Virus production

Abstract

Provided herein are methods for producing a virus. Also provided herein are bioreactors comprising virus-infected host cells containing the virus. In some aspects, the disclosure relates to the production of a recombinant oncolytic virus, e.g., a recombinant vaccinia virus. In one aspect, the disclosure includes: growing a culture comprising a plurality of virus-infected host cells in a bioreactor to produce at least about 50 plaque-forming units (PFU) to about 350 PFU of the recombinant oncolytic virus per host cell, wherein the bioreactor is about 0.2 m 2 Approximately 500m from 2 The method includes the step of forming a surface area of

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Application No. 62 / 770,577, filed November 21, 2018, which is incorporated herein by reference in its entirety. [Background technology]

[0002] background Viruses can be found for use in many fields, including therapy, such as oncolytic therapy. Oncolytic viruses hold great promise for the treatment of many types of cancer. In many cases, recombinant oncolytic viruses produced through genetic engineering of naturally occurring viruses have proven to be effective tools for eradicating cancer cells. However, therapeutic application of oncolytic viruses has high requirements in many aspects. For example, clinically applicable viruses may need to be highly pure. Also, high titers of viruses may be required to achieve therapeutic effects. In addition, how to scale up virus production for therapeutic applications remains a major challenge. Therefore, improved methods of producing viruses are desirable. Summary of the Invention [Means for solving the problem]

[0003] Abstract In one aspect, the disclosure includes: growing a culture comprising a plurality of virus-infected host cells in a bioreactor to produce at least about 50 plaque forming units (PFU) to about 350 PFU of recombinant oncolytic virus per host cell, wherein the bioreactor is about 0.2 m 2 Approximately 500m from 2 The method includes the steps of:

[0004] In one aspect, the disclosure provides a method for producing a population of recombinant oncolytic viruses comprising: (i) growing a culture comprising a plurality of virus-infected host cells in a bioreactor; and (ii) recovering a population of recombinant oncolytic viruses from said culture, said recovering comprising lysing said culture to produce a lysate comprising said population of recombinant oncolytic viruses; and (iii) clarifying said lysate to produce a population of purified recombinant oncolytic viruses, wherein said population of purified recombinant oncolytic viruses comprises at least about 50% to about 90% of said population recombinant oncolytic viruses in said lysate, wherein said bioreactor is about 0.2 m 2 Approximately 500m from 2 The method includes the steps of:

[0005] In one aspect, the disclosure provides: recovering a population of recombinant oncolytic viruses from a culture comprising a plurality of virus-infected host cells grown in a bioreactor, said population being greater than or equal to about 1.5×10 11 From about 5 × 10 13 PFU of the recombinant oncolytic virus, and the bioreactor is about 0.2 m 2 Approximately 500m from 2 The method includes the steps of:

[0006] In one aspect, the disclosure provides: recovering a population of recombinant oncolytic viruses from a culture comprising a plurality of virus-infected host cells grown in a bioreactor, said population being greater than or equal to about 1.5×10 virus / mL of said culture. 8 From about 2 × 10 10 The bioreactor contained a viral titer of 1000 PFU and was approximately 0.2 m 2 Approximately 500m from 2 The method includes the steps of:

[0007] In one aspect, the disclosure includes (i) growing a culture comprising a plurality of virus-infected host cells in a bioreactor; and (ii) recovering a population of recombinant oncolytic viruses from said culture, wherein said bioreactor is about 0.2 m2 Approximately 500m from 2 wherein said recovering is performed under alkaline conditions.

[0008] In one aspect, the disclosure provides a method for producing a population of recombinant oncolytic viruses by growing a culture comprising a plurality of virus-infected host cells in a bioreactor, wherein the plurality of virus-infected host cells are infected with the viruses at a multiplicity of infection (moi) of about 0.0001 to about 0.01 PFU / cell, and wherein the bioreactor is about 0.2 m 2 Approximately 500m from 2 The method includes the steps of:

[0009] In one aspect, the disclosure provides a method for producing a recombinant oncolytic virus comprising: (i) growing in a bioreactor a culture comprising a plurality of virus-infected host cells, wherein the plurality of virus-infected host cells are infected with a virus at a multiplicity of infection (moi) of about 0.0001 to about 0.01; and (ii) recovering from the culture a population of recombinant oncolytic viruses, wherein the population is about 1.5×10 11 From about 5 × 10 13 The present invention also provides a method for producing a PFU of said recombinant oncolytic virus.

[0010] In some embodiments, the bioreactor comprises a fixed bed. In some embodiments, the fixed bed comprises microcarriers. In some embodiments, the fixed bed comprises a volume of about 0.01 L to about 25 L. In some embodiments, the fixed bed comprises a packed density of at least about 80 g / L to about 144 g / L. In some embodiments, the fixed bed comprises a packed density of at least about 96 g / L.

[0011] In some embodiments, the plurality of virally infected host cells comprises at least about 1,000 cells / cm in the bioreactor. 2 to approximately 150,000 cells / cm 2In some embodiments, the culture comprising the virus-infected host cells is grown for about 24 hours to about 96 hours. In some embodiments, the culture comprising the virus-infected host cells is grown for about 48 hours to about 72 hours. In some embodiments, the culture comprising the virus-infected host cells comprises a dissolved oxygen tension (DOT) level of at least about 20% to about 100%. In some embodiments, the culture comprising the virus-infected host cells is maintained at a temperature of about 32° C. to about 38° C. In some embodiments, the culture comprising the virus-infected host cells is maintained at a pH of about 7.0 to about 7.5. In some embodiments, the plurality of virus-infected cells are infected with a virus at a multiplicity of infection (moi) of about 0.0005 to about 0.2.

[0012] In some embodiments, the method further comprises lysing the plurality of virus-infected host cells by incubating in an alkaline buffer. In some embodiments, the method further comprises lysing the plurality of virus-infected host cells by incubating with an enzyme for degrading cellular debris. In some embodiments, the method further comprises lysing the plurality of virus-infected host cells by incubating with an enzyme for degrading cellular debris and an alkaline buffer. In some embodiments, the recovering comprises lysing the plurality of virus-infected host cells by incubating in an alkaline buffer. In some embodiments, the recovering comprises lysing the plurality of virus-infected host cells by incubating with an enzyme for degrading cellular debris. In some embodiments, the recovering comprises lysing the plurality of virus-infected host cells by incubating with an enzyme for degrading cellular debris and an alkaline buffer. In some embodiments, a concentration of the enzyme during the incubation is from about 50 IU / mL to about 200 IU / mL. In some embodiments, the incubating occurs for about 2 hours to about 8 hours. In some embodiments, the incubating occurs at a temperature of about 22° C. to about 28° C. In some embodiments, the enzyme for degrading cellular debris comprises Benzonase®. In some embodiments, the alkaline buffer comprises a pH greater than 8. In some embodiments, the alkaline buffer comprises a pH greater than 8.0 to about 10.0. In some embodiments, the alkaline buffer comprises a pH of about 9.5. In some embodiments, the alkaline buffer comprises a Tris buffer. In some embodiments, the method further comprises monitoring the pH during said incubation.

[0013] In some embodiments, the method further comprises filtering to produce a filtered lysate. In some embodiments, the method further comprises treating the filtered lysate with an enzyme to degrade cellular debris. In some embodiments, the treating is performed under alkaline conditions. In some embodiments, the treating is performed for about 10 hours to about 24 hours. In some embodiments, the treating is followed by tangential flow filtration of the filtered lysate to produce a purified preparation of the recombinant oncolytic virus. In some embodiments, the tangential flow filtration comprises concentrating the filtered lysate, followed by at least a first and a second round of diafiltration. In some embodiments, the first round of diafiltration comprises a buffer exchange with a buffer comprising a pH of about 7.0 to about 7.5. In some embodiments, the first round of diafiltration comprises about 10 volumes of the buffer exchange. In some embodiments, the buffer comprising a pH of about 7.0 to about 7.5 comprises a Tris concentration of about 15 mM to about 40 mM. In some embodiments, the buffer with a pH of about 7.0 to about 7.5 further comprises at least about 5% to at least about 20% sucrose by volume. In some embodiments, the second round of diafiltration comprises a buffer exchange with a buffer with a pH of greater than 7.5. In some embodiments, the second round of diafiltration comprises about 6 volumes of the buffer exchange with a buffer with a pH of greater than 7.5. In some embodiments, the buffer with a pH of greater than 7.5 comprises a Tris concentration of about 10 mM to about 30 mM. In some embodiments, the buffer with a pH of greater than 7.5 further comprises at least about 5% to at least about 10% sucrose by volume.

[0014] In some embodiments, the purified preparation of the recombinant oncolytic virus comprises about 5 ng to about 100 ng of host cell DNA per unit dose of the recombinant oncolytic virus. In some embodiments, the purified preparation of the recombinant oncolytic virus comprises about 0.1 μg to about 10 μg of host cell protein per unit dose of the recombinant oncolytic virus. In some embodiments, the unit dose is about 1×10 9From about 1×10 13 In some embodiments, the purified preparation of the recombinant oncolytic virus comprises 5×10 PFU of the recombinant oncolytic virus. 9 In some embodiments, the purified preparation of the recombinant oncolytic virus comprises up to about 400 ng, about 200 ng, about 100 ng, about 50 ng, or about 40 ng of host cell DNA per PFU. 9 In some embodiments, the purified preparation of the recombinant oncolytic virus contains up to about 80 ng, about 40 ng, about 20 ng, about 10 ng, or about 8 ng of host cell DNA per PFU. 9 In some embodiments, the purified preparation of the recombinant oncolytic virus comprises up to about 7.5 μg, about 7 μg, about 6 μg, about 5 μg, or about 4 μg of host cell protein per PFU. 9 In some embodiments, the purified preparation of the recombinant oncolytic virus comprises up to about 1.5 μg, about 1.4 μg, about 1.2 μg, about 1 μg, or about 0.8 μg of host cell protein per PFU. 9 Per PFU, up to about 100 ng, about 60 ng, about 50 ng, about 30 ng, or about 25 ng of serum albumin is used in the culture.

[0015] In some embodiments, the host cell is selected from the group consisting of a HeLa cell, a 293 cell, and a Vero cell.In some embodiments, the recombinant oncolytic virus comprises a recombinant oncolytic vaccinia virus.

[0016] In one aspect, the disclosure provides a composition comprising a population of recombinant oncolytic viruses produced by the methods described herein.

[0017] In one aspect, the disclosure provides a bioreactor comprising a culture of host cells comprising at least about 50 PFU to about 350 PFU of a recombinant oncolytic virus per host cell.

