 40% to about 85%, about 40% to about 80%, about 40% to about 75%, or about 40% to about 70% after selecting appropriate loading conditions and appropriate elution conditions, indicating that the use of the process method of the present application can significantly improve the efficiency of poliovirus purification.

[0235] In the ion exchange chromatography method used in the present application, the RNA plasmid in the purified virus is at least about 1.00E+09 copies / ml, about 2.00E+09 copies / ml, about 3.00E+09 copies / ml, about 4.00E+09 copies / ml, about 5.00E+09 copies / ml, about 6.00E+09 copies / ml, about 7.00E+09 copies / ml, about 8.00E+09 copies / ml, about 9.00E+09 copies / ml, about 1.00E+10 copies / ml, about 2.00E+10 copies / ml, about 3.00E+10 copies / ml, about 4.00E+10 copies / ml, about 5.00E+10 copies / ml, about 6.00E+10 copies / ml, about 7.00E+10 copies / ml, about 8.00E+10 copies / ml, about 9.00E+10 copies / ml, about 1.00E+11 copies / ml, about 2.00E+11 copies / ml, about 3.00E+11 copies / ml, about 4.00E+11 copies / ml, or about 5.00E+11 copies / ml after selecting appropriate loading conditions and appropriate elution conditions, indicating that the use of the process method of the present application can effectively protect and purify the genetic material of poliovirus, ensuring the activity and integrity of the virus.

[0236] The ion exchange chromatography used in the present application can also remove DNA. Since the host cell DNA is negatively charged, it will bind to the functional groups on the surface of the chromatography carrier. However, compared to poliovirus, host DNA requires a higher ionic strength eluent to be eluted from the anion exchange column. Therefore, under the elution conditions suitable for poliovirus, the material eluted from the ion exchange chromatography does not include host DNA. Therefore, in one embodiment, no DNA nuclease needs to be added before or after the chromatography step.

[0237] In one embodiment, the chromatography step of the present application includes the use of size exclusion chromatography (SEC).

[0238] The size exclusion chromatography used in the present application can also achieve the effects of impurity removal and virus purification at the same time.

[0239] The improved molecular exclusion chromatography purification process provided herein provides one or more of the following advantages: increased viral yield, reduced purification time (i.e., the improved process is faster than previously known), and the ability to select the ideal compromise between product yield and purity (i.e., greater process flexibility). The reduction in purification time for the improved process is due, at least in part, to the reduction in the number of purification steps. The molecular exclusion chromatography process is also less complex and less costly, making it more suitable for large scale production.

[0240] For example, the molecular exclusion chromatography purification process used herein can be performed in 8 hours or less, or less than 8 hours (e.g., 4 to 8 hours, 4 to 6 hours, 6 to 8 hours, or 7 to 8 hours).

[0241] While in one embodiment, the recovery of virus is not significantly improved over traditional molecular exclusion chromatography purification processes, the viral batch-to-batch consistency is significantly improved, and both host cell DNA (HCD) and host cell protein (HCP) are significantly reduced, while the time for the purification step is significantly improved.

[0242] The molecular exclusion chromatography packing used herein can be a commercially available chromatography material known to be useful for SEC of biological materials. Chromatography packing of different base materials (natural and synthetic polymers) can be used herein. The SEC packing can have different particle sizes and pore sizes.

[0243] In one embodiment, the SEC packing is a gel.

[0244] For example, the SEC packing is a dextran gel. For example, the SEC packing is a polyacrylamide gel. For example, the SEC packing is a sepharose gel. For example, the SEC packing is a hydrophilic gel.

[0245] The molecular exclusion chromatography described herein also includes the use of buffers, including phosphate, citrate, phosphoric acid, and other biocompatible buffers. The use of stabilizers (e.g., detergents) to configure the buffers is also included herein. Compounds that prevent ionic interactions between the SEC packing and the poliovirus, such as sodium chloride, can also be added to the buffers.

[0246] The present application also provides a poliovirus purification process comprising ultrafiltration and chromatography processes.

[0247] In one embodiment, the ultrafiltration comprises the use of hollow fiber ultrafiltration concentration. The hollow fiber membranes used therein can be any commercially available hollow fiber membrane, such as the hollow fiber membrane material can be PVDF, PES, or PSF.

[0248] For example, the hollow fiber ultrafiltration concentration can comprise increasing the membrane area of the hollow fiber. For example, the membrane area is increased to at least 150% to 200% of the original membrane area.

[0249] The ultrafiltration and chromatography methods described in this application can be used at the laboratory level or at the pilot-scale and industrial production level.

[0250] In one embodiment, the chromatographic step of this application includes the use of affinity chromatography.

[0251] In affinity chromatography, viruses or viral vectors are selectively captured by binding to specific ligands on the stationary phase, and then eluted by changing the buffer conditions or adding specific eluents.

[0252] In one implementation, affinity chromatography can be based on specific receptor-ligand interactions. For example, poliovirus can specifically bind to mimics of its cell surface receptors, and this interaction can be used for viral affinity chromatography.

[0253] In one implementation, affinity chromatography can be peptide- or protein-based affinity chromatography that utilizes the interaction between the poliovirus and its key host factors, such as using accessory proteins or enzymes required by the virus as affinity ligands.

[0254] In one implementation, affinity chromatography can be nucleic acid-based affinity chromatography, for example, using complementary DNA or RNA sequences as ligands to capture the virus by specifically binding to the genetic material of the poliovirus.

[0255] Upstream process

[0256] On the other hand, this application provides an upstream preparation method for poliovirus, which includes the steps in the order shown:

[0257] a) Provide cells, which are present in a culture medium;

[0258] b) Adjust the cells to a suitable cell density, and then contact the poliovirus with the cells to obtain a mixed solution of poliovirus and cells;

[0259] c) Obtain the poliovirus harvest fluid from the mixed solution.

[0260] The cells used in this application are African green monkey kidney cells (Vero cells), and the methods for resuscitation, culture, and passage of these cells are any methods known to those skilled in the art.

[0261] For example, cryopreserved Vero cells are thawed in complete medium, which includes 90% DMEM and 10% FBS, and passaged when the cells are grown to 80-90% confluency, using 0.25% trypsin for digestion, followed by addition of complete medium to stop the digestion, and then passaging by centrifugation, discarding the supernatant, resuspension, and the like.

[0262] In certain embodiments, the application includes culturing cells using a serum- free culture method for the production of poliovirus.

[0263] The serum-free culture method used in the present application refers to the absence of serum in the culture medium used for cell growth and infection. In one embodiment, the serum-free culture method is performed in the absence of any component of direct animal origin (e.g., serum or serum components, etc.). In one embodiment, the serum-free culture method is performed in a chemically defined medium. In one embodiment, any additives supplemented to the culture medium during infection with the virus also do not contain components of animal origin. The serum-free culture can be performed in a batch, fed-batch, continuous culture system, etc., in a petri dish, a spinner flask, or a bioreactor. Serum-free culture media suitable for use in the present application can be well known to those skilled in the art and are generally readily available from commercial sources or prepared according to standard methods under conventional conditions. Serum-free culture media that can be used in the present application include, but are not limited to, custom serum-free media for Vero cells. In one embodiment, the present application uses a chemically defined serum-free culture medium, such as a medium suitable for Vero cell culture that does not contain animal-derived components, such as NutriVero Flex 10, CD Vero-S. In one embodiment, the present application can also use serum-free media, such as a specialized medium suitable for Vero cells, such as VERO SFM01, VERO SFM01 pro serum-free medium, Pesche series media, OPM-VERO SFM6C3. In one embodiment, the present application uses an ultra-low protein serum-free medium, such as VP-SFM AGT. In one embodiment, the present application uses a serum-free medium containing 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and sodium bicarbonate buffer, such as ProVero-1 NAO, etc.

[0264] In the methods of the present application, lipids and / or hydrolysates and / or other supplements can be supplemented to the serum-free culture medium to further improve the yield.

[0265] In other embodiments, the application includes culturing cells using a microcarrier culture method for the production of poliovirus.

[0266] The microcarriers suitable for this application are well known to those skilled in the art and are generally available in large quantities from commercial sources or prepared under conventional conditions according to standard methods. The microcarriers described in this application include, but are not limited to, commercially available microcarriers such as the Cytodex series, Cytopore, Cytoline, etc. In one embodiment, the microcarrier is the spherical microcarrier Cytodex-1.

[0267] The microcarrier dosage used in this application balances cost and viral infection titer, meaning the viral harvest titer is at least four times that of conventional methods. In some embodiments, the microcarrier dosage is controlled at about 2 g / L to about 6 g / L. In one embodiment, the microcarrier dosage is controlled at about 2 g / L to about 4 g / L. In another embodiment, the microcarrier dosage is controlled at about 3 g / L.

[0268] This application also includes a method for improving cell viability grown under microcarrier conditions, comprising adding a feed to a culture medium containing microcarriers, said feed such that the sugar content in the culture system is not less than about 0.5 g / L, about 1 g / L, about 1.5 g / L, about 2 g / L or higher.

[0269] In one embodiment, the feeding ensures that the sugar content in the culture medium is not less than 1 g / L.

[0270] In one embodiment, the feed used in this application is a fresh, complete culture medium.

[0271] The feeding method of this application can be any method known in the art, such as continuous feeding, intermittent feeding, semi-continuous liquid replacement feeding, or discontinuous liquid replacement feeding.

[0272] In one embodiment, the feed used in this application is added through discontinuous fluid replacement.

[0273] Compared with traditional cell factory processes, the microcarrier culture method used in this application increases the total number of cells before inoculation by nearly 30% while providing the same cell attachment surface area, increases the virus harvesting liquid titer by 4 times, and significantly reduces the harvesting liquid volume, which is more conducive to subsequent purification operations.

[0274] In some embodiments, this application also improves the virus infection parameters in the pilot-scale process and the scale-up process, respectively.

[0275] The method described in this application, which uses poliovirus to infect cells and / or to propagate the virus, is suitable for use at temperatures ranging from about 33°C to 38°C. When the cells have a cell density of about 1 × 10⁻⁶, the infection is most effective. 5 cells / ml, approximately 2×10 5 cells / ml, approximately 3×105 about 4 x 10 5 about 5 x 10 5 about 6 x 10 5 about 7 x 10 5 about 8 x 10 5 about 9 x 10 5 about 1 x 10 6 about 2 x 10 6 about 3 x 10

[0276] The cells are contacted with the poliovirus to allow the poliovirus to infect the cells and propagate. For example, a batch of poliovirus seed is added to pre-warmed virus maintenance medium, which is then added to the cell culture and allowed to adsorb to the cells, with constant agitation, to initiate the virus culture.

[0277] In the methods described herein, the poliovirus can infect the cells at a multiplicity of infection (MOI) of from about 0.001 to about 10. In certain embodiments, the infection can be at an MOI of from about 0.01 to about 0.5.

[0278] In one embodiment, the infection is at an MOI of from about 0.01 to about 0.1 after microcarrier culture.

[0279] For example, the infection is at an MOI of about 0.05 after microcarrier culture.

[0280] In one embodiment, the infection is at an MOI of from about 0.05 to about 0.2 after serum-free culture.

[0281] For example, the infection is at an MOI of about 0.05 after serum-free culture.

[0282] In the methods described herein, the poliovirus is harvested within about 0.5 days to about 10 days after infection.

[0283] In one embodiment, the poliovirus is harvested within about 3 days to about 6 days after infection of the cells after microcarrier culture.

[0284] For example, the poliovirus is harvested on day 4 after infection of the cells after microcarrier culture.

[0285] For example, the poliovirus is harvested on day 5 after infection of the cells after microcarrier culture.

[0286] In one embodiment, the poliovirus is harvested within about 5 days to about 7 days after infection of the cells following serum-free culture.

[0287] For example, the poliovirus is harvested on day 6 after infection of the cells following serum-free culture.

[0288] Following propagation of the poliovirus in the cells, the virus or components thereof are harvested from the cell culture. This can be accomplished by conventional methods known to those of ordinary skill in the art. Virus produced and released in the cell culture medium can be separated from the biological tissue of the cells by conventional methods such as centrifugation or filtration and harvested in the supernatant. In this case, centrifugation or filtration is the harvesting step. The virus can be harvested using conventional methods, for example, at the end of the culture by collecting the culture medium containing the virus suspension, which can be filtered using, for example, a 0.22 μm, 0.45 μm or 0.8 μm filter and optionally stored at 4°C.

[0289] In the methods described herein, the virus infection of the cells can be performed at the scale of one cell factory, ten cell factories or ten ten cell factories. The scale of the cell factories can also be adjusted according to the needs of the poliovirus, for example, scaled up to pilot plant scale or plant scale.

[0290] In the virus culture methods used herein, the virus titer in the harvest can be at least about 1.00E+06 CCID 50 / ml, about 2.00E+06 CCID 50 / ml, about 3.00E+06 CCID 50 / ml, about 4.00E+06 CCID 50 / ml, about 5.00E+06 CCID 50 / ml, about 6.00E+06 CCID 50 / ml, about 7.00E+06 CCID 50 / ml, about 8.00E+06 CCID 50 / ml, about 9.00E+06 CCID 50 / ml, 1.00E+07 CCID 50 / ml, about 2.00E+07 CCID 50 / ml, about 3.00E+07 CCID 50 / ml, about 4.00E+07 CCID 50 / ml, about 5.00E+07 CCID 50 / ml, 6.00E+07 CCID 50 / ml, 7.00E+07 CCID 50 / ml, 8.00E+07 CCID 50 / ml, 9.00E+07 CCID 50 / ml, 1.00E+08 CCID 50 / ml, 2.00E+08 CCID 50 / ml, 3.00E+08 CCID 50 / ml, 4.00E+08 CCID 50 / ml, 5.00E+08 CCID 50 / ml, 6.00E+08 CCID 50 / ml, 7.00E+08 CCID 50 / ml, 8.00E+08 CCID 50 / ml, 9.00E+08 CCID 50 / ml, 1.00E+09 CCID 50 / ml, 2.00E+09 CCID 50 / ml, 3.00E+09 CCID 50 / ml, 4.00E+09 CCID 50 / ml, or 5.00E+09 CCID 50 / ml, indicating that the virus culture method of the present application can obtain a better virus yield.

[0291] In one embodiment, the virus titer in the harvest fluid can be about 1.00E+06 CCID 50 / ml to 9.00E+08 CCID 50 / ml.

[0292] In one embodiment, the virus titer in the harvest fluid can be about 1.00E+06 CCID 50 / ml to 5.00E+08 CCID 50 / ml.

[0293] In one embodiment, the virus titer in the harvest fluid can be about 1.00E+06 CCID 50 / ml to 1.00E+08 CCID 50 / ml.

[0294] Downstream process

[0295] In another aspect, the present application provides a downstream purification method of poliovirus.

[0296] Any known upstream production process of poliovirus or variant thereof can be used to produce the starting material used in the purification process of the present application. Suitable sources of poliovirus or variant thereof are any eukaryotic cell that supports replication of poliovirus. The host cell is a mammalian host cell line that supports poliovirus infection and replication.

[0297] The application is described below by way of example with reference to specific embodiments, but the application is not limited to the described embodiments.

[0298] In one embodiment, the recombinant poliovirus is purified using ion exchange chromatography.

[0299] In one embodiment, the supernatant of the virus-containing mixture is pre-treated prior to the chromatography step. Typically, in the pre-treatment step, cell debris and other insoluble impurities are removed from the supernatant and it is adjusted to conditions suitable for chromatographic purification.

[0300] In one embodiment, a nuclease is added prior to the chromatography step. The nuclease can be a DNA-removing nuclease, and / or an RNA-removing nuclease.

[0301] For example, Benzonase Benzonuclease is added prior to the chromatography step.

[0302] For example, the virus-containing mixture obtained by any known means is purified using monolithic column ion exchange chromatography comprising a polymethyl methacrylate matrix.