[0018] In some embodiments, the bioreactor comprises a fixed bed, said fixed bed comprising said culture of host cells. In some embodiments, said culture of host cells is grown in microcarriers within said fixed bed. In some embodiments, the bioreactor comprises said culture of host cells, said culture comprising about 1,000 cells / cm. 2 Approximately 10,000 cells / cm from the fixed bed 2 In some embodiments, the fixed bed comprises a volume of about 0.01 L to about 25 L. In some embodiments, the fixed bed comprises a packed density of at least about 80 g / L to about 144 g / L. In some embodiments, the fixed bed comprises a packed density of about 0.2 m 2 Approximately 500m from 2 Includes a surface area of

[0019] In one aspect, the present disclosure provides a method for the preparation of a medicament comprising administering to a subject a therapeutically effective amount of 11 From about 5 × 10 13 A bioreactor is provided that includes PFU of a recombinant oncolytic virus. In some embodiments, the bioreactor includes a fixed bed, the fixed bed including a culture that includes the recombinant oncolytic virus. In some embodiments, the fixed bed includes a volume of about 0.01 L to about 25 L. In some embodiments, the fixed bed includes a packed density of at least about 80 g / L to about 144 g / L. In some embodiments, the fixed bed includes a packed density of at least about 0.2 m 2 Approximately 500m from 2 In some embodiments, the recombinant oncolytic virus comprises a recombinant oncolytic vaccinia virus.

[0020] In one aspect, the disclosure provides a method for producing a recombinant oncolytic virus, comprising: (i) contacting a culture of host cells with a vaccinia virus in a bioreactor at a multiplicity of infection of about 0.001 PFU / cell; (ii) growing the culture for about 72 hours to generate a further culture comprising a plurality of virus-infected host cells; (iii) recovering a population of recombinant oncolytic virus from the further culture comprising the plurality of virus-infected host cells, wherein the population is about 1.5×10 11 From about 5 × 10 13 PFU of the recombinant oncolytic virus, and the bioreactor is about 0.2 m 2 Approximately 500m from 2 wherein said recovering is performed under alkaline conditions.

[0021] In some embodiments, the bioreactor comprises a fixed bed. In some embodiments, the fixed bed comprises microcarriers. In some embodiments, the fixed bed comprises a volume of about 0.01 L to about 25 L. In some embodiments, the fixed bed comprises a packed density of at least about 80 g / L to about 144 g / L. In some embodiments, the fixed bed comprises a packed density of at least about 96 g / L. In some embodiments, the host cells are cultured in the bioreactor at a rate of at least about 1,000 cells / cm. 2 to approximately 150,000 cells / cm 2In some embodiments, the further culture comprises a dissolved oxygen tension (DOT) level of at least about 20% to about 100%. In some embodiments, the further culture comprises a dissolved oxygen tension (DOT) level of about 50%. In some embodiments, the further culture is maintained at a temperature of about 32° C. to about 38° C. In some embodiments, the further culture is maintained at a temperature of about 36° C. In some embodiments, the further culture is maintained at a pH of about 7.0 to about 7.5. In some embodiments, the further culture is maintained at a pH of about 7.2. In some embodiments, the recovering comprises lysing the plurality of virus-infected host cells by incubating with an enzyme for degrading cell debris and an alkaline buffer. In some embodiments, a concentration of the enzyme for degrading cell debris during the incubation is about 50 IU / mL to about 200 IU / mL. In some embodiments, the incubating is performed at a temperature of about 22° C. to about 28° C. In some embodiments, the incubating is performed at a temperature of about 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C., or up to about 30° C. In some embodiments, the incubating is performed at a temperature of about 25° C. In some embodiments, the incubating is performed at a temperature of about 27° C. In some embodiments, the enzyme for degrading cellular debris comprises Benzonase®. In some embodiments, the cellular enzyme for degrading cellular debris comprises Benzonase®, and the incubating is performed in the presence of Benzonase® at a concentration of about 150 IU / mL at a temperature of about 27° C. for about 5 hours. In some embodiments, the alkaline buffer comprises a pH greater than 8. In some embodiments, the alkaline buffer comprises a pH greater than 8.0 to about 10.0. In some embodiments, the alkaline buffer comprises a pH of about 9.5. In some embodiments, the alkaline buffer comprises a Tris buffer.

[0022] In some embodiments, the method further comprises monitoring the pH during said incubation.

[0023] In some embodiments, the method further comprises filtering to produce a filtered lysate. In some embodiments, the method further comprises treating the filtered lysate with a cytolytic enzyme. In some embodiments, the treating is performed under alkaline conditions. In some embodiments, the treating is performed for about 10 hours to about 24 hours. In some embodiments, the treating is performed at a temperature of about 4° C. for about 18 hours. In some embodiments, the treating is followed by tangential flow filtration of the filtered lysate to produce a purified preparation of the recombinant oncolytic virus. In some embodiments, the tangential flow filtration comprises concentrating the filtered lysate, followed by at least one and two rounds of diafiltration. In some embodiments, a second round of concentration may be performed. In some embodiments, the first round of diafiltration comprises a buffer exchange with a buffer comprising a pH of about 9.0 to about 9.5. In some embodiments, the first round of diafiltration comprises about 20 volumes of the buffer exchange. In some embodiments, the buffer with a pH of about 9.0 to about 9.5 comprises a Tris concentration of about 40 mM to about 100 mM. In some embodiments, the second round of diafiltration comprises a buffer exchange with a buffer with a pH of about 7.0 to about 8.0. In some embodiments, the second round of diafiltration comprises about 10 volumes of the buffer exchange. In some embodiments, the buffer with a pH of about 7.0 to about 8.0 comprises a Tris concentration of about 15 mM to about 30 mM. In some embodiments, the buffer with a pH of about 7.0 to about 8.0 further comprises at least about 5% to at least about 20% sucrose by volume. In some embodiments, the purified preparation of the recombinant oncolytic virus comprises about 5 ng to about 100 ng of host cell DNA in a unit dose of the recombinant oncolytic virus. In some embodiments, the purified preparation of the recombinant oncolytic virus comprises about 0.1 μg to about 10 μg of host cell protein in a unit dose of the recombinant oncolytic virus. In some embodiments, the unit dose is about 1×10 9 From about 1×10 13In some embodiments, the purified preparation of the recombinant oncolytic virus comprises 5×10 PFU of the recombinant oncolytic virus. 9 In some embodiments, the purified preparation of the recombinant oncolytic virus comprises up to about 400 ng, about 200 ng, about 100 ng, about 50 ng, or about 40 ng of host cell DNA per PFU. 9 In some embodiments, the purified preparation of the recombinant oncolytic virus comprises up to about 5×10 ng of the recombinant oncolytic virus, about 2000 ng, about 1000 ng, about 500 ng, about 250 ng, or about 200 ng of host cell DNA per PFU. 9 In some embodiments, the purified preparation of the recombinant oncolytic virus comprises up to about 7.5 μg, about 7 μg, about 6 μg, about 5 μg, or about 4 μg of host cell protein per PFU. 9 In some embodiments, the purified preparation of the recombinant oncolytic virus comprises up to about 5×10 of the recombinant oncolytic virus, about 37.5 μg, about 35 μg, about 30 μg, about 25 μg, or about 20 μg of host cell protein per PFU. 9 Per PFU, up to about 100 ng, about 60 ng, about 50 ng, about 30 ng, or about 25 ng of serum albumin is used in the culture.

[0024] In another aspect, the disclosure provides a method for producing a recombinant oncolytic virus, comprising: (i) contacting a culture of host cells with a vaccinia virus in a bioreactor at a multiplicity of infection of about 0.001 PFU / cell; (ii) growing the culture at a temperature of about 36° C. and a dissolved oxygen tension level of about 50% for about 72 hours to generate a further culture comprising a plurality of virus-infected host cells; and (iii) recovering a population of recombinant oncolytic virus from the further culture comprising the plurality of virus-infected host cells, wherein the recovering is performed in the presence of a buffer comprising a pH of about 9.5.

[0025] In another aspect, the disclosure provides a method for producing a recombinant oncolytic virus, comprising: (i) contacting a culture of host cells with a vaccinia virus in a bioreactor at a multiplicity of infection of about 0.003 PFU / cell; (ii) growing the culture at a temperature of about 35° C. and a dissolved oxygen tension level of about 45% for about 48 hours to generate a further culture comprising a plurality of virus-infected host cells; (iii) recovering a population of recombinant oncolytic virus from the further culture comprising the plurality of virus-infected host cells, wherein the bioreactor is about 0.2 m 2 Approximately 500m from 2 and said recovering is performed in the presence of a buffer comprising a pH of about 8.5.

[0026] In another aspect, the disclosure provides a method for producing a recombinant oncolytic virus, comprising: (i) contacting a culture of host cells with a vaccinia virus in a bioreactor at a multiplicity of infection of about 0.002 PFU / cell; (ii) growing the culture at a temperature of about 32° C. and a dissolved oxygen tension level of about 50% for about 24 hours to generate a further culture comprising a plurality of virus-infected host cells; (iii) recovering a population of recombinant oncolytic virus from the further culture comprising the plurality of virus-infected host cells, wherein the bioreactor is about 0.2 m 2 Approximately 500m from 2 and said recovering is performed in the presence of a buffer comprising a pH of about 8.0.

[0027] In another aspect, the disclosure provides a method for producing a recombinant oncolytic virus, comprising: (i) contacting a culture of host cells with a vaccinia virus in a bioreactor at a multiplicity of infection of about 0.002 PFU / cell; (ii) growing the culture at a temperature of about 37° C. and a dissolved oxygen tension level of about 50% for about 96 hours to generate a further culture comprising a plurality of virus-infected host cells; (iii) recovering a population of recombinant oncolytic virus from the further culture comprising the plurality of virus-infected host cells, wherein the bioreactor is about 0.2 m 2Approximately 500m from 2 and said recovering is performed in the presence of a buffer comprising a pH of about 7.8.

[0028] In another aspect, the disclosure provides a method for producing a recombinant oncolytic virus, comprising: (i) contacting a culture of host cells with a vaccinia virus in a bioreactor at a multiplicity of infection of about 0.005 PFU / cell; (ii) growing the culture at a temperature of about 37° C. and a dissolved oxygen tension level of about 50% for about 96 hours to generate a further culture comprising a plurality of virus-infected host cells; (iii) recovering a population of recombinant oncolytic virus from the further culture comprising the plurality of virus-infected host cells, wherein the bioreactor is about 0.2 m 2 Approximately 500m from 2 and said recovering is performed in the presence of a buffer comprising a pH of about 9.5.

[0029] In another aspect, the disclosure provides a method for producing a recombinant oncolytic virus, comprising: (i) contacting a culture of host cells with a vaccinia virus in a bioreactor at a multiplicity of infection of about 0.001 PFU / cell; (ii) growing the culture at a temperature of about 36° C. and a dissolved oxygen tension level of about 50% for about 72 hours to generate a further culture comprising a plurality of virus-infected host cells; (iii) extracting at least about 10 virus-infected host cells from the further culture comprising the plurality of virus-infected host cells; 12 and recovering a population of recombinant oncolytic viruses comprising PFU of said recombinant oncolytic viruses, said recovering being performed in the presence of a buffer comprising a pH of about 9.5.

[0030] In another aspect, the disclosure provides a method for producing a recombinant oncolytic virus, comprising: (i) contacting a culture of host cells with a vaccinia virus in a bioreactor at a multiplicity of infection of about 0.003 PFU / cell; (ii) growing the culture at a temperature of about 35° C. and a dissolved oxygen tension level of about 45% for about 48 hours to generate a further culture comprising a plurality of virus-infected host cells; (iii) extracting at least about 10 virus-infected host cells from the further culture comprising the plurality of virus-infected host cells; 12 and harvesting a population of recombinant oncolytic viruses comprising PFU of said recombinant oncolytic viruses, said bioreactor being about 0.2 m 2 Approximately 500m from 2 and said recovering is performed in the presence of a buffer comprising a pH of about 8.5.

[0031] In another aspect, the disclosure provides a method for producing a recombinant oncolytic virus, comprising: (i) contacting a culture of host cells with a vaccinia virus in a bioreactor at a multiplicity of infection of about 0.002 PFU / cell; (ii) growing the culture at a temperature of about 32° C. and a dissolved oxygen tension level of about 50% for about 24 hours to generate a further culture comprising a plurality of virus-infected host cells; (iii) extracting at least about 10 virus-infected host cells from the further culture comprising the plurality of virus-infected host cells; 12 and harvesting a population of recombinant oncolytic viruses comprising PFU of said recombinant oncolytic viruses, said bioreactor being about 0.2 m 2 Approximately 500m from 2 and said recovering is performed in the presence of a buffer comprising a pH of about 8.0.

[0032] In another aspect, the disclosure provides a method for producing a recombinant oncolytic virus, comprising: (i) contacting a culture of host cells with a vaccinia virus in a bioreactor at a multiplicity of infection of about 0.002 PFU / cell; (ii) growing the culture at a temperature of about 37° C. and a dissolved oxygen tension level of about 50% for about 96 hours to generate a further culture comprising a plurality of virus-infected host cells; (iii) extracting at least about 10 virus-infected host cells from the further culture comprising the plurality of virus-infected host cells; 12 and harvesting a population of recombinant oncolytic viruses comprising PFU of said recombinant oncolytic viruses, said bioreactor being about 0.2 m 2 Approximately 500m from 2 and said recovering is performed in the presence of a buffer comprising a pH of about 7.8.

[0033] In another aspect, the disclosure provides a method for producing a recombinant oncolytic virus, comprising: (i) contacting a culture of host cells with a vaccinia virus in a bioreactor at a multiplicity of infection of about 0.005 PFU / cell; (ii) growing the culture at a temperature of about 37° C. and a dissolved oxygen tension level of about 50% for about 96 hours to generate a further culture comprising a plurality of virus-infected host cells; (iii) extracting at least about 10 virus-infected host cells from the further culture comprising the plurality of virus-infected host cells; 12 and harvesting a population of recombinant oncolytic viruses comprising PFU of said recombinant oncolytic viruses, said bioreactor being about 0.2 m 2 Approximately 500m from 2 and said recovering is performed in the presence of a buffer comprising a pH of about 9.5.

[0034] In some embodiments, the method further comprises purifying the population of recombinant oncolytic viruses recovered in step (iii) to produce a purified preparation of the recombinant oncolytic viruses. In some embodiments, the purified preparation of the recombinant oncolytic viruses comprises at least about 2×10 11 From about 7 × 10 11 Contains PFU viruses. Incorporation by Reference

[0035] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0036] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings, in which: [Brief description of the drawings]

[0037] [Figure 1] FIG. 1 is a flow chart of an exemplary virus manufacturing process.

[0038] [Diagram 2] FIG. 2 is a flow chart of an exemplary virus manufacturing process.

[0039] [Diagram 3] FIG. 3 is a flow chart of an exemplary virus manufacturing process.

[0040] [Figure 4] FIG. 4 is a flow chart of an exemplary virus manufacturing process.

[0041] [Diagram 5] FIG. 5 is a flow chart of an exemplary virus manufacturing process.

[0042] [Figure 6] FIG. 6 is a flow chart of an exemplary downstream process for producing a virus.

[0043] [Figure 7]FIG. 7 is a flow chart of an exemplary downstream process for producing a virus.

[0044] [Figure 8] FIG. 8 is a flow chart of an exemplary downstream process for producing a virus.

[0045] [Figure 9] FIG. 9 is a flow chart of an exemplary downstream process for producing a virus.

[0046] [Figure 10] FIG. 10 shows a schematic diagram of an exemplary setup for the clarification process.

[0047] [Figure 11] FIG. 11 shows a schematic diagram of an exemplary setup for the TFF and diafiltration processes.

[0048] [Figure 12] FIG. 12 shows a schematic diagram of an exemplary setup for the final filtration process. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0049] Detailed Description A specific definition

[0050] The terminology used herein is for the purpose of describing particular instances only and is not intended to be limiting. As used herein, the singular forms "a," "an," and "the" may include the plural forms unless the context clearly indicates otherwise. Furthermore, to the extent the terms "contains," "containing," "including," "includes," "having," "has," "with," or variations thereof are used in any of the detailed description and / or claims, such terms are intended to be inclusive in a manner similar to the term "comprising."

[0051] The term "about" or "approximately" may mean within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" may mean within 1 or more than 1 standard deviation according to convention for a given value. When a particular value is described in this application and claims, unless otherwise stated, it should be assumed that the term "about" means an acceptable error range for the particular value, e.g., ±10% of the value modified by the term "about."

[0052] The terms "individual," "patient," or "subject" may be used interchangeably. None of the terms require or limit a situation to be characterized by the supervision (e.g., continuous or intermittent) of a medical professional (e.g., a physician, registered nurse, nurse practitioner, physician's assistant, orderly, or hospice worker). In some embodiments, a patient, subject, or individual may be under the supervision of a medical professional.

[0053] The term "mutation" as used herein may refer to a deletion, an insertion of a heterologous nucleic acid, an inversion or a substitution, and includes an open reading frame truncation mutation as commonly understood in the art.

[0054] The term "gene", as used herein, may refer to a segment of nucleic acid that encodes a particular protein or RNA (also referred to as a "coding sequence" or "coding region"), optionally together with associated regulatory regions, e.g., promoters, operators, terminators, etc., which may be located upstream or downstream of the coding sequence.

[0055] The terms "recombinant virus" and "modified virus" are used interchangeably herein and may refer to a virus that contains one or more mutations in its genome, including but not limited to deletions, insertions of heterologous nucleic acid, inversions, substitutions, or combinations thereof.

[0056] The term "oncolytic" as used herein may refer to the killing of cancer or tumor cells, for example by an agent, such as an oncolytic vaccinia virus, through direct lysis of the cells, by stimulating an immune response against the cells, apoptosis, expression of toxic proteins, shutting down autophagy and protein synthesis, inducing anti-tumor immunity, or any combination thereof. Direct lysis of an infected cancer or tumor cell by an agent, such as an oncolytic vaccinia virus, may be the result of viral replication within the cell. In certain instances, the term "oncolytic" refers to the killing of cancer or tumor cells without lysing the cells.

[0057] The term "subject" may refer to an animal, including, but not limited to, a primate (e.g., a human), cow, sheep, goat, horse, dog, cat, rabbit, rat, or mouse. The terms "subject" and "patient" are used interchangeably herein, e.g., in reference to a mammalian subject, e.g., a human subject.

[0058] The terms "treat", "treating" and "treatment" may be meant to include reducing or eliminating a disorder, disease, or condition; or one or more of the symptoms associated with the disorder, disease, or condition; or reducing or eradicating the cause of the disorder, disease, or condition itself. Desirable effects of treatment may include, but are not limited to, preventing the appearance or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or relieving the disease state, and ameliorating or improving prognosis.

[0059] One aspect of the present disclosure provides a method and system for producing viruses. In some cases, the method and system provided herein can be efficient and productive in producing viruses. For example, the method and system described herein can produce viruses at high titers per host cell. The method and system described herein can be applied to the production of viruses at industrial scale, for example, with high yield per production cycle, from, for example, infecting host cells, culturing virus-infected cells, to virus recovery and virus clarification and purification. The method provided herein can produce high purity virus preparations with limited or no protein or nucleic acid contamination. In some cases, the method and system provided herein can be applied to the production of recombinant viruses. In some cases, the method and system provided herein can be applied to the production of oncolytic viruses.

[0060] The method of producing a virus provided herein may include growing a cell culture comprising virus-infected host cells in a bioreactor. In some cases, the methods and systems provided herein may use a fixed-bed bioreactor to culture virus-infected host cells. The method of producing a virus may include infecting (inoculating) host cells to generate a cell culture comprising virus-infected host cells. The method of producing a virus may include harvesting a population of viruses, e.g., oncolytic viruses, e.g., recombinant oncolytic viruses, from the cell culture. In some cases, harvesting the population of viruses may be performed under alkaline conditions. The method may include clarification and purification of the cell lysate obtained from the harvest to generate a purified virus preparation. In some cases, the clarification and purification may include filtering the cell lysate. In some cases, the clarification and purification may include treating the cell lysate with an enzyme to degrade cell debris, followed by filtering. The method may also include filtering to generate a concentrated virus preparation of high purity. In some cases, the filtration may include tangential flow filtration.

[0061] The method of producing a virus provided herein may include (a) infecting (inoculating) a plurality of host cells to produce a cell culture comprising virus-infected host cells; (b) growing the cell culture comprising virus-infected host cells in a bioreactor; (c) harvesting a population of viruses, e.g., oncolytic viruses, e.g., recombinant oncolytic viruses, from the cell culture under alkaline conditions; and (d) clarification and purification of the cell lysate obtained from the harvesting to produce a purified virus preparation. In some cases, the clarification and purification may include: (i) treating the cell lysate with an enzyme to degrade cell debris; and (ii) filtering the treated cell lysate to produce a highly pure concentrated virus preparation.