[0303] For example, the monolithic column ion exchange chromatography comprising a polymethyl methacrylate matrix used in the present application further comprises QA functional groups.

[0304] For example, the monolithic column ion exchange chromatography comprising a polymethyl methacrylate matrix used in the present application further comprises DEAE functional groups.

[0305] For example, the virus-containing mixture used in the present application is clarified.

[0306] For example, the virus-containing mixture used in the present application is adjusted prior to loading on the monolithic column ion exchange chromatography such that the virus-containing mixture is capable of binding to the ion exchange chromatography functional groups.

[0307] For example, the pH of the virus-containing mixture is adjusted to between 8.5 and 9.5.

[0308] For example, the conductivity of the supernatant of the virus-containing mixture is adjusted to no more than about 15 ms / cm.

[0309] For example, the conductivity of the supernatant of the mixed solution containing the virus is adjusted to be not higher than about 10 ms / cm.

[0310] For example, the conductivity of the supernatant of the mixed solution containing the virus is adjusted to be about 2.0 to about 8.0 ms / cm.

[0311] For example, the conductivity of the supernatant of the mixed solution containing the virus is adjusted to be about 3.0 to about 7.0 ms / cm.

[0312] For example, the conductivity of the supernatant of the mixed solution containing the virus is adjusted to be about 5.0 to about 6.0 ms / cm.

[0313] For example, the ion exchange chromatography method used in the present application further comprises eluting the mixed solution containing the virus so that the virus particles and other impurities are separated. The elution needs to be performed under suitable conditions.

[0314] For example, the suitable elution conditions can be adjusting the elution pH.

[0315] For example, the elution pH can be about 5.0 to about 7.5.

[0316] For example, the elution pH can be about 5.5 to about 6.0.

[0317] For example, the suitable elution conditions can be adjusting the elution salt concentration.

[0318] For example, the elution salt concentration can be about 50 mM to about 300 mM.

[0319] For example, the elution salt concentration can be about 250 mM.

[0320] For example, the suitable elution conditions can be adjusting the elution mode.

[0321] For example, the elution mode can be one-step elution.

[0322] In one embodiment, the present application can further purify the poliovirus using anion exchange chromatography after the ion exchange chromatography purification, so that the content of HCP impurities is further reduced.

[0323]

[0324]

[0325] In one embodiment, the present application can further purify the poliovirus using anion exchange chromatography after the ion exchange chromatography purification, so that the content of HCP impurities is further reduced.

[0326] ​​For example, the anion exchange chromatography is further purified by a functional group of quaternary amino group.

[0327] For example, the eluent of the ion exchange chromatography is loaded into the anion exchange chromatography, and the eluent is adjusted to a suitable condition.

[0328] For another example, the suitable loading condition means that the pH of the eluent of the ion exchange chromatography can be adjusted to about 7.0 to about 8.0.

[0329] For another example, the suitable loading condition means that the conductivity of the eluent of the ion exchange chromatography can be adjusted to about 2.0 to about 8.0 ms / cm.

[0330] For another example, the suitable loading condition means that the conductivity of the eluent of the ion exchange chromatography can be adjusted to about 3.0 to about 7.0 ms / cm.

[0331] For another example, the suitable loading condition means that the conductivity of the eluent of the ion exchange chromatography can be adjusted to about 5.0 to about 6.0 ms / cm.

[0332] For example, the anion exchange chromatography used in the present application further purified method further includes further elution, and the further elution is adjusted to a suitable condition so that the HCP impurity content is greatly reduced.

[0333] For another example, the suitable further elution condition means that a salt solution with strong ionic strength can be selected.

[0334] For another example, the salt solution is a phosphate salt solution.

[0335] For another example, the suitable elution condition can be adjusting the elution pH.

[0336] For another example, the elution pH can be about 7.0 to about 8.0.

[0337] For another example, the suitable elution condition can be adjusting the elution salt concentration.

[0338] For another example, the elution salt concentration can be about 3 mM to about 50 mM, about 3 mM to about 40 mM, about 4 mM to about 40 mM, about 5 mM to about 40 mM, about 10 mM to about 40 mM, about 10 mM to about 30 mM, about 10 mM to about 20 mM.

[0339] For another example, the elution salt concentration can be about 4.7 mM, 20 mM or 40 mM.

[0340] After the poliovirus is purified by using the exemplary ion exchange chromatography method of the present application, the titer of the purified virus can be at least about 1.00E+07 CCID 50 / ml, about 2.00E+07 CCID50 about 3.00E+07 CCID 50 about 4.00E+06 CCID 50 about 5.00E+07 CCID 50 about 6.00E+07 CCID 50 about 7.00E+07 CCID 50 about 8.00E+07 CCID 50 about 9.00E+07 CCID 50 about 1.00E+08 CCID 50 about 2.00E+08 CCID 50 about 3.00E+08 CCID 50 about 4.00E+08 CCID 50 about 5.00E+08 CCID 50 about 6.00E+08 CCID 50 about 7.00E+08 CCID 50 about 8.00E+08 CCID 50 about 9.00E+08 CCID 50 about 1.00E+09 CCID 50 about 2.00E+09 CCID 50 about 3.00E+09 CCID 50 about 4.00E+09 CCID 50 about 5.00E+09 CCID 50 about 5.00E+09 CCID

[0341] In one embodiment, the purified virus titer can be about 1.00E+07 CCID 50 / ml to 5.00E+09 CCID 50 / ml. In one embodiment, the purified virus titer can be about 5.00E+07 CCID 50 / ml to 5.00E+09 CCID 50 / ml. In one embodiment, the purified virus titer can be about 5.00E+07 CCID 50 / ml to 1.00E+09 CCID 50 / ml.

[0342] The recovery of the purified virus after purifying the poliovirus using the exemplary ion exchange chromatography method of the present application can be at least about 40% to about 99%, about 40% to about 90%, about 40% to about 85%, about 40% to about 80%, about 40% to 75%, or about 40% to 70%, indicating that the use of the process of the present application can significantly improve the efficiency of purifying poliovirus.

[0343] The RNA plasmid in the purified virus after purifying the poliovirus using the exemplary ion exchange chromatography method of the present application can be at least about 1.00E+10 copies / ml, about 2.00E+10 copies / ml, about 3.00E+10 copies / ml, about 4.00E+10 copies / ml, about 5.00E+10 copies / ml, about 6.00E+10 copies / ml, about 7.00E+10 copies / ml, about 8.00E+10 copies / ml, about 9.00E+10 copies / ml, about 1.00E+11 copies / ml, about 2.00E+11 copies / ml, about 3.00E+11 copies / ml, about 4.00E+11 copies / ml, or about 5.00E+11 copies / ml, indicating that the use of the process of the present application can effectively protect and purify the genetic material of poliovirus, ensuring the activity and integrity of the virus.

[0344] In one embodiment, the poliovirus is purified using a molecular exclusion chromatography. The molecular exclusion chromatography can be a gel column chromatography.

[0345] For example, the gel column chromatography is used to purify the mixed solution containing the virus obtained by any known means.

[0346] For example, the mixed solution containing the poliovirus is pretreated before using the gel column chromatography.

[0347] For another example, the pretreatment refers to ultrafiltration concentration.

[0348] For further example, the ultrafiltration concentration includes hollow fiber ultrafiltration concentration. The hollow fiber membrane used therein can be any commercially available hollow fiber membrane, such as the hollow fiber membrane material can be PVDF, PES, or PSF.

[0349] For further example, the hollow fiber ultrafiltration concentration includes ultrafiltration concentration with increased hollow fiber membrane area. For example, the membrane area is increased to at least 150% to 200% of the original membrane area.

[0350] For another example, the gel column chromatography can be agarose gel. The agarose gel can be any commercially available agarose gel packing.

[0351] For example, the column height of the gel column chromatography used in this application is about 30-100 cm.

[0352] For example, the gel column chromatography used in this application requires only one or two loading cycles.

[0353] For example, the gel column chromatography loading volume used in this application is approximately 10%-40% CV.

[0354] For example, after using gel column chromatography, further elution can be performed using anion exchange chromatography, with the conditions adjusted to be suitable, which can significantly reduce the content of HCP impurities.

[0355] For example, the further suitable elution conditions refer to the use of a salt solution with a strong ionic strength.

[0356] For example, the salt solution is a phosphate solution.

[0357] For example, the suitable elution conditions could be adjusting the elution pH.

[0358] For example, the elution pH may be approximately 7.0 to approximately 8.0.

[0359] For example, the suitable elution conditions could be adjusting the concentration of the elution salt.

[0360] For example, the elution salt concentration can be approximately 3mM to approximately 50mM, approximately 3mM to approximately 40mM, approximately 4mM to approximately 40mM, approximately 5mM to approximately 40mM, approximately 10mM to approximately 40mM, approximately 10mM to approximately 30mM, or approximately 10mM to approximately 20mM.

[0361] For example, the elution salt concentration can be approximately 4.7 mM, 20 mM, or 40 mM.

[0362] After purifying poliovirus using the exemplary size exclusion chromatography method of this application, the purification step time is significantly improved (conventional size exclusion chromatography requires 6-10 cycles, while the improved method requires only 1-2 cycles), batch-to-batch viral stability is significantly improved, and both host cell DNA (HCD) and host cell protein (HCP) are substantially reduced. For example, HCD is reduced by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or more. For example, HCP is reduced by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, or more.

[0363] In the present application, the HCPs can be detected using detection methods including, but not limited to, electrophoresis, immunoassay (ELISA), and mass spectrometry (MS).

[0364] In the present application, the HCDs can be detected using detection methods including, but not limited to, electrophoresis (e.g., capillary electrophoresis), PCR, DNA probe hybridization, and fluorescent staining.

[0365] Purification process

[0366] More specifically, the present application also provides a method for preparing a poliovirus solution, comprising the following steps in the order shown:

[0367] a) providing cells, the cells being present in a culture medium, the culture medium being a serum-free culture medium;

[0368] b) adjusting the cells to a suitable cell density, and then contacting poliovirus with the cells to obtain a mixed solution of poliovirus and cells, the poliovirus being contacted with the cells for a time period of about 5-7 days.

[0369] c) centrifuging the mixed solution, and collecting the supernatant, the virus titer in the supernatant being at least about 1.00E+06 CCID 50 / ml, about 2.00E+06 CCID 50 / ml, about 3.00E+06 CCID 50 / ml, about 4.00E+06 CCID 50 / ml, about 5.00E+06 CCID 50 / ml, about 6.00E+06 CCID 50 / ml, about 7.00E+06 CCID 50 / ml, about 8.00E+06 CCID 50 / ml, about 9.00E+06 CCID 50 / ml, 1.00E+07 CCID 50 / ml, about 2.00E+07 CCID 50 / ml, about 3.00E+07 CCID 50 / ml, about 4.00E+07 CCID 50 / ml, about 5.00E+07 CCID 50 / ml, about 6.00E+07 CCID 50 / ml, about 7.00E+07 CCID 50 / ml, about 8.00E+07 CCID 50 / ml, about 9.00E+07 CCID 50 / ml, approximately 1.00E+08CCID 50 / ml, 2.00E+08CCID 50 / ml, 3.00E+08CCID 50 / ml, 4.00E+08CCID 50 / ml, 5.00E+08CCID 50 / ml, 6.00E+08CCID 50 / ml, 7.00E+08CCID 50 / ml, 8.00E+08CCID 50 / ml, 9.00E+08CCID 50 / ml, 1.00E+09CCID 50 / ml, 2.00E+09CCID 50 / ml, 3.00E+09CCID 50 / ml, 4.00E+09CCID 50 / ml or 5.00E+09CCID 50 / ml;

[0370] d) The supernatant is purified by a chromatographic step to obtain the poliovirus solution, wherein the chromatographic step includes the use of size exclusion chromatography and / or ion exchange chromatography.

[0371] The serum-free culture used in this scheme overcomes the batch-to-batch variability caused by the addition of serum in traditional processes, thereby increasing the stability and quality control of the production process, ensuring the safety of the production process, and reducing the possibility of external contamination. At the same time, through the optimization of the chromatographic method, the purification step time is significantly improved (traditional size exclusion chromatography requires 6-10 cycles, while the improved method requires only 1-2 cycles), the purification time is shorter, the batch-to-batch stability of the virus is significantly improved, and the host cell DNA (HCD) and host cell protein (HCP) are significantly reduced, making it easy to scale up and commercialize.

[0372] In step a), the serum-free medium includes, but is not limited to, custom serum-free medium for Vero cells. In one embodiment, the present application uses a chemically defined serum-free medium, such as NutriVero FlexlO, CD Vero-S. In one embodiment, the present application can also use VERO SFM01, VERO SFM01 pro serum-free medium, Pesche series medium, OPM-VERO SFM6C3. In one embodiment, the present application uses an ultra-low protein serum-free medium, such as VP-SFM AGT. In one embodiment, the present application uses a serum-free medium containing HEPES and sodium bicarbonate buffer, such as ProVero-1 NAO, etc.

[0373] In the method of the present application, lipids and / or hydrolysates and / or other supplements can be added to the serum-free medium to further improve the yield.

[0374] In step b), the appropriate cell density is a cell density of about 1 x 10 5 cells / ml, about 2 x 10 5 cells / ml, about 3 x 10 5 cells / ml, about 4 x 10 5 cells / ml, about 5 x 10 5 cells / ml, about 6 x 10 5 cells / ml, about 7 x 10 5 cells / ml, about 8 x 10 5 cells / ml, about 9 x 10 5 cells / ml, about 1 x 10 6 cells / ml, or about 2 x 10 6 cells / ml, at which time the cells are contacted with the poliovirus.

[0375] The contacting can be performed at an MOI of about 0.05 to about 0.2 when the poliovirus is contacted with the cells for about 5 to 7 days.

[0376] For example, the contacting can be performed at an MOI of about 0.05 when the poliovirus is contacted with the cells for about 5 to 7 days.

[0377] In step c), after the poliovirus has multiplied in the cells, the virus or a component thereof is harvested from the cell culture. This can be done using conventional methods also known to those skilled in the art. The virus produced and released in the cell culture medium can be separated from the biological tissue of the cells by conventional methods such as centrifugation or filtration and harvested in the supernatant. In this case, centrifugation or filtration is the harvesting step. Conventional methods can be used to harvest the virus, for example, by collecting the culture medium containing the virus suspension at the end of the culture, which can be filtered using filters such as 0.22 μm, 0.45 μm, or 0.8 μm and optionally stored at 4°C.

[0378] In one embodiment, the viral titer of the mixed solution is at least about 1.00E+06CCID. 50 / ml, approximately 2.00E+06CCID 50 / ml, approximately 3.00E+06CCID 50 / ml, approximately 4.00E+06CCID 50 / ml, approximately 5.00E+06CCID 50 / ml, approximately 6.00E+06CCID 50 / ml, approximately 7.00E+06CCID 50 / ml, approximately 8.00E+06CCID 50 / ml, approximately 9.00E+06CCID 50 / ml, 1.00E+07CCID 50 / ml, approximately 2.00E+07CCID 50 / ml, approximately 3.00E+07CCID 50 / ml, approximately 4.00E+07CCID 50 / ml, approximately 5.00E+07CCID 50 / ml, approximately 6.00E+07CCID 50 / ml, approximately 7.00E+07CCID 50 / ml, approximately 8.00E+07CCID 50 / ml, approximately 9.00E+07CCID 50 / ml, approximately 1.00E+08CCID 50 / ml, 2.00E+08CCID 50 / ml, 3.00E+08CCID 50 / ml, 4.00E+08CCID 50 / ml, 5.00E+08CCID 50 / ml, 6.00E+08CCID 50 / ml, 7.00E+08CCID 50 / ml, 8.00E+08 CCID 50 / ml or 9.00E+08 CCID 50 / ml.