[0062] The methods provided herein include growing a cell culture to deliver a virus, e.g., an oncolytic virus, e.g., a recombinant oncolytic virus, to at least about 50 PFU per host cell (PFU / cell), at least about 60 PFU / cell, at least about 70 PFU / cell, at least about 80 PFU / cell, at least about 90 PFU / cell, at least about 100 PFU / cell, at least about 110 PFU / cell, at least about 120 PFU / cell, at least about 140 PFU / cell, at least about 160 PFU / cell, at least about 180 PFU / cell, at least about 200 PFU / cell, at least about 220 PFU / cell, at least about 240 PFU / cell, at least about 250 PFU / cell, at least about 260 PFU / cell, at least about 270 PFU / cell, at least about 290 PFU / cell, at least about 300 PFU / cell, at least about 310 PFU / cell, at least about 320 PFU / cell, at least about 330 PFU / cell, at least about 340 PFU / cell, at least about 350 PFU / cell, at least about 360 PFU / cell, at least about 370 PFU / cell, at least about 380 PFU / cell, at least about 390 PFU / cell, at least about 400 PFU / cell, at least about 410 PFU / cell, at least about 420 PFU / cell, at least about 430 PFU / cell, at least about 440 PFU / cell, at least about 450 PFU / cell, at least about 460 PFU / cell, at least about 470 PFU / cell, at least about 480 PFU / cell, at least about 490 PFU / cell, at least about 500 PFU / cell, at least about In some embodiments, the method may include producing a titer of at least about 280 PFU / cell, at least about 290 PFU / cell, at least about 300 PFU / cell, at least about 320 PFU / cell, at least about 340 PFU / cell, at least about 350 PFU / cell, at least about 360 PFU / cell, at least about 380 PFU / cell, at least about 400 PFU / cell, at least about 450 PFU / cell, at least about 500 PFU / cell, at least about 600 PFU / cell, at least about 700 PFU / cell, at least about 800 PFU / cell, at least about 900 PFU / cell, at least about 1000 PFU / cell, at least about 2000 PFU / cell, or at least about 5000 PFU / cell. In some cases, the method may include growing the cell culture to produce the virus, e.g., an oncolytic virus, e.g., a recombinant oncolytic virus, at a titer of about 50 PFU / cell to about 350 PFU / cell.In some cases, the method includes growing the cell culture to inject the virus into the cell culture at about 100 PFU / cell to about 300 PFU / cell, 150 PFU / cell to about 300 PFU / cell, 200 PFU / cell to about 300 PFU / cell, 250 PFU / cell to about 300 PFU / cell, 100 PFU / cell to about 500 PFU / cell, 200 PFU / cell to about 500 PFU / cell, 250 PFU / cell to about 500 PFU / cell, 300 PFU / cell to about 500 PFU / cell, 400 PFU / cell to about 500 PFU / cell, This may include producing at a titer of 100 PFU / cell to about 1000 PFU / cell, 200 PFU / cell to about 1000 PFU / cell, 300 PFU / cell to about 1000 PFU / cell, 400 PFU / cell to about 1000 PFU / cell, 500 PFU / cell to about 1000 PFU / cell, 600 PFU / cell to about 1000 PFU / cell, 700 PFU / cell to about 1000 PFU / cell, 800 PFU / cell to about 1000 PFU / cell, or 900 PFU / cell to about 1000 PFU / cell.

[0063] The methods provided herein provide for a method for detecting approximately 1.5×10 11 From about 5 × 10 13 Optionally, the method includes recovering a population of viruses, e.g., oncolytic viruses, e.g., recombinant oncolytic viruses, from the culture containing the PFU of virus. In some cases ... 11 PFU, at least about 1.5 × 10 11 PFU, at least about 2 × 10 11 PFU, at least about 2.5 × 10 11 PFU, at least about 3 × 10 11 PFU, at least about 4 × 10 11 PFU, at least about 5 × 10 11 PFU, at least about 7.5 × 10 11 PFU, at least about 1 × 10 12 PFU, at least about 1.5 × 10 12 PFU, at least about 2 × 10 12 PFU, at least about 2.5 × 10 12 PFU, at least about 3 × 10 12PFU, at least about 4 × 10 12 PFU, at least about 5 × 10 12 PFU, at least about 7.5 × 10 12 PFU, at least about 1 × 10 13 PFU, at least about 1.5 × 10 13 PFU, at least about 2 × 10 13 PFU, at least about 2.5 × 10 13 PFU, at least about 3 × 10 13 PFU, at least about 4 × 10 13 PFU, at least about 5 × 10 13 PFU, at least about 7.5 × 10 13 PFU, or at least about 1 × 10 14 Optionally, the method includes harvesting the population of viruses from the culture containing PFU of virus. In some cases, the method includes harvesting the population of viruses from the culture containing PFU of virus. 11 From about 1×10 14 PFU, approx. 5×10 11 From about 1×10 14 PFU, approx. 1×10 12 From about 1×10 14 PFU, approx. 5×10 12 From about 1×10 14 PFU, approx. 1×10 13 From about 1×10 14 PFU, approx. 5×10 13 From about 1×10 14 PFU, approx. 1×10 11 From about 5 × 10 13 PFU, approx. 2×10 11 From about 5 × 10 13 PFU, approx. 5×10 11 From about 5 × 10 13 PFU, approx. 1×10 12 From about 5 × 10 13 PFU, approx. 2×10 12 From about 5 × 10 13 PFU, approx. 5×10 12 From about 5 × 10 13 PFU, or approximately 1 × 10 13 From about 5 × 10 13 This may include recovering the virus from a culture containing PFU of virus.

[0064] The methods provided herein provide for the production of approximately 1.5×10 8 From about 2 × 10 10 PFU, e.g., about 1.5×10 per mL of the culture 8 From about 5 × 10 8 PFU / mL, 2.5×10 8 From about 7.5 × 10 8 PFU / mL, 5×10 8 From about 2.5 × 10 9 PFU / mL, 1.5×10 9 From about 5 × 10 9 PFU / mL, 2.5×10 9 From about 1×10 10 PFU / mL, 5×10 9 From about 1.5 × 10 10 PFU / mL, or approximately 7.5 x10 9 From about 2 × 10 10 It may include recovering a population of virus, eg, an oncolytic virus, eg, a recombinant oncolytic virus, from a virus-containing culture at a virus titer of PFU.

[0065] The method provided herein may include recovering a population of viruses, such as oncolytic viruses, e.g., recombinant oncolytic viruses, from the culture, producing a lysate comprising the population of viruses, and clarifying the lysate to produce a purified population of viruses, which may comprise at least about 50% to about 90% of the population of viruses in the lysate. In some cases, the purified population of viruses may comprise at least about 40%, at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% of the population of viruses, such as oncolytic viruses, e.g., recombinant oncolytic viruses, in the cell culture. In some cases, the purified population of viruses is present in the cell culture at about 40% to about 99%, about 50% to about 99%, about 60% to about 99%, about 70% to about 99%, about 80% to about 99%, about 90% to about 99%, about 95% to about 99%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about 80% to about 90%, about 85% to about 99%, or about 100% to about 100%. The population of viruses, e.g., oncolytic viruses, e.g., recombinant oncolytic viruses, may comprise about 90%, about 40% to about 80%, about 50% to about 80%, about 60% to about 80%, about 70% to about 80%, about 75% to about 80%, about 40% to about 50%, about 40% to about 60%, about 40% to about 70%, about 50% to about 60%, or about 50% to about 70% of the virus, e.g., oncolytic viruses, e.g., recombinant oncolytic viruses.

[0066] The methods provided herein may include purifying the collected virus lysate to produce a purified preparation of virus. The purified preparation of virus produced according to the methods provided herein may contain at most about 2 ng, at most about 3 ng, at most about 4 ng, at most about 5 ng, at most about 6 ng, at most about 7 ng, at most about 8 ng, at most about 9 ng, at most about 10 ng, at most about 11 ng, at most about 12 ng, at most about 13 ng, at most about 14 ng, at most about 15 ng, at most about 16 ng, at most about 17 ng, at most about 19 ng, at most about 20 ng, at most about 21 ng, at most about 22 ng, at most about 23 ng, at most about 24 ng, at most about 25 ng, at most about 26 ng, at most about 27 ng, at most about 28 ng, at most about 29 ng, at most about 30 ng, at most about 31 ng, at most about 32 ng, at most about 33 ng, at most about 34 ng, at most about 35 ng, at most about 36 ng, at most about 37 ng, at most about 38 ng, at most about 39 ng, at most about 40 ng, at most about 41 ng, at most about 42 ng, at most about 43 ng, at most about 44 ng, at most about 45 ng, at most about 46 ng, at most about 47 ng, at most about 48 ng, at most about 49 ng, at most about 50 ng, at most about 51 ng, at most about 52 ng, at most about 53 ng, at most about 54 ng, at most about 55 ng, at most about The nucleic acid sequence may comprise 7 ng, at most about 18 ng, at most about 19 ng, at most about 20 ng, at most about 21 ng, at most about 22 ng, at most about 23 ng, at most about 24 ng, at most about 25 ng, at most about 28 ng, at most about 30 ng, at most about 50 ng, at most about 75 ng, at most about 100 ng, at most about 500 ng, at most about 750 ng, at most about 1 μg, or at most about 2 μg of host cell DNA. In some cases, a purified preparation of a virus produced according to the methods provided herein may contain about 2ng, about 3ng, about 4ng, about 5ng, about 6ng, about 7ng, about 8ng, about 9ng, about 10ng, about 11ng, about 12ng, about 13ng, about 14ng, about 15ng, about 16ng, about 17ng, about 18ng, about 19ng, about 20ng, about 21ng, about 22ng, about 23ng, about 24ng, about 25ng, about 28ng, about 30ng, about 50ng, about 82mg, about 100ng, about 200ng, about 500ng, about 750ng, about 1μg, or about 2μg of host cell DNA per unit dose of virus. In some cases, the host cell DNA in a purified preparation of a virus may be measured by DNA extraction and purification using a silica-based column or DNA extraction by standard methods. In some cases, host cell DNA in purified preparations of the virus may be measured by quantitative real-time PCR with a probe specific for the host cell genome after processing with QuickExtract™ or Qiagen® kits. As provided herein, a unit dose of virus is about 0.5×10 9 , about 1×10 9 , about 2×109 , about 5×10 9 , about 7.5×10 9 , about 1×10 10 , about 2×10 10 , about 5×10 10 , about 7.5×10 10 , about 1×10 11 , about 2×10 11 , about 5×10 11 , about 7.5×10 11 , about 1×10 12 , about 2×10 12 , about 5×10 12 , about 7.5×10 12 , about 1×10 13 , about 2×10 13 , about 5×10 13 , about 7.5×10 13 , about 1×10 14 , about 2×10 14 , about 5×10 14 , about 7.5×10 14 , about 1×10 15 , or approximately 1 × 10 16 In some cases, the unit dose of the virus may contain about 1×10 PFU of the virus. 9 From about 1×10 13 The unit dose of the virus may include about 1×10 9 From about 1×10 13 of viruses, e.g. 5 × 10 9 In some examples, the unit dose of virus may include mature intracellular viruses (IMV), intracellular enveloped viruses (IEV), cell-associated enveloped viruses (CEV), and extracellular enveloped viruses (EEV).