[0379] In one embodiment, the virus titer of the mixed solution is about 1.00E+06 CCID 50 / ml to 5.00E+08 CCID 50 / ml. In one embodiment, the virus titer of the mixed solution is about 1.00E+06 CCID 50 / ml to 9.00E+07 CCID 50 / ml.

[0380] In one embodiment, the virus titer of the supernatant is at least about 1.00E+06 CCID 50 / ml, about 2.00E+06 CCID 50 / ml, about 3.00E+06 CCID 50 / ml, about 4.00E+06 CCID 50 / ml, about 5.00E+06 CCID 50 / ml, about 6.00E+06 CCID 50 / ml, about 7.00E+06 CCID 50 / ml, about 8.00E+06 CCID 50 / ml, about 9.00E+06 CCID 50 / ml, 1.00E+07 CCID 50 / ml, about 2.00E+07 CCID 50 / ml, about 3.00E+07 CCID 50 / ml, about 4.00E+07 CCID 50 / ml, about 5.00E+07 CCID 50 / ml, about 6.00E+07 CCID 50 / ml, about 7.00E+07 CCID 50 / ml, about 8.00E+07 CCID 50 / ml, about 9.00E+07 CCID 50 / ml, about 1.00E+08 CCID 50 / ml, 2.00E+08 CCID 50 / ml, 3.00E+08 CCID 50 / ml, 4.00E+08 CCID 50 / ml, 5.00E+08 CCID 50 / ml, 6.00E+08 CCID50 / ml, 7.00E+08 CCID 50 / ml, 8.00E+08 CCID 50 / ml, or 9.00E+08 CCID 50 / ml.

[0381] In one embodiment, the virus titer of the supernatant obtained is at least about 5.00E+06 CCID 50 / ml to 9.00E+08 CCID 50 / ml. In one embodiment, the virus titer of the supernatant obtained is at least about 5.00E+06 CCID 50 / ml to 5.00E+08 CCID 50 / ml. In one embodiment, the virus titer of the supernatant obtained is at least about 5.00E+06 CCID 50 / ml to 9.00E+07 CCID 50 / ml.

[0382] In one embodiment, the supernatant of the virus mixture solution is pretreated before or after the chromatography step of d). Generally, in the pretreatment step, cell debris and other insoluble impurities are removed from the supernatant, which is adjusted to a condition suitable for chromatography purification.

[0383] In one embodiment, a nuclease is added before the chromatography step. The nuclease can be a DNA-removing nuclease or an RNA-removing nuclease.

[0384] For example, Benzonase® full power nuclease is added before the chromatography step.

[0385] In the step of d), in one embodiment, the chromatography step is ion exchange chromatography.

[0386] For example, the ion exchange chromatography includes monomethoxy polyethylene glycol methacrylate matrix-based monolith ion exchange chromatography.

[0387] For another example, the monomethoxy polyethylene glycol methacrylate matrix-based monolith ion exchange chromatography used in the present application also has QA functional groups.

[0388] For another example, the monomethoxy polyethylene glycol methacrylate matrix-based monolith ion exchange chromatography used in the present application also has DEAE functional groups.

[0389] For example, the virus-containing mixture solution used in the present application is subjected to suitable adjustment before being loaded into the ion exchange chromatography, so that the virus-containing mixture solution can be combined with the ion exchange chromatography functional groups.

[0390] For example, the pH of the virus-containing mixed solution is adjusted to about 8.5 to about 9.5.

[0391] For example, the conductivity of the supernatant of the virus-containing mixed solution is adjusted to about 2.0 to about 8.0 ms / cm.

[0392] For example, the conductivity of the supernatant of the virus-containing mixed solution is adjusted to about 3.0 to about 7.0 ms / cm.

[0393] For example, the conductivity of the supernatant of the virus-containing mixed solution is adjusted to about 5.0 to about 6.0 ms / cm.

[0394] For example, the monolithic column ion exchange chromatography method used in the present application further includes eluting the virus-containing mixed solution so that the virus particles and other impurities are separated. The elution needs to be performed under suitable conditions.

[0395] For example, the suitable elution conditions can be selected by using an eluent with a strong ionic strength.

[0396] For example, the eluent includes, but is not limited to, NaCl, KCl, Na2SO4, and / or Na3PO4, K2SO4, and / or K3PO4.

[0397] For example, the eluent is NaCl.

[0398] For example, the suitable elution conditions can be adjusting the elution pH.

[0399] For example, the elution pH can be about 5.0 to about 7.5.

[0400] For example, the elution pH can be about 5.5 to about 6.0.

[0401] For example, the suitable elution conditions can be adjusting the elution salt concentration.

[0402] For example, the elution salt concentration can be about 50 mM to about 300 mM.

[0403] For example, the elution salt concentration can be about 250 mM.

[0404] For example, the suitable elution conditions can be adjusting the elution mode.

[0405] For example, the elution mode can be one-step elution.

[0406] In one embodiment, the application can further purify the poliovirus after the ion exchange chromatography purification using anion exchange chromatography to further reduce the HCP impurity content, e.g., by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or more.

[0407] For example, the anion exchange chromatography has a functional group of quaternary amino group.

[0408] For example, the eluate of the ion exchange chromatography is loaded onto the anion exchange chromatography and the eluate is adjusted to a suitable condition.

[0409] For example, the suitable loading condition is that the pH of the eluate of the ion exchange chromatography can be adjusted to about 7.0 to about 8.0.

[0410] For example, the suitable loading condition is that the conductivity of the eluate of the ion exchange chromatography can be adjusted to about 2.0 to about 8.0 ms / cm.

[0411] For example, the suitable loading condition is that the conductivity of the eluate of the ion exchange chromatography can be adjusted to about 3.0 to about 7.0 ms / cm.

[0412] For example, the suitable loading condition is that the conductivity of the eluate of the ion exchange chromatography can be adjusted to about 5.0 to about 6.0 ms / cm.

[0413] For example, the further purification method using the anion exchange chromatography of the application further comprises further elution, which is adjusted to a suitable condition, so that the HCP impurity content is substantially reduced, e.g., by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or more.

[0414] For example, the suitable condition of the further elution is that a salt solution with higher ionic strength can be used.

[0415] For example, the salt solution is a phosphate salt solution.

[0416] For example, the suitable elution condition can be adjusting the elution pH.

[0417] For example, the elution pH can be about 7.0 to about 8.0.

[0418] For example, the suitable elution condition can be adjusting the elution salt concentration.

[0419] For example, the elution salt concentration can be approximately 3mM to approximately 50mM, approximately 3mM to approximately 40mM, approximately 4mM to approximately 40mM, approximately 5mM to approximately 40mM, approximately 10mM to approximately 40mM, approximately 10mM to approximately 30mM, or approximately 10mM to approximately 20mM.

[0420] For example, the elution salt concentration can be approximately 4.7 mM, 20 mM, or 40 mM.

[0421] In step d), in one embodiment, the chromatographic step is performed using size exclusion chromatography and ion exchange chromatography.

[0422] The size exclusion chromatography can be gel column chromatography.

[0423] For example, this application uses gel column chromatography to purify mixed solutions containing viruses obtained by any known method.

[0424] For example, pretreatment of a mixed solution containing poliovirus before using gel column chromatography.

[0425] For example, pretreatment refers to ultrafiltration concentration.

[0426] For example, ultrafiltration concentration includes hollow fiber ultrafiltration concentration. The hollow fiber membrane used can be any commercially available hollow fiber membrane, such as PVDF, PES, or PSF.

[0427] For example, hollow fiber ultrafiltration concentration includes ultrafiltration concentration that increases the area of ​​the hollow fiber membrane. For example, the membrane area is increased to at least 150%-200% of the original membrane area.

[0428] For example, gel column chromatography can use agarose gel. The agarose gel can be any commercially available agarose gel packing material.

[0429] For example, the column height of the gel column chromatography used in this application is about 30-100 cm.

[0430] For example, the gel column chromatography used in this application requires only one or two loading cycles.

[0431] For example, the gel column chromatography loading volume used in this application is approximately 10%-40% CV.

[0432] For example, after using gel column chromatography, further elution is performed using anion exchange chromatography, with the conditions adjusted to be suitable to significantly reduce the HCP impurity content, for example, by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or more.

[0433] For example, the further elution condition can be adjusting elution pH.

[0434] For example, the further elution condition can be adjusting elution pH.

[0435] For example, the further elution condition can be adjusting elution pH.

[0436] For example, the further elution condition can be adjusting elution pH.

[0437] For example, the further elution condition can be adjusting elution pH.

[0438] For example, the further elution condition can be adjusting elution pH.

[0439] For example, the further elution condition can be adjusting elution pH.

[0440] The present application also provides another method for preparing poliovirus solution, which comprises the following steps in the order shown:

[0441] a) providing cells, which are in culture medium, and the cells are cultured with microcarriers;

[0442] b) adjusting the cells to a proper cell density, and then contacting poliovirus with the cells to obtain a mixed solution of poliovirus and cells, and the contacting time is about 3-6 days;

[0443] c) centrifuging the mixed solution, and collecting the supernatant, and the virus titer in the supernatant is at least about 1.00E+06 CCID 50 / ml, about 2.00E+06 CCID 50 / ml, about 3.00E+06 CCID 50 / ml, about 4.00E+06 CCID 50 / ml, about 5.00E+06 CCID 50 / ml, about 6.00E+06 CCID 50 / ml, about 7.00E+06 CCID 50 / ml, about 8.00E+06 CCID 50 / ml, about 9.00E+06 CCID 50 / ml, 1.00E+07 CCID 50 / ml, about 2.00E+07 CCID50 about 3.00E+07 CCID / ml 50 about 4.00E+07 CCID / ml 50 about 5.00E+07 CCID / ml 50 about 6.00E+07 CCID / ml 50 about 7.00E+07 CCID / ml 50 about 8.00E+07 CCID / ml 50 about 9.00E+07 CCID / ml 50 about 1.00E+08 CCID / ml 50 2.00E+08 CCID / ml 50 3.00E+08 CCID / ml 50 4.00E+08 CCID / ml 50 5.00E+08 CCID / ml 50 6.00E+08 CCID / ml 50 7.00E+08 CCID / ml 50 8.00E+08 CCID / ml 50 9.00E+08 CCID / ml 50 1.00E+09 CCID / ml 50 2.00E+09 CCID / ml 50 3.00E+09 CCID / ml 50 4.00E+09 CCID / ml 50 5.00E+09 CCID / ml or 50 / ml.

[0444] d) purifying the supernatant by a chromatography step to obtain the poliovirus solution, the chromatography step comprising using molecular exclusion chromatography and / or ion exchange chromatography.

[0445] The microcarrier culture method used in this scheme reduces the contamination of exogenous microorganisms under the premise of providing the same cell attachment surface area compared with the traditional cell factory process, increases the total amount of cells before infection by nearly 30%, increases the titer of the virus harvest liquid by 4 times, significantly reduces the volume of the harvest liquid, and is more conducive to subsequent purification operations; at the same time, through the optimization of the chromatography method, the purification step time is significantly improved (the traditional molecular exclusion chromatography purification cycle number is 6-10 times, and the improved method purification cycle number is 1-2 times), the purification time is shorter, the virus batch stability is significantly improved, the host cell DNA (HCD) and host cell protein (HCP) are greatly reduced, and the scale-up and commercial production are easy.

[0446] The microcarriers used in step a) are well known to those skilled in the art and are generally available in large quantities from commercial sources or can be prepared according to standard methods under conventional conditions. The microcarriers described herein include, but are not limited to, the Cytodex series, Cytopore, Cytoline, and the like. In one embodiment, the microcarriers are the spherical microcarriers Cytodex-1.

[0447] The amount of microcarriers used in the present application balances the cost and viral infection titer, i.e., the viral harvest titer is at least 4-fold greater than that obtained by conventional methods. In certain embodiments, the amount of microcarriers used in the present application is controlled to be between about 2 g / L and about 6 g / L. In one embodiment, the amount of microcarriers used in the present application is controlled to be between about 2 g / L and about 4 g / L. In one embodiment, the amount of microcarriers used in the present application is controlled to be about 3 g / L.

[0448] The present application also includes a method for increasing the viability of cells grown in the presence of microcarriers, comprising adding a feed to the culture medium containing the microcarriers, the feed resulting in a sugar concentration in the culture system of no less than about 0.5 g / L, about 1 g / L, about 1.5 g / L, about 2 g / L, or more.

[0449] In one embodiment, the feed results in a sugar concentration in the culture medium of no less than 1 g / L.

[0450] In one embodiment, the feed used in the present application is fresh complete medium.

[0451] The method of feed addition in the present application can be any method known in the art, such as continuous feed, intermittent feed addition, semi-continuous exchange feed, or discontinuous exchange feed.

[0452] In one embodiment, the feed used in the present application is added by discontinuous exchange feed.

[0453] In step b), the appropriate cell density is a cell density of about 1 x 10 5 cells / ml, about 2 x 10 5 cells / ml, about 3 x 10 5 cells / ml, about 4 x 10 5 cells / ml, about 5 x 10 5 cells / ml, about 6 x 10 5 cells / ml, about 7 x 10 5 cells / ml, about 8 x 10 5 cells / ml, about 9 x 10 5 cells / ml, about 1 x 10 6 cells / ml, or about 2 x 106 The cell density is measured in cells / ml, at which point the cells come into contact with the poliovirus.

[0454] When the poliovirus is in contact with cells for approximately 3-6 days, the contact can be carried out under conditions of an MOI of approximately 0.01 to approximately 0.1.

[0455] For example, when the poliovirus is in contact with cells for about 5-7 days, the contact can be carried out under conditions where the MOI is about 0.05.

[0456] In step c), after the poliovirus has multiplied in the cells, the virus or a component thereof is harvested from the cell culture. This can be done using conventional methods also known to those skilled in the art. The virus produced and released in the cell culture medium can be separated from the biological tissue of the cells by conventional methods such as centrifugation or filtration and harvested in the supernatant. In this case, centrifugation or filtration is the harvesting step. Conventional methods can be used to harvest the virus, for example, by collecting the culture medium containing the virus suspension at the end of the culture, which can be filtered using filters such as 0.22 μm, 0.45 μm, or 0.8 μm and optionally stored at 4°C.

[0457] In one embodiment, the viral titer of the mixed solution is at least about 1.00E+06CCID. 50 / ml, approximately 2.00E+06CCID 50 / ml, approximately 3.00E+06CCID 50 / ml, approximately 4.00E+06CCID 50 / ml, approximately 5.00E+06CCID 50 / ml, approximately 6.00E+06CCID 50 / ml, approximately 7.00E+06CCID 50 / ml, approximately 8.00E+06CCID 50 / ml, approximately 9.00E+06CCID 50 / ml, 1.00E+07CCID 50 / ml, approximately 2.00E+07CCID 50 / ml, approximately 3.00E+07CCID 50 / ml, approximately 4.00E+07CCID 50 / ml, approximately 5.00E+07CCID 50 / ml, approximately 6.00E+07CCID 50 / ml, approximately 7.00E+07CCID 50 / ml, approximately 8.00E+07CCID 50 / ml, approximately 9.00E+07CCID50 / ml, about 1.00E+08 CCID 50 / ml, 2.00E+08 CCID 50 / ml, 3.00E+08 CCID 50 / ml, 4.00E+08 CCID 50 / ml, 5.00E+08 CCID 50 / ml, 6.00E+08 CCID 50 / ml, 7.00E+08 CCID 50 / ml, 8.00E+08 CCID 50 / ml, or 9.00E+08 CCID 50 / ml.

[0458] In one embodiment, the virus titer of the mixed solution is about 1.00E+06 CCID 50 / ml to 5.00E+08 CCID 50 / ml.

[0459] In one embodiment, the virus titer of the mixed solution is about 1.00E+06 CCID 50 / ml to 9.00E+07 CCID 50 / ml.