[0067] Purified preparations of virus produced according to the methods provided herein can contain up to 5×10 9In some cases, purified preparations of virus produced according to the methods provided herein may contain at most about 5 x 10 ng, at most about 10 ng, at most about 15 ng, at most about 20 ng, at most about 25 ng, at most about 28 ng, at most about 30 ng, at most about 33 ng, at most about 35 ng, at most about 40 ng, at most about 45 ng, at most about 50 ng, at most about 80 ng, at most about 100 ng, at most about 150 ng, at most about 200 ng, at most about 250 ng, at most about 300 ng, at most about 400 ng, or at most about 500 ng of host cell DNA in a dose of virus. 9 A purified preparation of virus produced according to the methods provided herein may contain about 5 ng, about 10 ng, about 15 ng, about 20 ng, about 25 ng, about 28 ng, about 30 ng, about 33 ng, about 35 ng, about 40 ng, about 45 ng, about 50 ng, about 80 ng, about 100 ng, about 150 ng, about 200 ng, about 250 ng, about 300 ng, about 400 ng, about 500 ng, about 1 μg, about 1.5 μg, about 2 μg, about 3 μg, or about 5 μg of host cell DNA in a dose of virus of 1×10 9 In some cases, purified preparations of virus produced according to the methods provided herein may contain at most about 1 ng, at most about 2 ng, at most about 3 ng, at most about 4 ng, at most about 5 ng, at most about 5.6 ng, at most about 6 ng, at most about 6.6 ng, at most about 7 ng, at most about 8 ng, at most about 9 ng, at most about 10 ng, at most about 16 ng, at most about 20 ng, at most about 30 ng, at most about 40 ng, at most about 50 ng, at most about 60 ng, at most about 80 ng, or at most about 100 ng, about 0.2 μg, about 0.3 μg, about 0.4 μg, or about 1 μg of host cell DNA in a dose of virus of 1×10 9 Approximately 51 ng of host cell DNA may be included in a single viral dose.

[0068] Purified preparations of virus produced according to the methods provided herein may contain at most about 0.1 μg, at most about 0.2 μg, at most about 0.3 μg, at most about 0.4 μg, at most about 0.5 μg, at most about 0.6 μg, at most about 0.7 μg, at most about 0.8 μg, at most about 0.9 μg, at most about 1 μg, at most about 1.1 μg, at most about 1.2 μg, at most about 1.3 μg, at most about 1.4 μg, at most about 1.5 μg, at most about 2 μg, at most about 5 μg, at most about 7.5 μg, at most about 10 μg, at most about 20 μg, at most about 30 μg, at most about 50 μg, or at most about 100 μg of host cell protein in a unit dose of virus. In some cases, the purified preparation of the virus produced according to the method provided herein may contain about 0.1 μg, about 0.2 μg, about 0.3 μg, about 0.4 μg, about 0.5 μg, about 0.6 μg, about 0.7 μg, about 0.8 μg, about 0.9 μg, about 1 μg, about 1.1 μg, about 1.2 μg, about 1.3 μg, about 1.4 μg, about 1.5 μg, about 2 μg, about 5 μg, about 7.5 μg, about 10 μg, about 20 μg, or about 30 μg of host cell protein in a unit dose of the virus. In some cases, the purified preparation of the virus produced according to the method provided herein may contain about 0.1 μg to about 7.5 μg of host cell protein in a unit dose of the recombinant oncolytic virus. In some cases, the content of host cell protein is determined by ELISA (enzyme-linked immunosorbent assay) targeting one or more host cell proteins.

[0069] In some cases, purified preparations of viruses produced according to the methods provided herein contain up to 5×10 9In some cases, purified preparations of virus produced according to the methods provided herein may contain at most about 5×10 host cell protein in a dose of virus of at most about 0.1 μg, at most about 0.5 μg, at most about 1 μg, at most about 1.5 μg, at most about 2 μg, at most about 2.5 μg, at most about 3 μg, at most about 3.5 μg, at most about 4 μg, at most about 4.5 μg, at most about 5 μg, at most about 5.5 μg, at most about 6 μg, at most about 6.5 μg, at most about 7 μg, at most about 7.5 μg, at most about 10 μg, or at most about 20 μg. 9 In some cases, purified preparations of virus produced according to the methods provided herein may contain about 1×10 host cell protein in a dose of virus of about 1×10. 9 In some cases, purified preparations of virus produced according to the methods provided herein may contain about 0.02 μg, at most about 0.1 μg, at most about 0.2 μg, at most about 0.3 μg, at most about 0.4 μg, at most about 0.5 μg, at most about 0.6 μg, at most about 0.7 μg, at most about 0.8 μg, at most about 0.9 μg, at most about 1 μg, at most about 1.1 μg, at most about 1.2 μg, at most about 1.3 μg, at most about 1.4 μg, at most about 1.5 μg, at most about 2 μg, or at most about 4 μg of host cell protein in a dose of virus. In some cases, purified preparations of virus produced according to the methods provided herein may contain about 5×10 9 In some embodiments, the virus dose may contain about 0.02 μg, about 0.1 μg, about 0.2 μg, about 0.3 μg, about 0.4 μg, about 0.5 μg, about 0.6 μg, about 0.7 μg, about 0.8 μg, about 0.9 μg, about 1 μg, about 1.1 μg, about 1.2 μg, about 1.3 μg, about 1.4 μg, about 1.5 μg, about 2 μg, or about 4 μg of host cell protein.

[0070] virus

[0071] The methods and systems provided herein may be applicable to the production of a variety of different viruses, such as oncolytic viruses, e.g., recombinant oncolytic viruses. In some cases, the methods and systems provided herein may be applicable to the production of vaccinia viruses, e.g., recombinant vaccinia viruses.

[0072] The exemplary oncolytic viruses that the methods and systems provided herein are applicable to include measles virus, poliovirus, poxvirus, vaccinia virus, adenovirus, adeno-associated virus, herpes simplex virus, vesicular stomatitis virus, reovirus, Newcastle disease virus, Seneca virus, lentivirus, mengovirus, and myxoma virus.In certain examples, the oncolytic virus may be vaccinia virus.In some cases, the methods and systems provided herein may be applicable to different strain variants of vaccinia virus, such as Lister, Dryvax, EM63, ACAM2000, modified vaccinia Ankara, LC16m8, CV-1, Western Reserve, Copenhagen, Connaught Laboratories, Wyeth, NYCBH, WRΔE3L, and Dairen I. The virus to which the method provided herein is applicable may contain genetic modification, such as deletion or mutation of one or more viral endogenous genes, or introduction of exogenous virus. In some cases, the virus may be a recombinant vaccinia virus. In some cases, the recombinant vaccinia virus may contain at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20 or even more modifications in the genome of the virus. The deletion of viral genes may include partial or complete deletion of viral genes. It should be noted that as used herein, "partial deletion" or "mutation" may refer to partial deletion or mutation of endogenous viral genes in situ, respectively. Alternatively, they may refer to replacing endogenous viral genes with other identical exogenous nucleic acids that lack part of the gene ("partial deletion") or have one or more nucleotide changes in the gene ("mutation").

[0073] In some cases, the virus that can be produced by the method disclosed herein may be a vaccinia virus that comprises one or more genetic modifications. In some cases, the vaccinia virus may comprise a TK (thymidine kinase) viral gene mutation, so as to be thymidine kinase negative, C12L (IL18 binding protein) viral gene deletion, TRIF gene insertion, HPGD gene insertion, and to have reduced surface glycosylation. In some cases, the vaccinia virus may comprise at least one of the following genetically engineered modifications: (i) C12L (IL18 binding protein) viral gene deletion; (ii) TRIF gene insertion; or (iii) HPGD gene insertion. In some cases, the vaccinia virus may comprise reduced surface glycosylation. In some cases, the reduced surface glycosylation may be reduced sialylation of the viral surface. In some cases, the vaccinia virus may have reduced glycosylation levels compared to modified vaccinia viruses. In some cases, the vaccinia virus may be treated with an agent that reduces the amount of glycosylation (e.g., sialylation) during or after the manufacturing process described herein, but before administration to the host. In some cases, the vaccinia virus may further comprise one or more modifications selected from the group of A34R Lysl51 to Glu mutation; full or partial deletion of B5R; A36R mutation / deletion, and / or A56R mutation / deletion. In certain embodiments, the vaccinia virus may be a deglycosylated VV that includes a full or partial deletion of B5R.

[0074] Any suitable host cell may be selected for producing viruses according to the present disclosure. In some cases, the host cell used in the methods provided herein may be an adherent cell. In other cases, the host cell may be cultured in suspension. In some cases, the host cell may be subcultured from an established cell line, such as, but not limited to, human embryonic kidney (HEK) cells, such as HEK293 cells, African green monkey kidney (AGMK) cells, such as Vero cells, Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells, and Hela cells. In some cases, the host cell may be a Hela cell. In some cases, the host (hots) cell may be a genetically modified cell, such as a Hela cell that can be genetically modified by the introduction of one or more exogenous genes, or the deletion or mutation of one or more endogenous genes. In some cases, the host cell may be a primary cell obtained from an organism, such as a human. In some cases, the host cell may be an immortalized cell that can be subcultured (or subcultured) indefinitely.

[0075] In some examples, the methods provided herein may include directly infecting a host cell with a virus, such as an oncolytic virus, such as a recombinant oncolytic virus. For example, the host cell may be directly contacted with the virus and infected. In some cases, the virus may be added to the culture medium of the host cell and inoculated. In some cases, the host cells may be infected with the virus at a multiplicity of infection (moi) of at least about 0.0005, at least about 0.00075, at least about 0.001, at least about 0.0015, at least about 0.002, at least about 0.003, at least about 0.005, at least about 0.0075, at least about 0.01, at least about 0.015, at least about 0.02, at least about 0.025, at least about 0.03, at least about 0.035, at least about 0.04, at least about 0.045, at least about 0.05, at least about 0.075, at least about 0.1, at least about 0.15, at least about 0.2, at least about 0.25, at least about 0.3, at least about 0.35, at least about 0.4, at least about 0.45, or at least about 0.5 PFU / cell. In some cases, the multiplicity of infection may be calculated as the plaque forming units of the virus used for infection divided by the number of host cells. In some cases, the host cells are at most about 0.0005, at most about 0.00075, at most about 0.001, at most about 0.0015, at most about 0.002, at most about 0.003, at most about 0.005, at most about 0.0075, at most about 0.01, at most about 0.015, at most about 0.02, at most about 0.025, at most about 0.03, at most about 0.006, at most about 0.008, at most about 0.009, at most about 0.10, at most about 0.11, at most about 0.12, at most about 0.13, at most about 0.14, at most about 0.15, at most about 0.16, at most about 0.17, at most about 0.18, at most about 0.19, at most about 0.20, at most about 0.21, at most about 0.22, at most about 0.23, at most about 0.24, at most about 0.25, at most about 0.26, at most about 0.27, at most about 0.28, at most about 0.29, at most about 0.30, at most about 0.31, at most about 0.32, at most about 0.33, at most about 0.34, at most about 0.35, at most about 0.36, at most about 0.37, at most about 0.38, at most about 0.39, at most about 0.40, at most about 0.41, at most about 0.42, at most about 0.43. The cells may be infected with the virus at an moi of about 0.035, at most about 0.04, at most about 0.045, at most about 0.05, at most about 0.075, at most about 0.1, at most about 0.15, at most about 0.2, at most about 0.25, at most about 0.3, at most about 0.35, at most about 0.4, at most about 0.45, or at most about 0.5 PFU / cell.In some cases, the virus may be infected at an moi of about 0.0005, about 0.00075, about 0.001, about 0.0015, about 0.002, about 0.003, about 0.005, about 0.0075, about 0.01, about 0.015, about 0.02, about 0.025, about 0.03, about 0.035, about 0.04, about 0.045, about 0.05, about 0.075, about 0.1, about 0.15, about 0.2, about 0.25, about 0.3, about 0.35, about 0.4, about 0.45, or about 0.5 PFU / cell. In some cases, the host cell may be infected with the virus at an moi of about 0.001 to about 0.02 PFU / cell.