[0460] In one embodiment, the virus titer of the supernatant is at least about 1.00E+06 CCID 50 / ml, about 2.00E+06 CCID 50 / ml, about 3.00E+06 CCID 50 / ml, about 4.00E+06 CCID 50 / ml, about 5.00E+06 CCID 50 / ml, about 6.00E+06 CCID 50 / ml, about 7.00E+06 CCID 50 / ml, about 8.00E+06 CCID 50 / ml, about 9.00E+06 CCID 50 / ml, 1.00E+07 CCID 50 / ml, about 2.00E+07 CCID 50 / ml, about 3.00E+07 CCID 50 / ml, about 4.00E+07 CCID 50 / ml, about 5.00E+07 CCID 50 / ml, about 6.00E+07 CCID 50 / ml, about 7.00E+07 CCID50 about 8.00E+07 CCID 50 about 9.00E+07 CCID 50 about 1.00E+08 CCID 50 about 2.00E+08 CCID 50 about 3.00E+08 CCID 50 about 4.00E+08 CCID 50 about 5.00E+08 CCID 50 about 6.00E+08 CCID 50 about 7.00E+08 CCID 50 about 8.00E+08 CCID 50 about 9.00E+08 CCID 50 about 1.00E+09 CCID

[0461] In one embodiment, the virus titer of the supernatant obtained is at least about 5.00E+06 CCID 50 / ml to 9.00E+08 CCID 50 / ml.

[0462] In one embodiment, the virus titer of the supernatant obtained is at least about 5.00E+06 CCID 50 / ml to 5.00E+08 CCID 50 / ml.

[0463] In one embodiment, the virus titer of the supernatant obtained is at least about 5.00E+06 CCID 50 / ml to 9.00E+07 CCID 50 / ml.

[0464] In one embodiment, the supernatant of the virus mixture solution is pretreated before or after the chromatography step of d). Typically, in the pretreatment step, cell debris and other insoluble impurities are removed from the supernatant, which is adjusted to a condition suitable for chromatography purification.

[0465] In one embodiment, a nuclease is added before the chromatography step. The nuclease can be a DNA-removing nuclease or an RNA-removing nuclease.

[0466] For example, Benzonase® all-purpose nuclease is added before the chromatography step.

[0467] In the step d), in one embodiment, the chromatography step is ion exchange chromatography.

[0468] For example, the ion exchange chromatography includes monolith ion exchange chromatography with a polymethylmethacrylate matrix.

[0469] For another example, the monolith ion exchange chromatography with a polymethylmethacrylate matrix used in the present application also has QA functional groups.

[0470] For another example, the monolith ion exchange chromatography with a polymethylmethacrylate matrix used in the present application also has DEAE functional groups.

[0471] For example, the virus-containing mixed solution used in the present application is subjected to suitable adjustment before being loaded into the ion exchange chromatography, so that the virus-containing mixed solution can bind to the functional groups of the ion exchange chromatography.

[0472] For another example, the pH of the virus-containing mixed solution is adjusted to about 7.0 to about 8.0.

[0473] For another example, the conductivity of the supernatant of the virus-containing mixed solution is adjusted to about 2.0 to about 8.0 ms / cm.

[0474] For another example, the conductivity of the supernatant of the virus-containing mixed solution is adjusted to about 3.0 to about 7.0 ms / cm.

[0475] For another example, the conductivity of the supernatant of the virus-containing mixed solution is adjusted to about 5.0 to about 6.0 ms / cm.

[0476] For example, the monolith ion exchange chromatography method used in the present application also includes eluting the virus-containing mixed solution so that the virus particles and other impurities are separated. The elution needs to be carried out under suitable conditions.

[0477] For another example, the suitable elution conditions can be to select an eluent with strong ionic strength.

[0478] For another example, the eluent includes, but is not limited to, NaCl, KCl, Na2SO4 and / or Na3PO4, K2SO4 and / or K3PO4.

[0479] For another example, the eluent is NaCl.

[0480] For another example, the suitable elution conditions can be to adjust the elution pH.

[0481] For another example, the elution pH can be about 5.0 to about 7.5.

[0482] For another example, the elution pH can be about 5.5 to about 6.0.

[0483] For another example, the suitable elution conditions can be to adjust the elution salt concentration.

[0484] For another example, the elution salt concentration can be about 50 mM to about 300 mM.

[0485] For another example, the elution salt concentration can be about 250 mM.

[0486] For another example, the suitable elution condition can be adjusting the elution mode.

[0487] For another example, the elution mode can be one-step elution.

[0488] In one embodiment, the application can further purify the poliovirus after the ion exchange chromatography purification using anion exchange chromatography, so that the HCP impurity content is further reduced, for example, by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or more.

[0489] For example, the anion exchange chromatography has a functional group of quaternary amino group.

[0490] For example, the eluate of the ion exchange chromatography is loaded into the anion exchange chromatography, and the eluate is adjusted to a suitable condition.

[0491] For another example, the suitable loading condition means that the pH of the eluate of the ion exchange chromatography can be adjusted to about 7.0 to about 8.0.

[0492] For another example, the suitable loading condition means that the conductivity of the eluate of the ion exchange chromatography can be adjusted to about 2.0 to about 8.0 ms / cm.

[0493] For another example, the suitable loading condition means that the conductivity of the eluate of the ion exchange chromatography can be adjusted to about 3.0 to about 7.0 ms / cm.

[0494] For another example, the suitable loading condition means that the conductivity of the eluate of the ion exchange chromatography can be adjusted to about 5.0 to about 6.0 ms / cm.

[0495] For example, the further purification method using the anion exchange chromatography used in the application further includes further elution, and the further elution is adjusted to a suitable condition, so that the HCP impurity content is greatly reduced, for example, by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or more.

[0496] For another example, the suitable condition of the further elution means that a salt solution with strong ionic strength can be selected.

[0497] For another example, the salt solution is a phosphate solution.

[0498] For another example, the suitable elution condition can be adjusting elution pH.

[0499] For yet another example, the elution pH can be about 7.0 to about 8.0.

[0500] For another example, the suitable elution condition can be adjusting elution salt concentration.

[0501] For yet another example, the elution salt concentration can be about 3 mM to about 50 mM, about 3 mM to about 40 mM, about 4 mM to about 40 mM, about 5 mM to about 40 mM, about 10 mM to about 40 mM, about 10 mM to about 30 mM, about 10 mM to about 20 mM.

[0502] For yet another example, the elution salt concentration can be about 4.7 mM, 20 mM or 40 mM.

[0503] In step d), in one embodiment, the chromatography step is using size exclusion chromatography and ion exchange chromatography.

[0504] The size exclusion chromatography can be gel column chromatography.

[0505] For example, the gel column chromatography used in the present application purifies the mixed solution containing viruses obtained by any known means.

[0506] For example, the mixed solution containing poliovirus is pretreated before using the gel column chromatography.

[0507] For another example, the pretreatment refers to ultrafiltration concentration.

[0508] For yet another example, the ultrafiltration concentration includes hollow fiber ultrafiltration concentration. The hollow fiber membrane used therein can be any commercially available hollow fiber membrane, such as the hollow fiber membrane material can be PVDF, PES or PSF.

[0509] For yet another example, the hollow fiber ultrafiltration concentration includes ultrafiltration concentration with increased hollow fiber membrane area. For example, the membrane area is increased to at least 150% to 200% of the original membrane area.

[0510] For another example, the gel column chromatography can be agarose gel. The agarose gel can be any commercially available agarose gel packing.

[0511] For example, the gel column chromatography used in the present application has a column height of about 30 to 100 cm.

[0512] For example, the gel column chromatography used in the present application requires only one or two sample loading cycles.

[0513] For example, the gel column chromatography used in the present application has a sample loading amount of about 10% to 40% CV.

[0514] For example, further elution after using gel column chromatography followed by anion exchange chromatography further elution, the further elution conditions are adjusted to be suitable, so that the HCP impurity content is greatly reduced, for example, reduced by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or more. For another example, the further elution suitable conditions refer to the salt solution with stronger ionic strength can be selected.

[0515] For another example, the salt solution is a phosphate solution.

[0516] For another example, the suitable elution conditions can be adjusting the elution pH.

[0517] For another example, the elution pH can be about 7.0 to about 8.0.

[0518] For another example, the suitable elution conditions can be adjusting the elution salt concentration.

[0519] For another example, the elution salt concentration can be about 3 mM to about 50 mM, about 3 mM to about 40 mM, about 4 mM to about 40 mM, about 5 mM to about 40 mM, about 10 mM to about 40 mM, about 10 mM to about 30 mM, about 10 mM to about 20 mM.

[0520] For another example, the elution salt concentration can be about 4.7 mM, 20 mM or 40 mM.

[0521] After using the method of the present application to purify poliovirus, the purified virus titer can be at least about 1.00E+07 CCID 50 / ml, about 2.00E+07 CCID 50 / ml, about 3.00E+07 CCID 50 / ml, about 4.00E+06 CCID 50 / ml, about 5.00E+07 CCID 50 / ml, about 6.00E+07 CCID 50 / ml, about 7.00E+07 CCID 50 / ml, about 8.00E+07 CCID 50 / ml, about 9.00E+07 CCID 50 / ml, about 1.00E+08 CCID 50 / ml, 2.00E+08 CCID 50 / ml, 3.00E+08 CCID 50 / ml, 4.00E+08 CCID 50 / ml, 5.00E+08 CCID50 / ml, 6.00E+08 CCID 50 / ml, 7.00E+08 CCID 50 / ml, 8.00E+08 CCID 50 / ml, 9.00E+08 CCID 50 / ml, 1.00E+09 CCID 50 / ml, 2.00E+09 CCID 50 / ml, 3.00E+09 CCID 50 / ml, 4.00E+09 CCID 50 / ml, or 5.00E+09 CCID 50 / ml, indicating that the process of the present application can significantly increase the purity of poliovirus.

[0522] After poliovirus is purified using the method of the present application, the recovery rate of the purified virus can be at least about 40% to about 99%, about 40% to about 90%, about 40% to about 85%, about 40% to about 80%, about 40% to 75%, or about 40% to about 70%, indicating that the process of the present application can significantly improve the purification efficiency of poliovirus.

[0523] After poliovirus is purified using the method of the present application, the RNA plasmid in the purified virus is at least about 1.00E+09 copies / ml, about 2.00E+09 copies / ml, about 3.00E+09 copies / ml, about 4.00E+09 copies / ml, about 5.00E+09 copies / ml, about 6.00E+09 copies / ml, about 7.00E+09 copies / ml, about 8.00E+09 copies / ml, about 9.00E+09 copies / ml, about 1.00E+10 copies / ml, about 2.00E+10 copies / ml, about 3.00E+10 copies / ml, about 4.00E+10 copies / ml, about 5.00E+10 copies / ml, about 6.00E+10 copies / ml, about 7.00E+10 copies / ml, about 8.00E+10 copies / ml, about 9.00E+10 copies / ml, about 1.00E+11 copies / ml, about 2.00E+11 copies / ml, about 3.00E+11 copies / ml, about 4.00E+11 copies / ml, or about 5.00E+11 copies / ml, indicating that the process of the present application can effectively protect and purify the genetic material of poliovirus, ensuring the activity and integrity of the virus.

[0524] The present application also provides the following embodiments:

[0525] 1. A method for preparing a poliovirus solution, comprising the steps in the order shown:

[0526] a) providing cells, said cells being present in a culture medium;

[0527] b) adjusting the cells to a suitable cell density, and then contacting poliovirus with the cells to obtain a mixed solution of poliovirus and cells;

[0528] c) subjecting the mixed solution to centrifugation, and collecting the supernatant;

[0529] d) purifying the supernatant by a chromatographic step to obtain the poliovirus solution.

[0530] 2. The method according to embodiment 1, wherein the culture medium in a) comprises a serum-containing culture medium.

[0531] 3. The method according to any one of embodiments 1-2, wherein the cells in a) are cultured using microcarriers.

[0532] 4. The method according to embodiment 3, wherein the cells further comprise an addition of a fed-batch culture, such that the glucose content in the cell culture system is not less than about 1 g / L.

[0533] 5. The method according to embodiment 4, wherein the feeding regime of the fed-batch culture comprises a discontinuous feeding regime.

[0534] 6. The method according to embodiment 5, wherein the discontinuous feeding regime is a daily medium exchange from the second day of culture.

[0535] 7. The method according to embodiment 1, wherein the culture medium in a) further comprises a serum-free culture medium.

[0536] 8. The method according to embodiment 7, wherein the serum-free culture medium comprises a chemically defined serum-free culture medium.

[0537] 9. The method according to embodiment 2, wherein the serum-containing culture medium comprises serum from at least one animal source selected from the group consisting of bovine, equine, chicken, ovine and human.

[0538] 10. The method according to embodiment 9, wherein the serum comprises fetal bovine serum, calf serum, bovine serum, equine serum, chicken serum, ovine serum or human serum.

[0539] 11. The method according to any one of embodiments 1-10, wherein the suitable cell density in b) is about 1 x 10 5

[0540] cells / ml - approximately 2 × 10 6 cells / ml.

[0541] 12. The method according to any one of embodiments 1-11, wherein the multiplicity of infection (MOI) of contact with poliovirus as described in b) is about 0.01 to about 0.5.

[0542] 13. The method according to embodiment 12, wherein the multiple of infection upon contact with the poliovirus is...

[0543] Under (MOI) conditions, the contact time is approximately 3-6 days.

[0544] 14. The method according to embodiment 13, wherein the multiple of infection (MOI) of the contact with poliovirus is...

[0545] It is approximately 0.01 to approximately 0.1.

[0546] 15. The method according to embodiment 12, wherein the multiple of infection upon contact with the poliovirus is...

[0547] The contact time under (MOI) conditions is approximately 5-7 days.

[0548] 16. The method according to embodiment 15, wherein the multiple of infection (MOI) of the contact with poliovirus is...

[0549] It ranges from approximately 0.05 to approximately 0.2.

[0550] 17. The method according to any one of embodiments 1-16, wherein the mixed solution in b) comprises poliovirus.

[0551] Toxins, cells, cell debris, and / or culture medium.

[0552] 18. The method according to any one of embodiments 1-17, wherein the viral titer in the supernatant described in c) is at least [missing information].

[0553] 1.00E+06CCID 50 / ml, 2.00E+06CCID 50 / ml, 3.00E+06CCID 50 / ml, 4.00E+06CCID 50 / ml, 5.00E+06CCID 50 / ml, 6.00E+06CCID 50 / ml, 7.00E+06CCID 50 / ml, 8.00E+06CCID 50 / ml, 9.00E+06 CCID 50 / ml, 1.00E+07 CCID 50 / ml, 2.00E+07 CCID 50 / ml, 3.00E+07 CCID 50 / ml, 4.00E+06 CCID 50 / ml, 5.00E+07 CCID 50 / ml, 6.00E+07 CCID 50 / ml, 7.00E+07 CCID 50 / ml, 8.00E+07 CCID 50 / ml, 9.00E+07 CCID 50 / ml, 1.00E+08 CCID 50 / ml, 2.00E+08 CCID 50 / ml, 3.00E+08 CCID 50 / ml, 4.00E+08 CCID 50 / ml, 5.00E+08 CCID 50 / ml, 6.00E+08 CCID 50 / ml, 7.00E+08 CCID 50 / ml, 8.00E+08 CCID 50 / ml, 9.00E+08 CCID 50 / ml, 1.00E+09 CCID 50 / ml, 2.00E+09 CCID 50 / ml, 3.00E+09 CCID 50 / ml, 4.00E+09 CCID 50 / ml or 5.00E+09

[0554] CCID 50 / ml.

[0555] 19. The method according to any one of embodiments 1-18, wherein the chromatography step in d) comprises using ion exchange chromatography, affinity chromatography, and / or molecular exclusion chromatography.

[0556] 20. The method according to any one of embodiments 1-19, wherein the chromatography step in d) comprises using gel column chromatography, monolithic column chromatography and / or anion exchange chromatography.

[0557] 21. The method according to any one of embodiments 1-20, wherein the purification in d) further comprises a pre-treatment, said pre-purification being performed prior to the chromatography step in d).

[0558] 22. The method of embodiment 21, wherein the pre-treatment comprises addition of a nuclease and / or filtration to remove bacteria, further comprising ultrafiltration.

[0559] 23. The method of any one of embodiments 1-22, wherein the purification in d) is performed at a pH of the supernatant in c) of about

[0560] 6.0-10.0.

[0561] 24. The method of any one of embodiments 1-23, wherein the chromatography step in d) further comprises elution using an eluent.

[0562] 25. The method of embodiment 24, the elution comprising using linear gradient elution and / or step elution.

[0563] 26. The method of any one of embodiments 24-25, the eluent having a pH of about 5.0-8.0.

[0564] 27. The method of any one of embodiments 24-25, the eluent comprising an elution salt, the elution salt comprising

[0565] NaCl, KCl, Na2SO4, Na3PO4, K2SO4, and / or K3PO4.

[0566] 28. The method of embodiment 27, wherein the elution salt is NaCl.

[0567] 29. The method of any one of embodiments 27-28, wherein the elution salt concentration is about 10-500 mM.

[0568] 30. The method of embodiment 20, the anion exchange chromatography used after the gel column and / or monolith column chromatography.

[0569] 31. The method of any one of embodiments 1-30, wherein the cells are Vero cells.

[0570] 32. The method of any one of embodiments 1-31, the poliovirus comprising a wild-type poliovirus and / or a mutant thereof, and the mutant comprising one or more substitutions, mutations, insertions, and / or deletions of a gene.

[0571] 33. A poliovirus solution having a viral titer of at least 1.00E+06 CCID50 / mL prepared by the method of any one of embodiments 1-32. 50 / ml, 2.00E+06 CCID 50 / ml, 3.00E+06

[0572] CCID 50 / ml, 4.00E+06 CCID 50 / ml, 5.00E+06 CCID 50 / ml, 6.00E+06 CCID 50 / ml, 7.00E+06

[0573] CCID 50 / ml, 8.00E+06 CCID 50 / ml, 9.00E+06 CCID 50 / ml, 1.00E+07 CCID 50 / ml, 2.00E+07

[0574] CCID 50 / ml, 3.00E+07 CCID 50 / ml, 4.00E+06 CCID 50 / ml, 5.00E+07 CCID 50 / ml, 6.00E+07

[0575] CCID 50 / ml, 7.00E+07 CCID 50 / ml, 8.00E+07 CCID 50 / ml, 9.00E+07 CCID 50 / ml, 1.00E+08

[0576] CCID 50 / ml, 2.00E+08 CCID 50 / ml, 3.00E+08 CCID 50 / ml, 4.00E+08 CCID 50 / ml, 5.00E+08

[0577] CCID 50 / ml, 6.00E+08 CCID 50 / ml, 7.00E+08 CCID 50 / ml, 8.00E+08 CCID 50 / ml, 9.00E+08

[0578] CCID 50 / ml, 1.00E+09 CCID 50 / ml, 2.00E+09 CCID 50 / ml, 3.00E+09CCID 50 / ml, 4.00E+09

[0579] CCID 50 / ml or 5.00E+09CCID 50 / ml.

[0580] 34. A method for preparing poliovirus harvest fluid, comprising the steps in the order shown:

[0581] a) Provide cells, which are present in a culture medium;

[0582] b) Adjust the cells to a suitable cell density, and then contact the poliovirus with the cells to obtain a mixed solution of poliovirus and cells;

[0583] c) Obtain the poliovirus harvest fluid from the mixed solution.

[0584] 35. The method according to embodiment 34, wherein the poliovirus harvesting fluid comprises poliovirus,

[0585] Cells and / or cell debris.

[0586] 36. The method according to any one of embodiments 34-35, wherein the culture medium described in a) is a serum-containing culture medium.

[0587] 37. The method according to any one of embodiments 34-36, wherein the cells described in a) are cultured using microcarriers.

[0588] 38. The method according to embodiment 37, wherein the cells further include fed culture to ensure that the glucose content in the cell culture system is not less than about 1 g / L.

[0589] 39. The method according to embodiment 38, wherein the fluid replacement method for replenishment includes discontinuous fluid replacement.

[0590] 40. The method according to embodiment 39, wherein the discontinuous medium change begins with changing the culture medium daily from the second day of culture.

[0591] 41. The method according to embodiment 34, wherein the culture medium described in a) further includes a serum-free culture medium.

[0592] 42. The method according to embodiment 41, wherein the serum-free culture medium comprises a serum-free culture medium with a clearly defined chemical composition.

[0593] 43. The method of embodiment 36, wherein said serum-containing medium comprises at least a serum of animal origin selected from the group consisting of bovine, equine, chicken, ovine and human.

[0594] 44. The method of embodiment 43, wherein said serum comprises fetal bovine serum, calf serum, bovine serum, equine serum, chicken serum, ovine serum or human serum.

[0595]

[0596] 45. The method of any one of embodiments 34-44, wherein said suitable cell density in b) is about 1 x 10 5

[0597] cells / ml to about 2 x 10 6 cells / ml.

[0598] 46. The method of any one of embodiments 34-45, wherein said multiplicity of infection (MOI) with poliovirus in b) is about 0.01 to about 0.5.

[0599] 47. The method of embodiment 46, wherein said multiplicity of infection (MOI) with poliovirus is contacted for a period of time of about 3 to 6 days.

[0600]

[0601] 48. The method of embodiment 47, wherein said multiplicity of infection (MOI) with poliovirus is about 0.01 to about 0.1.

[0602]

[0603] 49. The method of embodiment 46, wherein said multiplicity of infection (MOI) with poliovirus is contacted for a period of time of about 5 to 7 days.

[0604]

[0605] 50. The method of embodiment 49, wherein said multiplicity of infection (MOI) with poliovirus is about 0.05 to about 0.2.

[0606]

[0607] 51. The method of any one of embodiments 34-50, wherein said mixture in b) comprises poliovirus, cells, cell debris and / or medium.

[0608]

[0609] ​​​​​​52. The method of any of embodiments 34-51, wherein the viral titer of the poliovirus harvest of c) is at least 1.00E+06 CCID50 50 / ml, 2.00E+06 CCID 50 / ml, 3.00E+06 CCID 50 / ml, 4.00E+06

[0610] CCID 50 / ml, 5.00E+06 CCID 50 / ml, 6.00E+06 CCID 50 / ml, 7.00E+06 CCID 50 / ml, 8.00E+06

[0611] CCID 50 / ml, 9.00E+06 CCID 50 / ml, 1.00E+07 CCID 50 / ml, 2.00E+07 CCID 50 / ml, 3.00E+07

[0612] CCID 50 / ml, 4.00E+06 CCID 50 / ml, 5.00E+07 CCID 50 / ml, 6.00E+07 CCID 50 / ml, 7.00E+07

[0613] CCID 50 / ml, 8.00E+07 CCID 50 / ml, 9.00E+07 CCID 50 / ml, 1.00E+08 CCID 50 / ml, 2.00E+08

[0614] CCID 50 / ml, 3.00E+08 CCID 50 / ml, 4.00E+08 CCID 50 / ml, 5.00E+08 CCID 50 / ml, 6.00E+08

[0615] CCID 50 / ml, 7.00E+08 CCID 50 / ml, 8.00E+08 CCID 50 / ml, 9.00E+08 CCID 50 / ml, 1.00E+09

[0616] CCID 50 / ml, 2.00E+09 CCID 50 / ml, 3.00E+09 CCID 50 / ml, 4.00E+09 CCID 50 / ml, or

[0617] 5.00E+09 CCID 50 / ml.

[0618] 53. The method of any one of embodiments 34-52, wherein the cells are Vero cells.

[0619] 54. The method of any one of embodiments 34-53, the poliovirus comprises a wild-type poliovirus and / or a mutant thereof, and the mutant comprises a substitution, mutation, insertion, and / or deletion of one or more genes.

[0620] 55. A method of purifying poliovirus, comprising performing a chromatography step on a mixed solution of poliovirus and cells.

[0621] 56. The method of embodiment 55, wherein the mixed solution comprises a viral titer of at least 1.00E+06 CCID 50 / ml,

[0622] 2.00E+06 CCID 50 / ml, 3.00E+06 CCID 50 / ml, 4.00E+06 CCID 50 / ml, 5.00E+06 CCID 50 / ml, 6.00E+06 CCID 50 / ml, 7.00E+06 CCID 50 / ml, 8.00E+06 CCID 50 / ml, 9.00E+06 CCID 50 / ml, 1.00E+07 CCID 50 / ml, 2.00E+07 CCID 50 / ml, 3.00E+07 CCID 50 / ml, 4.00E+06 CCID 50 / ml, 5.00E+07 CCID 50 / ml, 6.00E+07 CCID 50 / ml, 7.00E+07 CCID 50 / ml, 8.00E+07 CCID50 / ml, 9.00E+07 CCID 50 / ml, 1.00E+08 CCID 50 / ml, 2.00E+08 CCID 50 / ml, 3.00E+08 CCID 50 / ml, 4.00E+08 CCID 50 / ml, 5.00E+08 CCID 50 / ml, 6.00E+08 CCID 50 / ml, 7.00E+08 CCID 50 / ml, 8.00E+08 CCID 50 / ml, 9.00E+08 CCID 50 / ml, 1.00E+09 CCID 50 / ml, 2.00E+09 CCID 50 / ml,

[0623] 3.00E+09 CCID 50 / ml, 4.00E+09 CCID 50 / ml, or 5.00E+09 CCID 50 / ml of poliovirus.

[0624] 57. The method of any one of embodiments 55-56, wherein the mixed solution comprises poliovirus, cells, cell debris, and / or media.

[0625] 58. The method of embodiment 57, wherein the cells comprise cells obtained using serum culture, serum-free culture, and / or microcarrier culture.

[0626] 59. The method of any one of embodiments 55-58, further comprising centrifuging the poliovirus mixed solution to obtain a supernatant.

[0627] 60. The method of embodiment 59, further comprising a pH of the supernatant is about 6.0-10.0 when the chromatography step is performed.

[0628] 61. The method of any one of embodiments 55-60, wherein the chromatography step comprises using ion exchange chromatography, affinity chromatography, and / or size exclusion chromatography.

[0629] 62. The method of any one of embodiments 55-61, wherein the purification further comprises a pre-treatment.

[0630] 63. The method of embodiment 62, wherein the pre-treatment comprises addition of a nuclease and / or filtration to remove bacteria, further comprising ultrafiltration.

[0631] 64. The method of any one of embodiments 55-63, wherein the chromatography step comprises use of gel column chromatography, monolith column chromatography, and / or anion exchange chromatography.

[0632] 65. The method of any one of embodiments 55-64, wherein the chromatography step further comprises use of an elution buffer for elution.

[0633] 66. The method of embodiment 65, wherein the elution buffer is used for linear gradient elution and / or step elution.

[0634] 67. The method of any one of embodiments 65-66, wherein the elution buffer has a pH of about 5.0-8.0.

[0635] 68. The method of any one of embodiments 65-67, wherein the elution buffer comprises an elution salt, the elution salt comprising NaCl, KC1, Na2S04, and / or Na3P04, K2S04, and / or K3P04.

[0636] 69. The method of embodiment 68, wherein the elution salt is NaCl.

[0637] 70. The method of any one of embodiments 68-69, wherein the elution salt has a concentration of about 10-500 mM.

[0638] 71. The method of embodiment 64, wherein the anion exchange chromatography is used after the gel column and / or monolith column chromatography.

[0639] 72. The method of any one of embodiments 57-71, wherein the cell is a Vero cell.

[0640] 73. The method of any one of embodiments 56-72, wherein the poliovirus comprises a wild-type poliovirus and / or a mutant thereof, and the mutant comprises one or more substitutions, mutations, insertions, and / or deletions of a gene.

[0641] 74. The method of any one of embodiments 55-73, wherein the purified poliovirus has a viral titer of at least 1.00E+06 CCID 50 / ml, 2.00E+06 CCID 50 / ml, 3.00E+06 CCID 50 / ml, 4.00E+06

[0642] CCID 50 / ml, 5.00E+06 CCID 50 / ml, 6.00E+06 CCID 50 / ml, 7.00E+06 CCID 50 / ml, 8.00E+06 CCID 50 / ml, 9.00E+06 CCID 50 / ml, 1.00E+07 CCID 50 / ml, 2.00E+07 CCID 50 / ml, 3.00E+07 CCID 50 / ml, 4.00E+06 CCID 50 / ml, 5.00E+07 CCID 50 / ml, 6.00E+07 CCID 50 / ml, 7.00E+07 CCID 50 / ml, 8.00E+07 CCID 50 / ml, 9.00E+07 CCID 50 / ml, 1.00E+08 CCID 50 / ml, 2.00E+08 CCID 50 / ml, 3.00E+08 CCID 50 / ml, 4.00E+08 CCID 50 / ml, 5.00E+08 CCID 50 / ml, 6.00E+08 CCID 50 / ml, 7.00E+08 CCID 50 / ml, 8.00E+08 CCID 50 / ml, 9.00E+08 CCID 50 / ml, 1.00E+09 CCID 50 / ml, 2.00E+09 CCID 50 / ml, 3.00E+09 CCID 50 / ml, 4.00E+09 CCID 50 / ml, or

[0643] 5.00E+09 CCID 50 / ml.

[0644] 75. A pharmaceutical composition comprising a poliovirus solution prepared using the method of any one of embodiments 1-32, a poliovirus harvest prepared using the method of any one of embodiments 34-54, and / or a purified poliovirus prepared using the method of any one of embodiments 55-74, and optionally a pharmaceutically acceptable adjuvant.

[0645] 76. Use of a poliovirus solution prepared using the method of any one of embodiments 1-32, a poliovirus harvest prepared using the method of any one of embodiments 34-54, and / or a purified poliovirus prepared using the method of any one of embodiments 55-74, and / or the pharmaceutical composition of embodiment 75, for the preparation of a medicament for the prevention, alleviation and / or treatment of a disease and / or a disorder.

[0646] 77. A method for the treatment, alleviation and / or prevention of a disease and / or a disorder, the method comprising administering to a patient in need thereof a therapeutic amount of a poliovirus solution prepared using the method of any one of embodiments 1-32, a poliovirus harvest prepared using the method of any one of embodiments 34-54, and / or a purified poliovirus prepared using the method of any one of embodiments 55-74, and / or the pharmaceutical composition of embodiment 75.

[0647] 78. A poliovirus solution prepared using the method of any one of embodiments 1-32, a poliovirus harvest prepared using the method of any one of embodiments 34-54, and / or a purified poliovirus prepared using the method of any one of embodiments 55-74, and / or the pharmaceutical composition of embodiment 75, for use in the treatment, alleviation and / or prevention of a disease and / or a disorder.

[0648]

[0649] Without wishing to be bound by any theory, the examples below are merely to illustrate the preparation and purification process of the poliovirus of the present application and are not intended to limit the scope of the present application.