[0076] Cultivation of virus-infected cells

[0077] The method provided herein may include growing a culture comprising a plurality of virus-infected host cells in a bioreactor. The method provided herein may produce viruses at high titers as described above. The bioreactor used in the method provided herein may be an integrated mixing system that provides uniform distribution of culture medium. In some cases, the bioreactor may be capable of culturing cells at high density, resulting in high production yields. In some cases, the bioreactor may have linear scalability, for example, the configuration of the bioreactor may be suitable for both bench-scale and industrial-scale production as needed.

[0078] In some cases, the bioreactor may include a fixed bed reactor. The methods and systems provided herein may include a fixed bed reactor that includes a vessel filled or packed with a carrier material that can be used as a support for immobilizing cells. The fixed bed reactor may also include a conditioning vessel that includes a culture medium. In some cases, the vessel with the carrier material and the conditioning vessel may be connected via a circulation system. The circulation system may be a circulation loop. The circulation system may include an oxygen-enriched medium that can be pumped back from the conditioning vessel through the fixed bed. In some cases, oxygen may be directly supplied to the fixed bed. In a fixed bed reactor, the cells can be held in the fixed bed. The medium in the conditioning vessel can be exchanged batchwise or continuously, so that the medium is depleted and the medium containing the product can be replaced with fresh medium. In some cases, the medium may be exchanged continuously. The fixed bed reactor may be an axial flow-based fixed bed system, and axial pumping of the medium through the fixed bed can be used to provide fresh medium and oxygen to the cells. In some cases, the fixed bed reactor may be a radial flow-based fixed bed system, where the radial pumping of the medium passes through the fixed bed, for example, the medium may be pumped radially from a central point through the fixed bed toward the periphery of the fixed bed. The fixed bed may include microcarriers. The microcarriers may be made of suitable materials. In some cases, the microcarriers may be made of PET (polyethylene terephthalate). In some cases, the microcarriers may be made of synthetic materials, natural materials, or both. Synthetic materials include, but are not limited to, glass (e.g., natron, borosilicate), DEAE-dextran, polypropylene, polyurethane, ceramic, acrylamide, polyhydroxyethyl methacrylate, polystyrene, and polyacrylamide. Natural materials include, but are not limited to, collagen, cellulose, gelatin, fibrin, chitin and its derivatives, chitosan, alginate, polysaccharides, and polyglycans.

[0079] In some cases, the cell culture including the virus-infected host cells may be grown in a fixed-bed bioreactor. The fixed-bed bioreactor may include microcarriers that can provide a relatively large surface-to-volume ratio, exposing the cells to sufficient oxygen and nutrients. The fixed-bed bioreactor may have a short discharge time, for example, the time required to fully discharge the culture medium in the bioreactor. In some cases, the discharge time may be less than about 3 hours, less than about 2 hours, less than about 1 hour, less than about 30 minutes, less than about 20 minutes, less than about 15 minutes, less than about 14 minutes, less than about 13 minutes, less than about 12 minutes, less than about 11 minutes, or less than about 10 minutes. In some cases, the discharge time may be about 80 minutes, about 50 minutes, about 30 minutes, about 12.5 minutes, about 12 minutes, about 11.5 minutes, about 11 minutes, or about 10.5 minutes. The fixed-bed bioreactor may have a small residual volume, for example, the residual volume of the culture medium, when the medium can be fully discharged. In some cases, the bioreactor may have a residual volume of less than about 1500 ml, less than about 1200 ml, less than about 1000 ml, less than about 900 ml, less than about 800 ml, less than about 700 ml, less than about 600 ml, less than about 500 ml, less than about 400 ml, less than about 300 ml, less than about 200 ml, less than about 150 ml, or less than about 100 ml. In some cases, the bioreactor may have a residual volume of about 1150 ml, about 1100 ml, about 1050 ml, about 1000 ml, about 950 ml, about 900 ml, about 850 ml, about 800 ml, about 750 ml, about 700 ml, about 650 ml, about 600 ml, about 550 ml, about 500 ml, about 450 ml, about 400 ml, about 350 ml, about 300 ml, about 250 ml, about 200 ml, or about 150 ml.

[0080] In some cases, the method may include growing the culture in a fixed bed comprising a volume of about 0.01L to about 25L. In some cases, the fixed bed may comprise a volume of at least about 0.005L, at least about 0.01L, at least about 0.05L, at least about 0.1L, at least about 0.5L, at least about 1L, at least about 2L, at least about 3L, at least about 4L, at least about 5L, at least about 6L, at least about 7L, at least about 8L, at least about 9L, at least about 10L, at least about 12L, at least about 14L, at least about 15L, at least about 16L, at least about 18L, at least about 20L, at least about 21L, at least about 22L, at least about 23L, at least about 24L, at least about 25L, at least about 26L, at least about 27L, at least about 28L, at least about 29L, at least about 30L, at least about 35L, at least about 40L, at least about 50L, at least about 75L, or at least about 100L. In some cases, the fixed bed may comprise a volume of about 0.005L, about 0.01L, about 0.05L, about 0.1L, about 0.5L, about 1L, about 2L, about 3L, about 4L, about 5L, about 6L, about 7L, about 8L, about 9L, about 10L, about 12L, about 14L, about 15L, about 16L, about 18L, about 20L, about 21L, about 22L, about 23L, about 24L, about 25L, about 26L, about 27L, about 28L, about 29L, about 30L, about 35L, about 40L, about 50L, about 60L, about 65L, about 70L, about 75L, or about 100L. In some cases, the fixed bed may comprise a volume of about 1L. In some cases, the fixed bed may comprise a volume of about 70L.In some cases, the fixed bed may comprise from about 0.005 L to about 25 L, from about 0.01 L to about 25 L, from about 0.05 L to about 25 L, from about 0.1 L to about 25 L, from about 0.15 L to about 25 L, from about 0.2 L to about 25 L, from about 0.5 L to about 25 L, from about 1 L to about 25 L, from about 5 L to about 25 L, from about 10 L to about 25 L, from about 15 L to about 25 L, from about 20 L to about 25 L, from about 0.005 L to about 15 L, from about 0.05 L to about 15 L, from about 0.5 L to about The volume may comprise 15L, about 5L to about 15L, about 10L to about 15L, about 0.005L to about 50L, about 0.5L to about 50L, about 5L to about 50L, about 10L to about 50L, about 15L to about 50L, about 25L to about 50L, about 30L to about 50L, about 1L to about 100L, about 5L to about 100L, about 10L to about 100L, about 20L to about 100L, about 50L to about 100L, or about 75L to about 100L.

[0081] In some cases, the method may include growing the culture in a fixed bed comprising a packed density of at least about 80 g / L to about 144 g / L. In some cases, the fixed bed may comprise a compressed density of at least about 20 g / L, at least about 30 g / L, at least about 40 g / L, at least about 50 g / L, at least about 60 g / L, at least about 70 g / L, at least about 80 g / L, at least about 90 g / L, at least about 91 g / L, at least about 92 g / L, at least about 93 g / L, at least about 94 g / L, at least about 95 g / L, at least about 96 g / L, at least about 97 g / L, at least about 98 g / L, at least about 99 g / L, at least about 100 g / L, at least about 110 g / L, at least about 120 g / L, at least about 130 g / L, at least about 140 g / L, at least about 150 g / L, or at least about 160 g / L. In some cases, the fixed bed may comprise a compressed density of at least about 96 g / L. In some cases, the fixed bed may comprise a packed density of about 60 g / L, about 70 g / L, about 80 g / L, about 90 g / L, about 100 g / L, about 110 g / L, about 120 g / L, about 130 g / L, about 140 g / L, about 141 g / L, about 142 g / L, about 143 g / L, about 144 g / L, about 145 g / L, about 146 g / L, about 147 g / L, about 148 g / L, about 149 g / L, about 150 g / L, about 155 g / L, or about 160 g / L.