[0650] Examples

[0651] Example 1. Preparation of recombinant poliovirus using a serum-containing process

[0652] Example 1.1 Cell recovery and subculturing

[0653] ​In a biological safety cabinet, a 37°C water bath-melted Vero working bank cell (Lot#20190916) was inoculated into a T-75 cell culture flask, and preheated complete culture medium (90% DMEM + 10% FBS) was added. The culture flask was transferred to a CO2 incubator for overnight culture at 37°C, 5% CO2, until the cells were completely adherent. In the biological safety cabinet, the old culture medium in the T-75 was discarded, and the cells were washed once with DPBS buffer, and then recombinant trypsin was added, and incubated at 37°C until the cells were rounded and detached. The cell suspension was collected, centrifuged at 200g for 5 min, the supernatant was discarded, and the cells were resuspended in fresh complete culture medium and counted. The T-75 culture flask was inoculated at a density of 1.5-2.0 x 10 4 cells / cm 2 , and the inoculated T-75 culture flask was transferred to a CO2 incubator for continuous culture at 37°C, 5% CO2, for 3-4 days until the cell confluence reached more than 85%. The cells were subcultured and expanded for 3-4 times in succession. After the last subculture and expansion, all cell culture flasks were transferred to a CO2 incubator for continuous culture at 37°C, 5% CO2, for 3-4 days until the cell confluence reached more than 85%. All cell culture flasks were transferred to a biological safety cabinet, the culture medium in the culture flasks was discarded, the cells were washed once with DPBS buffer, and then recombinant trypsin was added, and incubated at 37°C until the cells were rounded and detached. The cell suspension was collected, centrifuged at 200g for 5 min, the supernatant was discarded, and the cells were resuspended in fresh complete culture medium and counted. The T-75 culture flask was inoculated at a density of 1.5-2.0 x 10 4 cells / cm 2 , and the inoculated T-75 culture flask was transferred to a CO2 incubator for continuous culture at 37°C, 5% CO2, for 3-4 days until the cell confluence reached more than 85%. The cells were subcultured and expanded for 3-4 times in succession. After the last subculture and expansion, all cell culture flasks were transferred to a CO2 incubator for continuous culture at 37°C, 5% CO2, for 3-4 days until the cell confluence reached more than 85%. All cell culture flasks were transferred to a biological safety cabinet, the culture medium in the culture flasks was discarded, the cells were washed once with DPBS buffer, and then recombinant trypsin was added, and incubated at 37°C until the cells were rounded and detached. The cell suspension was collected, centrifuged at 200g for 5 min, the supernatant was discarded, and the cells were resuspended in fresh complete culture medium and counted. The T-75 culture flask was inoculated at a density of 1.5-2.0 x 10

[0654] Example 1.2 Virus inoculation and culture

[0655] When the cell confluence reaches more than 85%, the T-150 culture flask is transferred to a biosafety cabinet. The culture solution in the T-150 culture flask is discarded, and the cells are washed once with DPBS buffer solution, and then recombinant trypsin is added, and the cells are incubated at 37°C until the cells are rounded and detached. The cell suspension is collected, centrifuged at 200g for 5min, the supernatant is discarded, the cells are resuspended with fresh complete culture medium and counted; the number of viable cells per unit culture area of the T-150 culture flask (culture area 150cm 2 ) is calculated, and this value represents the number of viable cells per unit culture area in a one-layer cell factory (culture area 636cm 2 ) and a ten-layer cell factory (culture area 6360cm 2 ), and then the total number of viable cells in each one-layer cell factory and ten-layer cell factory can be calculated.

[0656] The total number of cells in a one-layer cell factory (cells) = T-150 cell counting density (cells / ml) x cell resuspension volume (ml) / 150 x 636

[0657] The total number of cells in a ten-layer cell factory (cells) = T-150 cell counting density (cells / ml) x cell resuspension volume (ml) / 150 x 6360

[0658] The recombinant poliovirus stock (Lot#20180107) is taken out from the -80°C refrigerator and thawed in a 27°C water bath. According to MOI = 0.05, the volume of virus stock required is calculated based on the total number of cells in a one-layer cell factory and a ten-layer cell factory:

[0659] The volume of virus stock required for a one-layer cell factory (ml) = total number of cells in a one-layer cell factory (cells) x MOI (0.05) / virus titer (CCID 50 / ml)

[0660] The volume of virus stock required for a ten-layer cell factory (ml) = total number of cells in a ten-layer cell factory (cells) x MOI (0.05) / virus titer (CCID 50 / ml)

[0661] The one-layer cell factory and the ten-layer cell factory are taken out from the incubator, the culture medium supernatant is discarded, an equal volume of DPBS is added for washing, discarded, and an equal volume of virus maintenance medium DMEM / F12 is added. Then, according to the calculated volume of virus stock, the virus stock is added to the cells in the one-layer cell factory and the ten-layer cell factory, respectively. After thorough mixing, the cell factories are transferred to a CO2 incubator and cultured at 33°C, 5% CO2.

[0662] Example 1.3 Virus Harvesting

[0663] The cells in one layer of cell factory were observed for cytopathic effect (CPE) daily, and when CPE reached more than 90%, the virus was ready to be harvested. Ten layers of cell factories were taken out of the incubator, and the culture supernatant was transferred into clean 50 ml centrifuge tubes, and centrifuged at 4000 g for 30 min. After centrifugation, the supernatant was collected, and the volume was about 1.25 L. MgCl2 solution and Benzonase full-enzyme were added to the supernatant, respectively, so that the final concentration of MgCl2 was 1 mM, and the final concentration of Benzonase full-enzyme was 25 U / ml. Then the supernatant was placed at room temperature for 18-22 h for Benzonase full-enzyme digestion. After digestion, it was filtered through a 0.22 um filter membrane into a clean storage bottle for downstream purification.

[0664] Several batches of small-scale upstream harvest liquid were prepared by this process, and the yield was relatively stable, and the batch consistency was good. The titer data are shown in Table 1.

[0665] Table 1 Titer determination results of three batches of small-scale upstream harvest liquid prepared by serum-containing process

[0666] Lot number Harvested droplet concentration (CCID 50 / ml) Lot#20180504-2 4.87E+07 Lot#20180505-2 2.74E+07 Lot#20180601-2 4.22E+07

[0667] Example 2 Preparation of recombinant poliovirus by microcarrier roller bottle culture process

[0668] Example 2.1 Cell preparation

[0669] A liquid nitrogen tank of Vero cell bank (Lot#20109016, cell passage P132) was taken out and quickly thawed in a 37°C water bath, and then transferred to a T-75 cell culture flask pre-added with preheated complete culture medium (90% DMEM + 10% FBS). The flask was placed in a 37°C, 5% CO2 cell incubator and cultured overnight until the cells were completely adherent. The next day, the old culture medium was discarded, and an equal volume of fresh complete culture medium was added for continuous culture until the cell confluence reached more than 85%. Then, the culture medium supernatant was discarded, and the cells were rinsed once with an equal volume of DPBS buffer, and then discarded. Preheated 0.25% trypsin was added to digest the cells until the cells became round. Complete culture medium was added to terminate the digestion process, and the cells digested were transferred to a clean 50 ml centrifuge tube, centrifuged at 200 g for 5 min at room temperature, and the supernatant was discarded. Complete culture medium was added to resuspend the cells, and the cell count was determined by a cell counter. The cells were diluted to 1.5-2.0 x 10 4 / cm 2The density of the cells was determined and the cells were subcultured. The subculture was repeated several times until the cell state was stable. After the last subculture, the cells in the culture bottle were digested with 0.25% trypsin. When the cells were rounded, the digestion was stopped by adding complete medium. The cells were transferred to a clean 50 ml centrifuge tube, centrifuged at 200 g for 5 min at room temperature, the supernatant was removed, and the cells were resuspended with complete medium. The cells were counted using a cell counter and were ready for use.

[0670] Example 2.2 Preparation of microcarriers

[0671] The spherical microcarriers Cytodex-1 (Cytiva, Cat. No. 17-0448-02) were weighed with an analytical balance, added to a siliconized glass reagent bottle, and covered with PBS. After washing thoroughly, the PBS was discarded and the microcarriers were soaked or swelled in PBS. After that, the microcarriers were sterilized in an autoclave at 121 °C for 30 min. After cooling, the PBS was carefully discarded in a biosafety cabinet, and the microcarriers were washed twice with complete medium. Finally, the microcarriers were resuspended in an appropriate amount of complete medium and were ready for use.

[0672] Example 2.3 Cell seeding and spinner flask culture

[0673] The spinner flask (Corning, Cat. No. 3152) was used for the microcarrier culture of Vero cells. The culture volume in the spinner flask was 100 ml, and the amount of microcarriers used was 3 g / L. The cell seeding density was 2.5E+05 cells / ml. The prepared cell and microcarrier suspension were added to the spinner flask, which was placed on a magnetic stirrer with a rotation speed of 60 rpm. The spinner flask was continuously stirred and cultured in a cell incubator at 37 °C and 5% CO2. The sugar content in the culture medium was detected daily during the continuous culture, and the medium was supplemented with fresh complete medium. The principle of the feeding was to ensure that the sugar content in the medium was not less than 1 g / L. The cell density was detected daily.

[0674] Example 2.4 Inoculation culture

[0675] When the cell density reached the maximum value and the daily sugar consumption was basically stable, the virus was prepared for inoculation. The recombinant poliovirus stock (Lot#20191226) was taken out from the -80 °C refrigerator and thawed in a 27 °C water bath. According to the calculated cell density, the amount of virus needed for inoculation was calculated at MOI = 0.05, as follows:

[0676] Volume of virus (ml) = cell density (cells / ml) x culture volume (100 ml) x MOI (0.05) / virus titer (CCID 50 / ml)

[0677] According to the calculation results, take the corresponding volume of the virus into the preheated virus maintenance medium (DMEM / F12), mix well for standby. Transfer the spinner flask from the incubator to the biosafety cabinet, carefully aspirate the supernatant after the microcarriers settle, wash 1-2 times with the same volume of DPBS, and add the virus and medium mixture prepared earlier to the spinner flask. Transfer the flask to a 33°C, 5% CO2 cell incubator, adjust the stirring speed to 30 rpm, and start the virus culture.

[0678] Example 2.5 Virus Harvesting

[0679] Daily sampling to determine the virus titer of the culture supernatant, continuous culture to the 4th day, the virus titer reaches the maximum value, the spinner flask is transferred to the biosafety cabinet, the supernatant is carefully aspirated into a 50ml centrifuge tube, 4000g centrifugation for 30min, the supernatant is collected and frozen.

[0680] The cell growth state under different cell seeding densities and different microcarrier amounts was explored, and the experimental groups are shown in Table 2. The total cell count was performed daily, and the results are shown in Table 4. Figure 1 .

[0681] Table 2 Microcarrier cell culture group parameters

[0682] Group 1 Group 2 Group 3 Group 4 Carrier quality 0.5 g 0.5g 0.3g 0.3g Carrier theoretical surface area 2200 cm 2 ]] 2200 cm 2 ]] 1320 cm 2 ]] 1320 cm 2 ]]> Cell inoculation density 1 x 10 6 cells / ml]] <![CDATA[2.5x10 5 cells / ml]]> 1 x 10 6 cells / ml 2.5 x 10 5 cells / ml]]

[0683] From Figure 1 It can be seen that under different seeding densities, the cells eventually reach a similar total amount (192h), so the cell seeding amount has little effect on the final cell growth state. The more microcarriers used, the higher the total cell count, but the improvement is not significant, and it will increase the cost of microcarrier use. Therefore, considering comprehensively, the final process is determined as cell seeding amount 2.5×10 5 / ml, microcarrier amount 3g / L.

[0684] The effect of feeding on cell growth during cell culture was explored, and the experimental design is shown in Table 3. The total cell count is shown in Table 4. The results show that daily medium replacement has a significant promoting effect on cell growth. At 96h and 120h, the total cell count of group 2 is 22.2% and 17.3% higher than that of group 1, respectively. The feeding process is finally determined for cell culture.

[0685] Table 3 Microcarrier cell culture feeding parameters

[0686] Group 1 Group 2 Carrier quality 0.3g 0.3g Carrier theoretical surface area 1320 cm 2 ]] 1320 cm 2 ]] Cell inoculation density 2.5 x 10 5 cells / ml]] 2.5 x 10 5 / ml Feeding mode No feeding Daily replacement of 25 ml fresh medium from the second day of culture

[0687] Table 4 Comparison of cell counts between microcarrier cell culture feeding groups and non-feeding groups

[0688]

[0689]

[0690] Next, the inoculation process was optimized, and three different inoculation MOIs were explored: 0.01, 0.05, and 0.1. The experimental design is shown in Table 5. From the titer determination results of the collected virus ( Figure 2 All groups reached their highest viral titers on day 3 (MOI 0.1) or day 4 (MOI 0.01, MOI 0.05). In terms of titer values, the group with MOI 0.05 achieved the highest titer (4.22E+07CCID). 50 / ml), the highest titers in the other two groups were 3.16E+07CCID. 50 / ml and 3.65E+07CCID 50 / ml. If the virus is collected at its highest titer time point, the titers are all higher than those of the T-75 control group (2.72E+07CCID). 50 / ml). From this perspective, the microcarriers, by increasing cell density, can indeed improve the virus titer at harvest to some extent. Looking at the titer trends in the harvested virus solution, all three groups showed a process of initial increase followed by a decrease after inoculation, reaching a peak around 3-6 days. The MOI 0.05 group consistently had the highest daily titer among the three groups. The inoculation process was determined to be MOI = 0.05, with harvest time between 3-6 days after inoculation, reaching a peak around day 4.

[0691] Table 5 Summary of Microcarrier Infection Parameters

[0692]

[0693] Recombinant polio oncolytic virus prepared by microcarrier culture technology, compared with traditional cell factory technology, provides the same cell attachment surface area (6360 cm²). 2 Under the premise of ) the total number of cells before inoculation increased by nearly 30%, the viral harvest liquid titer increased by 4 times (Table 6), and the harvest liquid volume was significantly reduced, which is more conducive to subsequent purification operations.

[0694] Table 6 Comparison of key parameters and harvest droplets in microcarrier culture and cell engineering culture processes.

[0695]

[0696]

[0697] Example 3: Preparation of Recombinant Poliovirus Harvesting Fluid Using a Serum-Free Process

[0698] Example 3.1 Serum-free Vero cell domestication process and preparation of working cell bank

[0699] A Vero cell bank (Lot#20170929, cell passage P126) stored in a liquid nitrogen tank was quickly thawed in a 37°C water bath, and the cell freezing medium contained 10% FBS. It was transferred to a T-75 cell culture flask pre-added with preheated complete culture medium (90% DMEM + 10% FBS), and the culture flask was placed in a 37°C, 5% CO2 cell incubator and cultured overnight until the cells were completely adherent. The next day, the old culture medium was discarded, and an equal volume of fresh complete culture medium was added for continuous culture until the cell confluence reached more than 85%. Then, the culture medium supernatant was discarded, and the cells were rinsed once with an equal volume of DPBS buffer, which was then discarded. The cells were digested with preheated recombinant trypsin until the cells became round. The digestion process was terminated by adding complete culture medium, and all the digested cells were transferred to a clean 50ml centrifuge tube, which was centrifuged at 200g for 5min at room temperature. The supernatant was removed, and the cells were resuspended with complete culture medium. The cell count was performed using a cell counter. The cells were subcultured and expanded at a density of 1.5-3.0 x 10 4 / cm 2 After 3-5 continuous passages until the cell state was stable, the old culture medium was discarded the next day after the last passage (about 24h after the passage), and the cells were cultured in serum-free culture medium OPM-VERO SFM6C3 (Opmay, item number C216306) until the cell confluence reached more than 85%. The cells were digested with recombinant trypsin until the cells became round, and the trypsin was diluted in OPM-VERO SFM6C3 to terminate the digestion process. All the digested cells were transferred to a clean 50ml centrifuge tube, which was centrifuged at 200g for 5min at room temperature. The supernatant was removed, and the cells were resuspended with OPM-VERO SFM6C3. The cell count was performed using a cell counter. The cells were subcultured and expanded at a density of 1.5-3.0 x 10 4 / cm 2The density of the cells was adjusted to about 1.0E+06 cells / ml. According to 1 ml per cell cryopreservation tube, it was divided and pre-frozen in a program cooling box at -80°C refrigerator, and then transferred to a liquid nitrogen tank the next day for storage. This was the serum-free Vero cell working cell bank (Lot#20220812, P131).