[0082] In some cases, the method may include seeding the host cells in the bioreactor and growing them for a certain period of time before inoculating with the virus. In other cases, the method may include seeding the virus-infected host cells in the bioreactor and growing the culture. In some cases, the host cells prior to inoculation with the virus are at least about 1,000 cells / cm in the bioreactor. 2 to approximately 50,000 cells / cm 2 In some cases, the host cells prior to inoculation with the virus may be seeded at a density of at least about 10,000 cells / cm. 2 to approximately 400,000 cells / cm 2In some cases, the host cells prior to inoculation with the virus may be seeded at a density of at least about 1,000 cells / cm. 2 , at least about 1,500 cells / cm 2 , at least about 2,000 cells / cm 2 , at least about 2,500 cells / cm 2 , at least about 3,000 cells / cm 2 , at least about 3,500 cells / cm 2 , at least about 4,000 cells / cm 2 , at least about 4,500 cells / cm 2 , at least about 5,000 cells / cm 2 , at least about 10,000 cells / cm 2 , at least about 20,000 cells / cm 2 , at least about 40,000 cells / cm 2 , at least about 50,000 cells / cm 2 , at least about 60,000 cells / cm 2 , at least about 70,000 cells / cm 2 , at least about 80,000 cells / cm 2 , at least about 90,000 cells / cm 2 , at least about 100,000 cells / cm 2 , at least about 110,000 cells / cm 2 , at least about 120,000 cells / cm 2 , at least about 130,000 cells / cm 2 , at least about 140,000 cells / cm 2 , at least about 150,000 cells / cm 2 , at least about 160,000 cells / cm 2 , at least about 170,000 cells / cm 2 , at least about 180,000 cells / cm 2 , at least about 190,000 cells / cm 2 , at least about 200,000 cells / cm 2 , at least about 210,000 cells / cm 2 , at least about 220,000 cells / cm 2 , at least about 240,000 cells / cm 2, at least about 260,000 cells / cm 2 , at least about 280,000 cells / cm 2 , at least about 300,000 cells / cm 2 , at least about 340,000 cells / cm 2 , at least about 360,000 cells / cm 2 , at least about 380,000 cells / cm 2 , at least about 400,000 cells / cm 2 , at least about 450,000 cells / cm 2 , or at least about 500,000 cells / cm 2 In some cases, the host cells may be seeded at a density of about 2,500 cells / cm. 2 In some cases, the host cells prior to inoculation with the virus may be seeded at a density of about 1,500 cells / cm. 2 , approximately 1,600 cells / cm 2 , approximately 1,800 cells / cm 2 , about 2,200 cells / cm 2 , about 2,200 cells / cm 2 , approximately 2,400 cells / cm 2 , approximately 2,600 cells / cm 2 , about 2,800 cells / cm 2 , about 3,000 cells / cm 2 , about 3,500 cells / cm 2 , about 4,000 cells / cm 2 , approximately 4,500 cells / cm 2 , about 5,000 cells / cm 2 , about 6,000 cells / cm 2 , about 7,000 cells / cm 2 , about 8,000 cells / cm 2 , about 9,000 cells / cm 2 , about 10,000 cells / cm 2 , about 15,000 cells / cm 2 , about 20,000 cells / cm 2 , about 30,000 cells / cm 2 , about 40,000 cells / cm 2 , about 50,000 cells / cm 2 , about 60,000 cells / cm 2 , about 70,000 cells / cm2 , about 80,000 cells / cm 2 , about 90,000 cells / cm 2 , about 100,000 cells / cm 2 , about 110,000 cells / cm 2 , about 120,000 cells / cm 2 , about 130,000 cells / cm 2 , about 140,000 cells / cm 2 , about 150,000 cells / cm 2 , about 160,000 cells / cm 2 , about 170,000 cells / cm 2 , about 180,000 cells / cm 2 , about 190,000 cells / cm 2 , about 200,000 cells / cm 2 , about 210,000 cells / cm 2 , about 220,000 cells / cm 2 , about 240,000 cells / cm 2 , about 260,000 cells / cm 2 , about 280,000 cells / cm 2 , about 300,000 cells / cm 2 , about 340,000 cells / cm 2 , about 360,000 cells / cm 2 , about 380,000 cells / cm 2 , about 400,000 cells / cm 2 , about 450,000 cells / cm 2 , or approximately 500,000 cells / cm 2 The seeds may be sown at a density of 100-200 μg / ml.

[0083] In some cases, the methods described herein may include growing the host cells in a bioreactor for a determined period of time, and then infecting the host cells at a determined density in the bioreactor. In some cases, the host cells may be grown in the bioreactor for about 5 days to about 15 days, such as about 5 days to about 8 days, about 7 days to about 10 days, about 8 days to about 12 days, or about 10 days to about 15 days. In some cases, the host cells may be grown in the bioreactor for about 5, 6, 7, 8, 9, or 10 days.

[0084] In some aspects, the present disclosure provides a bioreactor that includes a culture of host cells that includes a large number of viruses. In some cases, the bioreactor may be a bioreactor that can be used to culture virus-infected host cells according to the methods provided herein. An exemplary bioreactor may include a culture of host cells that includes at least about 50 PFU to about 350 PFU of recombinant oncolytic virus per host cell. Another exemplary bioreactor can include at least about 1.5×10 11 From about 5 × 10 13 The bioreactor may comprise a culture of host cells containing PFU of the recombinant oncolytic virus. In some cases, the bioreactor may comprise a fixed bed, and the fixed bed may comprise a culture of host cells. In some cases, the culture of host cells may be grown in microcarriers within the fixed bed. In some cases, the culture may be at about 2,000 cells / cm. 2 to approximately 10,000 cells / cm 2 In some cases, the fixed bed may comprise a volume of about 0.01 L to about 25 L. In some cases, the fixed bed may comprise a packed density of at least about 80 g / L to about 144 g / L. In some cases, the fixed bed may comprise a packed density of at least about 0.2 m 2 Approximately 500m from 2 , for example, about 0.2 m 2 About 0.5m from 2 , about 0.4m 2 About 1m from 2 , approx. 0.8m 2 About 1.5m from 2 , about 1m 2 Approximately 5m from 2 , about 4m 2 Approximately 6m from 2 , about 5m 2 Approximately 20m from 2 , about 20m 2 About 50m from 2 , about 50m 2 About 100m from 2 , about 100m 2 Approximately 200m from 2 , about 200m2 Approximately 400m from 2 , about 250m 2 Approximately 500m from 2 In some cases, the fixed bed may comprise a surface area of ​​about 4 m 2 In some cases, the fixed bed may comprise a surface area of ​​about 500 m 2 Without wishing to be bound by any particular theory, the scale of virus production may depend on the surface area of ​​the fixed bed used in the methods described herein. For example, a surface area of ​​about 4 m 2 When virus-infected cells were cultured using a fixed bed with a surface area of ​​4 × 10 11 From 1×10 12 PFU of virus can be produced using the methods provided herein. In some cases, about 500 ml 2 When virus-infected cells were cultured using a fixed bed with a surface area of ​​1 × 10 13 From 2.5 × 10 14 PFU's of virus can be produced using the methods provided herein.

[0085] The method provided herein may include growing the culture for a certain period of time, during which the virus can replicate.The method provided herein may include growing the culture for a sufficient period of time to reach a desired virus titer in the culture.In some cases, the culture may be grown for about 24 hours to about 96 hours. In some cases, the culture may be grown for about 20 hours, about 22 hours, about 24 hours, about 26 hours, about 28 hours, about 30 hours, about 32 hours, about 34 hours, about 36 hours, about 38 hours, about 40 hours, about 42 hours, about 44 hours, about 46 hours, about 48 hours, about 50 hours, about 52 hours, about 54 hours, about 56 hours, about 58 hours, about 60 hours, about 62 hours, about 64 hours, about 66 hours, about 68 hours, about 70 hours, about 72 hours, about 74 hours, about 76 hours, about 78 hours, about 80 hours, about 82 hours, about 84 hours, about 86 hours, about 88 hours, about 90 hours, about 92 hours, about 94 hours, about 96 hours, about 100 hours, about 120 hours, about 150 hours, about 160 hours, or about 180 hours. In some cases, the culture may be grown for about 72 hours. In some cases, the culture may be grown for at least about 20 hours, at least about 24 hours, at least about 28 hours, at least about 30 hours, at least about 32 hours, at least about 36 hours, at least about 40 hours, at least about 42 hours, at least about 44 hours, at least about 46 hours, at least about 48 hours, at least about 52 hours, at least about 56 hours, at least about 60 hours, at least about 62 hours, at least about 64 hours, at least about 66 hours, at least about 68 hours, at least about 70 hours, at least about 72 hours, at least about 76 hours, at least about 80 hours, at least about 84 hours, at least about 88 hours, at least about 92 hours, at least about 94 hours, at least about 96 hours, at least about 100 hours, or at least about 120 hours.In some cases, the culture may be grown for at most about 24 hours, at most about 28 hours, at most about 32 hours, at most about 36 hours, at most about 40 hours, at most about 44 hours, at most about 48 hours, at most about 52 hours, at most about 56 hours, at most about 60 hours, at most about 64 hours, at most about 68 hours, at most about 72 hours, at most about 76 hours, at most about 80 hours, at most about 84 hours, at most about 88 hours, at most about 92 hours, at most about 96 hours, at most about 100 hours, at most about 120 hours, at most about 150 hours, at most about 160 hours, or at most about 180 hours. In some cases, the culture may be grown for about 3 days.

[0086] The methods provided herein may include growing the culture at a dissolved oxygen tension (DOT) level of at least about 20% to about 100%. In some cases, the culture may include a DOT level of at least about 20%, at least about 30%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%. In some cases, the culture may comprise a DOT level of about 20%, about 30%, about 40%, about 42%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 65%, about 70%, about 80%, about 90%, about 95%, or about 100%. In some cases, the culture may comprise a DOT level of about 50%. In some cases, the culture may contain a DOT level of at most about 25%, at most about 30%, at most about 40%, at most about 50%, at most about 60%, at most about 70%, at most about 80%, at most about 90%, at most about 95%, or at most about 100%. In some cases, the culture may contain a DOT level of about 50%.

[0087] The methods provided herein may include growing a culture comprising virus-infected host cells at a temperature of about 32° C. to about 38° C. The methods provided herein may include growing a culture comprising virus-infected host cells at a maintained temperature. In some cases, the maintained temperature may be about 32° C., about 33° C., about 34° C., about 35° C., about 36° C., about 36.2° C., about 36.4° C., about 36.6° C., about 36.8° C., about 37° C., about 37.2° C., about 37.4° C., about 37.6° C., about 37.8° C., or about 38° C. In some cases, the temperature of the culture may be changed from time to time, for example, at a first maintained temperature for a first period and at a second maintained temperature for a second period, depending on the life cycle of the virus, the growth stage of the host cells, or both. In some cases, the maintained temperature can be controlled very precisely, for example, with a variation of less than ±1.5°C, less than ±1.2°C, less than ±1.0°C, less than ±0.9°C, less than ±0.8°C, less than ±0.7°C, less than ±0.6°C, less than ±0.5°C, less than ±0.4°C, less than ±0.3°C, less than ±0.2°C, less than ±0.1°C, or less than ±0.05°C. The precision with which the temperature of the culture can be maintained may be adjusted depending on a number of factors, such as the life cycle of the virus, the temperature sensitivity of the virus and host cells, and the culture medium. In some cases, the culture may be maintained at about 37°C.

[0088] In some cases, the method provided herein may include growing the culture comprising virus-infected host cells at a pH of about 7.0 to about 7.5. In some cases, the pH of the culture may be maintained at a certain level, for example, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, or about 8.0. In some cases, the maintained pH may be at least about 8.0, or up to about 7.0, depending on the type of host cell and virus. In some cases, the pH of the culture may be maintained at about 7.2. In some cases, the method may include growing the culture at a pH maintained with a fluctuation of less than about 1.2, less than about 1.1, less than about 1.0, less than about 0.9, less than about 0.8, less than about 0.7, less than about 0.6, less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.2, less than about 0.1, less than about 0.08, less than about 0.06, less than about 0.05, less than about 0.02, or less than about 0.01. During cell culture, the pH of the culture containing the virus-infected host cells may be adjusted by replacing the medium with fresh medium that can be sufficiently pH adjusted. The pH of the culture may be maintained by a pH buffer. In some cases, the pH may be adjusted to a desired level by controlling CO2 gas or by supplementing the culture medium with acid or alkali, such as adding HCl to reduce the pH or adding NaOH to increase the pH. In some embodiments, the pH may also be modified by adding CO2.