[0700] Example 3.2 Exploration of infection process parameters

[0701] Example 3.2.1 Serum-free culture of Vero cells

[0702] A cell was taken from the serum-free Vero cell working cell bank, quickly thawed in a 37°C water bath, and added to a cell culture flask containing preheated OPM-VERO SFM6C3. Then it was transferred to a 37°C, 5% CO2 cell incubator and cultured overnight until the cells were almost completely adherent. The next day, the old culture medium was discarded and an equal volume of fresh OPM-VERO SFM6C3 was added for continuous culture. The cells successfully recovered and the final confluence reached more than 85%. Then, the supernatant was discarded after rinsing the cells once with an equal volume of DPBS buffer, and preheated recombinant trypsin was added to digest the cells until the cell morphology was round. OPM-VERO SFM6C3 was added to dilute the trypsin and stop the digestion. Then the liquid in the culture flask was transferred to a centrifuge tube, centrifuged at 200g for 5min at room temperature, the supernatant was removed, and OPM-VERO SFM6C3 was added to resuspend the cells completely, and the cells were counted. According to 1.5-3.0x10 4 / cm 2 The density of the cells was adjusted to about 1.0E+06 cells / ml. According to 1 ml per cell cryopreservation tube, it was divided and pre-frozen in a program cooling box at -80°C refrigerator, and then transferred to a liquid nitrogen tank the next day for storage. This was the serum-free Vero cell working cell bank (Lot#20220812, P131). 4 / cm 2 The density of the cells was adjusted to about 1.0E+06 cells / ml. According to 1 ml per cell cryopreservation tube, it was divided and pre-frozen in a program cooling box at -80°C refrigerator, and then transferred to a liquid nitrogen tank the next day for storage. This was the serum-free Vero cell working cell bank (Lot#20220812, P131).

[0703] Example 3.2.2 Exploration of infection process parameters

[0704] Under a microscope, once the cell confluence was greater than 85%, the T-75 cells were removed from the incubator for inoculation. The working stock of recombinant poliovirus was removed from a -80°C freezer and thawed in a 27°C water bath. One T-75 cell culture was taken, and after washing the cells once with DPBS, cell counting was performed. Inoculation was then performed at MOIs of 0.05, 0.1, and 0.2, and the inoculation volumes were calculated as follows:

[0705] Required seed volume (ml) for MOI = 0.05: Total T-75 cell count (cells) × MOI (0.05) / Seed titer (CCID) 50 / ml)

[0706] MOI = 0.1 required virus volume (ml): Total T-75 cell count (cells) × MOI (0.1) / virus titer (CCID) 50 / ml)

[0707] Required seed volume (ml) for MOI=0.2: Total T-75 cell count (cells) × MOI(0.2) / Seed titer (CCID) 50 / ml)

[0708] The old T-75 medium to be inoculated was discarded, and the culture was rinsed once with an equal volume of DPBS. Then, an equal volume of fresh, preheated OPM-VERO SFM6C3 medium was added, followed by the previously calculated volume of virus inoculum. After mixing, the culture was incubated at 33°C under 5% CO2 conditions. From day 2 of incubation, CPE was observed daily, and samples were taken to measure the supernatant titer until day 6. The data are summarized in Table 7. The results showed that, under the condition of MOI = 0.05, the highest harvest titer (3.16E+07CCID) was obtained when the virus was maintained for 5 days. 50 The titer ( / ml) was more than twice that of the MOI=0.1 and MOI=0.2 groups, and the titer levels were not significantly different over 5-6 days.

[0709] Subsequently, a simulated production process was employed using a single-layer cell factory with a fixed virus inoculation rate of MOI = 0.05, and the harvest titer was measured at different maintenance culture times. The results showed that after 5-7 days of virus maintenance culture, the harvest titer remained at a high level, while the cytopathic effect did not reach its maximum value (70-85%) (Table 8). Based on the above studies using culture flasks and cell factory scale, the inoculation process parameters for preparing recombinant poliovirus using serum-free culture were preliminarily confirmed: inoculation MOI = 0.05, and the harvest titer reached its maximum value 5-7 days after inoculation.

[0710] Table 7 Results of the exploratory study on serum-free poliovirus inoculation process.

[0711]

[0712] Table 8 Results of the investigation of the number of days of virus maintenance culture for poliovirus prepared by the serum-free process

[0713] Days post infection CPE Titer (CCID 50 / ml) D2 ＜5％ 1.33E+05 D3 About 20% 4.87E+06 D4 About 40% 1.78E+07 D5 70-80％ 3.65E+07 D6 80-85％ 2.37E+07 D7 85-90％ 2.05E+07 D8 About 90% 1.78E+07

[0714] Example 3.2.3: Inoculation culture at the cell culture flask scale

[0715] After the Vero cells cryopreserved in serum-free medium were recovered and subcultured continuously, the cells were inoculated into T-75 flasks at a density of 1.5-3.0 x 10 4 / cm 2 After the cells were confluent, the virus seed of recombinant poliovirus was inoculated at an MOI of 0.05, and the supernatant was collected 5-7 days after inoculation for titer determination. The results were compared with the virus containing serum control group, and are shown in Table 9. The titer data show that the virus harvest titer prepared by the serum-free and serum-containing processes is at the same order of magnitude at the flask scale.

[0716] Table 9 Comparison of the supernatant titer of the harvest after inoculation by the serum-free and serum-containing processes (flask scale)

[0717] Group Serum-containing control OPM-VERO SFM6C3 Lot number Lot#20230629 Lot#20230629 Harvested droplet concentration (CCID 50 / ml) 2.05E+07 1.00E+07

[0718] Example 3.3 Process verification at the pilot scale (ten-layer cell factory, 1.25 L)

[0719] Example 3.3.1 Serum-free culture of Vero cells

[0720] A cell was taken from the serum-free Vero cell working cell bank, quickly thawed in a 37°C water bath, and added to a cell culture flask containing preheated OPM-VERO SFM6C3. Then, the flask was transferred to a 37°C, 5% CO2 cell incubator and incubated overnight until the cells were completely adherent. The next day, the old culture medium was discarded, and an equal volume of fresh OPM-VERO SFM6C3 was added for continued culture until the cell confluence reached more than 85%. Then, the culture medium supernatant was discarded, and the cells were rinsed once with an equal volume of DPBS buffer, which was then discarded. Preheated recombinant trypsin was added to digest the cells until the cell morphology was round. OPM-VERO SFM6C3 was added to dilute the trypsin and stop the digestion. Then, the liquid in the flask was transferred to a centrifuge tube, which was centrifuged at 200g for 5 min at room temperature to remove the supernatant. OPM-VERO SFM6C3 was added to resuspend the cells completely, and the cells were counted. The cells were subcultured at a density of 1.5-3.0 x 10 4 / cm 2 After continuous subculturing for several times, the cells were inoculated at a density of 1.0-2.0 x 10 4 / cm 2The density of the cells was passaged into one T-150 cell culture flask (surface area 150 cm 2 ), one one-layer cell factory (surface area 636 cm 2 ), and one ten-layer cell factory (surface area 6360 cm 2 ). The cells were cultured in a 37°C, 5% CO2 cell incubator until the confluence reached more than 85%.

[0721] Example 3.3.2 Virus inoculation culture

[0722] The working strain of recombinant poliovirus was taken out from the -80°C refrigerator and thawed in a 27°C water bath. The T-150 culture flask was taken out from the cell incubator, the cells were washed once with DPBS and then digested and counted. The total cell number of the one-layer cell factory and the ten-layer cell factory was calculated according to the following formula:

[0723] Total cell number of one-layer cell factory (cells) = T-150 cell counting density (cells / ml) x cell resuspension volume (ml) / 150 x 636

[0724] Total cell number of ten-layer cell factory (cells) = T-150 cell counting density (cells / ml) x cell resuspension volume (ml) / 150 x 6360

[0725] According to MOI = 0.05, the volume of the virus strain needed to be added was calculated according to the total cell number of the one-layer cell factory and the ten-layer cell factory:

[0726] Volume of virus strain needed for one-layer cell factory (ml) = total cell number of one-layer cell factory (cells) x MOI (0.05) / virus titer (CCID 50 / ml)

[0727] Volume of virus strain needed for ten-layer cell factory (ml) = total cell number of ten-layer cell factory (cells) x MOI (0.05) / virus titer (CCID 50 / ml)

[0728] The one-layer cell factory and the ten-layer cell factory were taken out from the cell incubator, the supernatant of the culture medium was discarded, an equal volume of DPBS was added for washing, discarded, and an equal volume of OPM-VERO SFM6C3 was added. Then, according to the calculated volume of the virus strain, the virus strain was added to the cells in the one-layer cell factory and the ten-layer cell factory, respectively. After mixing thoroughly, they were placed in the cell incubator and cultured at 33°C, 5% CO2.

[0729] Example 3.3.3 Virus harvesting

[0730] After 5-7 days of infection, the cytopathic effect (CPE) was observed, and the CPE should be about 70-90%. The ten-layer cell factories were taken out of the incubator, and the culture supernatant was transferred to a clean 50-ml centrifuge tube. The supernatant was centrifuged at 4000g for 30 min at room temperature. After centrifugation, the supernatant was collected, and the volume was about 1.25 L. MgCl2 solution and Benzonase were added to the supernatant, respectively, so that the final concentration of MgCl2 was 1 mM, and the final concentration of Benzonase was 25 U / ml. Then the supernatant was placed at room temperature for 18-22 h for Benzonase digestion. After digestion, the supernatant was filtered through a 0.22-um filter membrane into a clean storage bottle for downstream purification.

[0731] Three batches of small-scale upstream harvest liquid were prepared, and the titers were basically consistent with the serum-containing culture process (Table 10), and the process reproducibility was good. Compared with the serum-containing process, the serum-free culture cost is lower, and the risk of biological source materials in the biological drug declaration process is avoided.

[0732] Table 10 Comparison of virus titers of upstream harvest liquid of serum-free and serum-containing processes (cell factory scale)

[0733] Lot number Vero cell culture process Harvested droplet concentration (CCID 50 / ml) Lot#20221024 Serum-free 3.16E+07 Lot#20230209-1 Serum-free 8.66E+06 Lot#20230209-2 Serum-free 9.00E+07 Lot#20221014 Serum-containing (control) 3.65E+07

[0734] Example 4 Gel column chromatography process development

[0735] Processes 1-3 have basically the same process steps, all of which use gel column chromatography, and the gel filler is Sephrose 6FF, but the size and size of the chromatographic column are different. The virus harvest liquid after gel column purification is further purified by subsequent anion exchange chromatography such as SuperQ650M anion exchange to further remove process-related impurities such as host proteins HCP and host nucleic acids HCD, and the material liquid after anion exchange chromatography is finally concentrated by ultrafiltration, and sterile filtration to obtain the final virus stock solution (DS).

[0736] Experimental method:

[0737] Column chromatography process route 1: According to the method of CN 108291210 A, the clarified virus harvest liquid is not subjected to the ultrafiltration concentration step and is directly subjected to gel column chromatography. The sample amount is 30% CV of the column volume, the chromatographic column used is XK50 / 60, the gel column height is 90 cm, the column volume is 800 mL, the volume of the virus harvest liquid for small-scale process development is 1.25 L, and the purification is performed in 5 times, and the treatment amount is 250 ml of virus harvest liquid each time; then the material liquid purified by the gel column for 5 times is combined, and the SuperQ650M chromatographic column with a column size of 1.6x30 cm is used to remove nucleic acids and HCP and other impurities, and finally the membrane area is 146 cm 2Ultrafiltration concentration and buffer exchange was performed using hollow fibers with a membrane area of 146 cm

[0738] Column chromatography process route 2: The volume of the clarified virus harvest was still 1.25 L, and ultrafiltration concentration and buffer exchange was performed using hollow fibers with a membrane area of 146 cm 2 The 5-fold volume concentrated harvest, i.e. 250 ml, was used as the starting material for gel column chromatography. The Sepharose 6FF gel column had a size of XK50 / 100, and the height of the gel column was 80-90 cm, and the column volume was 1570 ml. Only one loading was required, and the purified material was passed through a Super Q 650M column with a size of 2.6 x 35 cm to remove nucleic acids and HCP, and finally the material was prepared using hollow fibers with a membrane area of 146 cm 2

[0739] Column chromatography process route 3: The volume of the clarified virus harvest was still 1.25 L, and ultrafiltration concentration and buffer exchange was performed using hollow fibers with a membrane area of 290 cm 2 The 5-fold volume concentrated harvest, i.e. 250 ml, was used as the starting material for gel column chromatography. The Sepharose 6FF gel column had a size of XK50 / 60, and the height of the gel column was reduced from 90 cm to 40 cm, and the column volume was 800 ml. Only one loading was required, and the purified material was passed through a Super Q 650M column with a size of 2.6 x 15 cm to remove nucleic acids and HCP, and finally the material was prepared using hollow fibers with a membrane area of 290 cm 2

[0740] Results of the experiments:

[0741] Column chromatography process route 1

[0742] Item Lot#1611009 Lot#1611010 Lot#1611011 Titer (TCID 50 )]]> 1.2 x 10 8 / mL 1.2 x 10 8 / mL 5.1 x 10 8 / mL Host cell protein (HCP) < 40 ng / mL < 40 ng / mL < 40 ng / mL Host cell DNA (HCD) <0.1 pg / mL <0.1 pg / mL <0.1 pg / mL Viral genome copy number 2.6 x 10 11 ]]> 1.9 x 10 11 ]]> 6.3 x 10 11 ]]> Titer recovery rate (%) 15％ 30％ 55％

[0743] Column chromatography process route 2

[0744] Item Lot#20180504-2 Lot#20180505-2 Lot#20180601-2 Titer (TCID 50 / mL) 4.87 x 10 7 / mL 4.22 x 10 7 / mL 8.37 x 10 7 / mL Host cell protein (HCP) < 100 ng / mL < 100 ng / mL < 100 ng / mL Host cell DNA (HCD) <1.65 pg / mL <1.65 pg / mL <1.65 pg / mL Viral genome copy number <![CDATA[1.62×10 10 ]]> 7.16 x 10 10 ]] 1.52 x 10 11 ]]> Titer recovery rate 5.36％ 8.38％ 10.95％

[0745] Column chromatography process route 3

[0746]

[0747]

[0748] ​​In combination with the process flow and virus detection results, the improved molecular exclusion chromatography purification method provided herein provides one or more of the following advantages: 1. an ideal compromise between product yield and purity (i.e. greater process flexibility); 2. a reduction in purification time, i.e. the improved method (Process 2 and Process 3) is faster than the previously known (Process 1), with the purification time of the improved method being reduced to 8 hours, or even shorter, e.g. to 4 hours; 3. improved batch-to-batch consistency of the purified virus. The molecular exclusion chromatography method is less complex and less costly, and is more suitable for large-scale production.

[0749] Example 5 Ion exchange column chromatography process development

[0750] Example 5.1 CIMmultus DEAE monolithic column virus capture process development

[0751] The CIMmultus DEAE monolithic column was selected for virus capture, and the operating process was as follows: the sample loading amount of the harvest liquid was 20 ml, which was diluted to 3 times the original volume with the equilibration buffer, the sample conductivity was reduced to 5.0-6.0 ms / cm, and then the pH was adjusted to 8.8-9.0 with 50 mM NaOH before loading, to ensure complete capture of the virus. The virus was then separated from the impurities by using elution buffers with different conductivities and pH values.

[0752] Experimental results:

[0753] Table 11. Virus purification results for Lot #20230209-1 batch

[0754]

[0755] Table 12. Virus purification results for Lot #20230209-2 batch

[0756]

[0757]

[0758] The above results (Tables 11-12) show that the CIMmultus DEAE monolithic column capture process unit operation is simple, the purification steps are fewer, the purification time is shorter, it is easy to scale up and commercialize production, the virus activity is well maintained, and the HCP residual amount is also low; the virus activity recovery rate of the process is further improved, and the batch-to-batch robustness is good.

[0759] Example 5.2 Concentration of virus harvest liquid using anion exchange column chromatography packing

[0760] Example 5.2.1 Virus capture using Super Q 650M anion exchange packing

[0761] Super Q 650M column chromatography capture operating conditions: Super Q 650M column chromatography equilibration buffer adjusted to 25 mM Tris, pH 8.5, sample diluted to 3 volumes with equilibration buffer before loading.

[0762] Table 13. Super Q 650M anion exchange chromatography purification results

[0763]

[0764] The above results show (Table 13) that the Super Q 650M packing material still cannot completely adsorb the virus, and a large part of the virus exists in the flow-through, resulting in a low virus yield for the capture process and failing to achieve the purpose of concentrating the virus.

[0765] Example 5.2.2. Capture using Avantor BAKERBOND poly QUAT strong anion composite column chromatography packing material

[0766] Avantor BAKERBOND poly QUAT strong anion composite column chromatography capture operating conditions: column chromatography equilibration buffer adjusted to 25 mM Tris, pH 8.5, sample diluted to 3 volumes with equilibration buffer before loading.

[0767] Table 14. Avantor BAKERBOND poly QUAT strong anion composite column chromatography purification results

[0768]

[0769]

[0770] The above results show (Table 14) that the strong anion exchange composite packing material of AVANTOR behaves similarly to the Super Q 650M packing material, and a large part of the virus exists in the flow-through.

[0771] Example 5.2.3. Reverse operation: let the virus flow through and the impurities be adsorbed to the packing material

[0772] Verify the effect of NW Rose Viral M and Cytiva Core 400 packing materials on HCP removal and RNA copy number yield. The NW Rose Viral M and Cytiva Core 400 packing materials are composite mode packing materials, which have an inert shell to block the virus outside and flow out from the pores, while the HCP, DNA (cutting), endotoxin, enzymes, etc. can enter the pores and be combined with the strong anion adsorption ligand, quaternary amine group, fixed on the inner core to achieve the purpose of purification, which is not affected by the type and pH of the buffer.

[0773] The results of the experiments are shown in Tables 15 and 16, and both fillers performed with a certain proportion of virus present in the later elutions, rather than being washed through the column.

[0774] Table 15. RNA copy yield results for NW Rose Viral M purification process

[0775]

[0776] Table 16. RNA copy yield results for Cytiva Core 400 purification process

[0777]

[0778]

[0779] Example 5.3 CIMmultus QA Monolith Capture Virus Process Development

[0780] The CIMmultus QA Monolith operating conditions were as follows: lot 20210625 batch of virus harvest was taken 20-40 mL, diluted with equilibration Buffer to 3 times the original volume, then pH was adjusted to 8.8-9.0 with 50 mM NaOH before loading, loading volume was 60-120 mL, elution was performed with 50-250 mM NaCl, elution pH was 6.0-7.0, eluate was collected, product qPCR and CCID50 Titer were detected.

[0781] Table 17. CCID50 and qPCR yield results for DOE experiments

[0782]

[0783] The results of the experiments (Table 17) show that when the CIMmultus QA Monolith was used for capture, the highest CCID50 activity recovery and plasmid copy number recovery were obtained at a loading volume of 60 mL and a NaCl concentration of 250 mM in the elution buffer, pH 6.00, which were 70.43% and 60.58%, respectively. The recovery of this step was comparable to or even slightly higher than that of the original process (two-step operation of hollow fiber TFF and Sepharose 6FF chromatography).

[0784] Example 5.4 Further Exploration of Monolith Capture Virus Process

[0785] Monolith Capture Process Pre-experiment

[0786] Based on the previous CIMmultus QA Monolithic Column and CIMmultus DEAE Monolithic Column experimental results, two kinds of monolithic column virus capture process experimental research were carried out. According to the previous experimental data, the sample loading was 20 mL of harvest liquid, which was diluted to 3 times the original volume with equilibration buffer, the sample conductivity was reduced to 5.0-6.0 ms / cm, and then the pH was adjusted to 8.8-9.0 with 50 mM NaOH before loading, to ensure the complete capture of the virus. Then the virus was separated from the impurities by using different conductivity and pH eluents. While ensuring the virus yield, the impurities were removed to the greatest extent.

[0787] CIMmultus QA 1ml Monolithic Cloumn verification "40mMPB, 250mM NaCl, pH6.0, 25CV" one-step elution and linear gradient elution two ways to affect the recovery rate of virus RNA copy number. CIMmultus DEAE 1ml Monolithic Cloumn verification 3 kinds of elution mode affect the recovery rate of virus RNA copy number. Respectively 40mM PB, pH6.0, 25CV one-step elution, 40mM PB, 250mM NaCl, pH6.0, 25CV one-step elution and linear gradient elution.

[0788] Experimental method

[0789] Experiment 1: According to the DOE experimental results of CIMmultus QA 1ml Monolithic Cloumn, the virus harvest liquid was 20ml, which was diluted to 3 times the original volume with equilibration buffer, and then loaded after adjusting the pH to 8.8-9.0 with 50mM NaOH, one-step elution, collection of eluent, detection of product qPCR and CCID50 Titer.

[0790] Experiment 2: Virus harvest liquid 20ml, diluted to 3 times the original volume with equilibration buffer, then loaded after adjusting the pH to 8.8-9.0 with 50mM NaOH, using 40mMPB, 250mM NaCl, pH6.0, 25CV, linear gradient elution, collection of eluent, detection of product qPCR and CCID50 Titer.

[0791] Experiment 3: According to the data of CIMmultus DEAE 1ml Monolithic Cloumn, the virus harvest liquid was diluted to 3 times the original volume with equilibration buffer, and then loaded after adjusting the pH to 8.8-9.0 with 50mM NaOH, the loading amount was 60-90ml (i.e. 20-30ml of harvest liquid), one-step elution, collection of eluent, detection of product qPCR and CCID50 Titer.

[0792] Experiment 4: According to the characteristics of CIMmultus DEAE 1ml Monolithic Cloumn, weak anion exchange, the virus harvest was diluted with equilibration buffer to 3 times the original volume, then adjusted to pH 9.0 with 50mM NaOH before loading, the loading amount was 60-90ml (i.e. 20-30ml harvest), linear gradient elution was used for purification, the eluate was collected, and the product qPCR and CCID50 Titer were detected.

[0793] Experiment 5: The virus harvest was 20ml, diluted with equilibration buffer to 3 times the original volume, then adjusted to pH 9.0 with 50mM NaOH before loading, 40mM PB, 250mM NaCl, pH6.0, 25CV, linear gradient elution was used for purification, the eluate was collected, and the product qPCR and CCID50 Titer were detected.

[0794] Experimental results

[0795] QA monolithic column used 40mM PB, 250mM NaCl, pH6.0 for one-step elution and linear gradient elution, one-step elution RNA copy number recovery rate was 74.14%, linear gradient elution RNA copy number recovery rate was 83.65%. DEAE monolithic column used 40mM PB, 250mM NaCl, pH6.0 for one-step elution and linear gradient elution, one-step elution RNA copy number recovery rate was 60.14%, linear gradient elution RNA copy number recovery rate was 64.54%; when the elution buffer was adjusted to 40mM PB, pH6.0 for pH gradient elution, the RNA copy number recovery rate was 47.83%. The results showed that CIMmultus QA monolithic column and CIMmultus DEAE monolithic column could purify virus under appropriate conditions, and the virus activity and recovery rate were very high. As shown in Tables 18-20 below.

[0796] Table 18. CIMmultus QA monolithic column purification result statistics

[0797]

[0798] Table 19. CIMmultus DEAE monolithic column purification result statistics

[0799]

[0800]

[0801] Table 20. CIMmultus DEAE monolithic column purification result statistics

[0802]

[0803] Example 5.5 Process further exploration of virus capture by DEAE monolithic column

[0804] Taking the CIMmultus DEAE 1ml Monolithic Cloumn purification process as an example, the difference between elution pH 5.5 and pH 6.0 was compared. Under different elution pH conditions, there were two elution peaks, peak 1 and peak 2, but the RNA copy number distribution was different under the two conditions. Under the pH 5.5 elution condition, the main peak was in peak 1, and the recovery rate was 32.17%, while under the pH 6.0 elution condition, the main peak was in peak 2, and the recovery rate was 31.52%, and the recovery rates of the two were close. Considering that the elution ability of pH 5.5 buffer is stronger than that of pH 6.0 buffer, which is beneficial to eliminate the fluctuation caused by the difference in feed properties between batches, therefore, the elution condition of pH 5.5 buffer is temporarily determined for the subsequent purification and process confirmation of CIMmultus DEAE monolithic column. The experimental results are shown in Tables 21-22:

[0805] Table 21. CIMmultus DEAE monolithic column purification result statistics

[0806]

[0807] Table 22. CIMmultus DEAE monolithic column purification result statistics

[0808]

[0809]

[0810] Three batches of samples were purified using the determined CIMmultus DEAE 1ml Monolithic Cloumn purification process. The PCR copy number recovery rate of lot#20221020 batch was 94.83%, and the CCID50 recovery rate was 88.90%, the experimental results are shown in Table 23; the PCR copy number recovery rate of lot#20221021 batch was 46.59%, and the CCID50 recovery rate was 37.47%, the experimental results are shown in Table 24; the PCR copy number recovery rate of lot#20221022 batch was 60.55%, and the CCID50 recovery rate was 50.00%, and the HCP concentration was 3065.66 ng / ml, the experimental results are shown in Table 25.

[0811] The experimental results are as follows:

[0812] Table 23. CIMmultus DEAE monolithic column purification results of lot#20221020 batch sample

[0813]

[0814] Table 24. Lot#20221021 batch sample CIMmultus DEAE Monolithic Column purification results

[0815]

[0816]

[0817] Table 25. Lot#20221022 batch sample CIMmultus DEAE Monolithic Column purification results

[0818]

[0819] Example 5.6 Post Monolithic Column Capture Chromatography Process Exploration

[0820] Take the CIMmultus DEAE 1ml Monolithic Column purification process as an example, on this basis, combined with the anion exchange Super Q 650M step of process 1-3, the sample after monolithic column purification is further purified by Super Q 650M chromatography, and the feasibility of the process is explored.

[0821] The operating conditions are as follows: dilute with equilibration buffer to 3 times the original volume, adjust the pH to 9.0 with 50mM NaOH before loading, adjust the elution buffer to 20mM PB, 20mM Citrate, pH 5.5, and elute. The harvest liquid eluted by CIMmultus DEAE purification is adjusted to pH 7.5, loaded on Super Q 650M column chromatography for further purification, and the product qPCR and CCID50Titer are detected.

[0822] Table 26. CIMmultus DEAE Monolithic Column HCP content and activity results

[0823]

[0824] The results of Table 26 show that the harvest HCP concentration of Lot#20221014 batch was 42210.64 ng / ml, the HCP concentration of CIMmultus DEAE 1 ml Monolithic Column collection solution was 311.41 ng / ml, and the HCP concentration of Super Q 650M collection solution was 38.23 ng / ml, which was significantly lower than the original process of 100-500 ng / ml. The total recovery rate of DS RNA copy number was 40.16%, and the total recovery rate of CCID50 was 45.58%, both of which were within the acceptable range. Therefore, the overall column CIMmultus DEAE combined with Super Q 650M chromatography method solved the problems often encountered in the existing process, and provided a sample with high activity and low impurity content.

[0825] Example 6 Chromatography process flow verification and scale-up

[0826] Taking the monolithic column chromatography purification process as an example, the virus capture purification process was established and scale-up verification was performed, and the specific operation was as follows:

[0827]

[0828] The experimental results are shown in the following table:

[0829] Table 27. Purification results of Lot#20230209-2 batch

[0830]

[0831] Lot#20230209-2 batch was purified, CIMmultus DEAE was eluted at pH 5.5, and Super Q 650M was eluted at pH 7.5, as shown in Table 27, the total recovery rate of RNA copy number was 85.43%, and the total recovery rate of CCID50 was 45.07%, indicating that the process is easy to scale up and commercialize, and can be applied to pilot and industrial production levels.

Claims

1. A method for preparing a poliovirus solution, comprising the steps in the order shown: a) Provide cells, which are present in a culture medium; b) Adjust the cells to a suitable cell density, and then contact the poliovirus with the cells to obtain a mixed solution of poliovirus and cells; c) Centrifuge the mixture and collect the supernatant; d) The supernatant was purified by chromatography to obtain the poliovirus solution.

2. The method according to claim 1, wherein the culture medium in a) comprises a serum culture medium.

3. The method according to any one of claims 1-2, wherein the cells described in a) are cultured using microcarriers.

4. The method according to claim 3, wherein the culture medium further comprises feeding such that the glucose content in the culture medium is not less than about 1 g / L.

5. The method according to claim 4, wherein the fluid replacement method for replenishment includes discontinuous fluid replacement.

6. The method according to claim 5, wherein the discontinuous medium change begins with changing the culture medium daily from the second day of culture.

7. The method according to claim 1, wherein the culture medium in a) further comprises serum-free culture medium.

8. The method according to any one of claims 1-7, wherein the suitable cell density described in b) is about 1 × 10⁻⁶. 5 cells / ml - approximately 2 × 10 6 cells / ml.

9. The method according to any one of claims 1-8, wherein the multiplicity of infection (MOI) of contact with poliovirus as described in b) is about 0.01 to about 0.

5.

10. The method of claim 9, wherein the exposure time under the said multiple of infection (MOI) condition for poliovirus exposure is about 3-6 days, and the multiple of infection (MOI) for poliovirus exposure is about 0.01 to about 0.

1.

11. The method of claim 9, wherein the exposure time under the said multiple of infection (MOI) condition for poliovirus exposure is about 5-7 days, and the multiple of infection (MOI) for poliovirus exposure is about 0.05 to about 0.

2.

12. The method according to any one of claims 1-11, wherein the viral titer in the supernatant described in c) is approximately 1.00E+06CCID. 50 / ml to 5.00E+09CCID 50 / ml.

13. The method according to any one of claims 1-12, wherein the chromatographic step in d) comprises using gel column chromatography, monolithic column chromatography and / or anion exchange chromatography.

14. The method according to any one of claims 1-13, wherein the purification in d) further comprises a pretreatment performed prior to the chromatographic step in d), the pretreatment comprising adding nuclease and / or filtration for sterilization.

15. The method according to any one of claims 1-14, wherein the purification in d) further comprises a pretreatment performed prior to the chromatographic step in d), the pretreatment further comprising ultrafiltration.

16. The method according to any one of claims 1-15, wherein the purification in d) is carried out when the pH of the supernatant in c) is about 6.0-10.

0.

17. The method according to any one of claims 1-16, wherein the chromatographic step in d) further comprises eluting with an eluent, the pH of which is preferably about 5.0-8.

0.

18. The method according to claim 17, wherein the eluent comprises an eluent salt, the eluent salt comprising NaCl, KCl, Na2SO4, Na3PO4, K2SO4 and / or K3PO4.

19. The method of claim 18, wherein the concentration of the elution salt is about 10-500 mM.

20. The method according to any one of claims 1-19, wherein the cells are African green monkey kidney (Vero) cells.

21. The method according to any one of claims 1-20, wherein the poliovirus comprises wild-type poliovirus and / or its mutants, and the mutants comprise substitutions, mutations, insertions and / or deletions of one or more genes.

22. A poliovirus solution prepared by any one of claims 1-21, wherein the poliovirus solution has a viral titer of approximately 1.00E+06CCID. 50 / ml to 5.00E+09CCID 50 / ml.

23. A pharmaceutical composition comprising the poliovirus solution of claim 22, and optionally a pharmaceutically acceptable adjuvant.

24. Use of the poliovirus solution of claim 22 in the preparation of a medicament for the prevention, relief and / or treatment of diseases and / or symptoms.

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