[0089] Virus recovery

[0090] Some aspects of the present disclosure provide a method for recovering a virus, such as an oncolytic virus, such as a recombinant oncolytic virus, from a culture comprising a plurality of virus-infected host cells. Recovering the virus may include lysing the virus-infected host cells, thereby releasing the virus into a lysate, and collecting the lysate. Recovering the virus may also include pre-harvesting washing of the bioreactor. In some cases, pre-harvesting washing may be performed to remove contaminants (e.g., proteins, nucleic acids, consumable products, and other molecules in the culture medium) from the lysate.

[0091] After culturing the virus-infected host cells for a certain period of time as described above, the bioreactor, e.g., the carrier, may be washed with a suitable washing solution in preparation for subsequent virus recovery. In some cases, the washing solution may be a buffer, e.g., a Tris buffer. In some cases, the washing solution may include 75 mM Tris and a pH of 7.3. The washing step may be performed at any suitable temperature and for any suitable period of time. In some cases, the parameters of the washing step, e.g., the washing solution (e.g., ion concentration, pH), temperature, washing period, may be sufficiently adjusted so that the host cells are hardly lysed in this step. In some cases, the carrier may be washed with 75 mM Tris solution (pH 7.3, 25°C) for 30 minutes.

[0092] In some embodiments, the present disclosure provides a pH shift method for recovering a virus from a culture. The recovering according to some embodiments of the present disclosure may include incubating the virus-infected host cells in an alkaline buffer. In some cases, the recovering may also include incubating the virus-infected host cells with an enzyme for degrading cell debris. In some cases, the recovering may include incubating the virus-infected host cells with an enzyme for degrading cell debris and an alkaline buffer. The alkaline buffer may include a pH of at least about 8. In some cases, the alkaline buffer may comprise a pH of at least about 8.0, at least about 8.2, at least about 8.4, at least about 8.5, at least about 8.6, at least about 8.8, at least about 9.0, at least about 9.1, at least about 9.2, at least about 9.3, at least about 9.4, at least about 9.5, at least about 9.6, at least about 9.7, at least about 9.8, at least about 9.9, at least about 10.0, at least about 10.4, at least about 10.8, or at least about 11.0. In some cases, the alkaline buffer may comprise a pH of about 8.0, about 8.2, about 8.4, about 8.5, about 8.6, about 8.8, about 8.9, about 9.0, about 9.1, about 9.2, about 9.3, about 9.4, about 9.5, about 9.6, about 9.7, about 9.8, about 9.9, about 10.0, about 10.2, about 10.4, about 10.6, about 10.8, or about 11.0. In some cases, the alkaline buffer may comprise Tris. In some cases, the alkaline buffer may comprise a pH of about 9.5. In some cases, the alkaline buffer may comprise a pH of about 9.6. In some cases, the alkaline buffer may have a pH determined with a variation of less than about 0.2, less than about 0.1, less than about 0.08, less than about 0.06, less than about 0.05, less than about 0.02, or less than about 0.01. In one example, the alkaline buffer may comprise 75 mM Tris, 2 mM MgCl2, and a pH of 9.5. In another example, the alkaline buffer may comprise 90 mM Tris, 2 mM MgCl2, and a pH of 9.5.In one example, the alkaline buffer may include 75 mM Tris, 2 mM MgCl2, and a pH of 9.6. In one example, the alkaline buffer may include 75 mM Tris, 2.5 mM MgCl2, and a pH of 9.6. Without wishing to be bound by any particular theory, the high pH of the lysis buffer may contribute to better lysis of the host cells, the host cell endosomes, or both, which may result in more readily exposed viruses that may have been "trapped" therein, resulting in a higher yield of viruses.

[0093] In some cases, enzymes can be used to degrade cell debris in the methods provided herein.The enzymes can include nucleases, such as endonucleases, for removing free nucleic acids in cell lysates.In some cases, enzymes can include proteases, such as TrypLE or trypsin.

[0094] Nucleases may refer to enzymes that can cleave phosphodiester bonds between nucleic acid monomers. Nucleases can produce single-stranded and double-stranded breaks in their target molecules. Different nucleases, such as exonucleases and endonucleases, can be used in the methods provided herein to act on different loci of the nucleic acid target. Exonucleases can digest nucleic acids from the ends, while endonucleases can act on the central region of the target molecule. Nucleases, as used herein, can be naturally occurring nucleases or recombinant (engineered) nucleases whose amino acid sequence is derived from a naturally occurring nuclease. For example, genetically engineered Serratia nuclease (e.g., Benzonase®) can be used for this purpose. In some cases, the method can include incubating virus-infected host cells with an enzyme for degrading cell debris, and the enzyme can be about 50 IU / mL to about 200 IU / mL. In some cases, during incubation for virus recovery, the enzyme for degrading cell debris may be about 20 IU / mL, about 30 IU / mL, about 50 IU / mL, about 75 IU / mL, about 100 IU / mL, about 125 IU / mL, about 130 IU / mL, about 140 IU / mL, about 150 IU / mL, about 160 IU / mL, about 170 IU / mL, about 180 IU / mL, about 190 IU / mL, about 200 IU / mL, about 220 IU / mL, about 240 IU / mL, about 260 IU / mL, about 280 IU / mL, about 300 IU / mL, or about 400 IU / mL.In some cases, during incubation for virus recovery, the enzyme for degrading cell debris may be at least about 20 IU / mL, at least about 30 IU / mL, at least about 50 IU / mL, at least about 75 IU / mL, at least about 100 IU / mL, at least about 125 IU / mL, at least about 130 IU / mL, at least about 140 IU / mL, at least about 150 IU / mL, at least about 160 IU / mL, at least about 170 IU / mL, at least about 180 IU / mL, at least about 190 IU / mL, at least about 200 IU / mL, at least about 220 IU / mL, or at least about 240 IU / mL. In some cases, during incubation for virus recovery, the enzyme for degrading cell debris may be at most about 50 IU / mL, at most about 75 IU / mL, at most about 100 IU / mL, at most about 125 IU / mL, at most about 130 IU / mL, at most about 140 IU / mL, at most about 150 IU / mL, at most about 160 IU / mL, at most about 170 IU / mL, at most about 180 IU / mL, at most about 190 IU / mL, at most about 200 IU / mL, at most about 220 IU / mL, at most about 240 IU / mL, at most about 260 IU / mL, at most about 280 IU / mL, at most about 300 IU / mL, or at most about 400 IU / mL. In some embodiments of the methods provided herein, recovering the virus can include incubating the virus-infected host cells with Benzonase® at about 150 IU / mL.

[0095] Incubation of virus-infected host cells in cell lysis buffer to recover virus may be continued for a suitable time. In some cases, incubation may be for about 2 hours to about 8 hours. In some cases, incubation may be for about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 15 hours, or about 20 hours. In some cases, incubation may be for at least about 2 hours to at least about 8 hours. In some cases, incubation may be for at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 12 hours, or at least about 15 hours. In some cases, incubation may be for about 2 hours to about 8 hours. In some cases, the incubation may be for at most about 2 hours, at most about 3 hours, at most about 4 hours, at most about 5 hours, at most about 6 hours, at most about 7 hours, at most about 8 hours, at most about 9 hours, at most about 10 hours, at most about 12 hours, at most about 15 hours, or at most about 20 hours. In some embodiments of the methods provided herein, harvesting the virus may include incubating the virus-infected host cells in a cell lysis buffer for about 4 hours.

[0096] Incubation of virus-infected host cells in cell lysis buffer to recover virus may be at any suitable temperature, for example, about 22°C to about 28°C. In some cases, the temperature for incubation to recover virus may be about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, or about 32°C. In some cases, the temperature may be maintained at a certain temperature. In some cases, the maintained temperature may be controlled very precisely, for example, with a variation of less than ±1.5°C, less than ±1.2°C, less than ±1.0°C, less than ±0.9°C, less than ±0.8°C, less than ±0.7°C, less than ±0.6°C, less than ±0.5°C, less than ±0.4°C, less than ±0.3°C, less than ±0.2°C, less than ±0.1°C, or less than ±0.05°C. In some embodiments of the methods provided herein, recovering the virus can include incubating the virus-infected host cells at about 25°C.

[0097] Clarification

[0098] In some cases, the methods provided herein may further include performing a clarification step to produce a filtered lysate. The clarification step may be performed to eliminate or reduce contaminants and purify the virus. In some cases, the clarification step provided herein may include filtration and nuclease treatment. In some cases, the clarification step may include purification of the virus by centrifugation.

[0099] The clarification step may include filtering. In some cases, the lysate collected by the method provided herein may be filtered through a filter to produce a filtered lysate. As provided herein, the filter may have a pore size that retains particles with a size larger than the pore size and allows particles with a size smaller than the pore size to pass through. Any suitable filter may be selected to perform the filtering step for the purpose of isolating and purifying viruses. In some cases, the filter may have an average filter size that may be at least about 0.15 μm, at least about 0.18 μm, at least about 0.2 μm, at least about 0.22 μm, at least about 0.25 μm, at least about 0.28 μm, at least about 0.3 μm, at least about 0.35 μm, at least about 0.4 μm, at least about 0.45 μm, at least about 0.5 μm, at least about 0.55 μm, at least about 0.6 μm, at least about 0.65 μm, at least about 0.7 μm, at least about 0.75 μm, at least about 0.8 μm, at least about 0.85 μm, at least about 0.9 μm, at least about 0.95 μm, or at least about 1 μm. In some cases, the filter may have an average filter size that may be at most about 0.18 μm, at most about 0.2 μm, at most about 0.22 μm, at most about 0.25 μm, at most about 0.28 μm, at most about 0.3 μm, at most about 0.35 μm, at most about 0.4 μm, at most about 0.45 μm, at most about 0.5 μm, at most about 0.55 μm, at most about 0.6 μm, at most about 0.65 μm, at most about 0.7 μm, at most about 0.75 μm, at most about 0.8 μm, at most about 0.85 μm, at most about 0.9 μm, at most about 0.95 μm, at most about 1 μm, or at most about 1.5 μm. In some cases, filtration may be performed through more than one filter, for example, 2, 3, 4, 5, 6 or more filters. In some cases, the more than one filter may have the same pore size or different sizes, for example, in some cases, the lysate may be filtered through a filter having an average size of about 5 μm, followed by a filter having an average size of about 1.2 μm.In some examples, the lysate may be filtered through a filter having an average size of about 5 μm, followed by a filter having an average size of about 2.4 μm, and then through a filter of about 1 μm.

[0100] As illustrated in FIG. 10, the clarification process may be performed by flushing the lysate through a filter with a flush buffer. In some cases, the flow rate for the clarification process may be about 50 to about 500 mL / min, e.g., about 50 to about 100 mL / min, about 75 to about 120 mL / min, about 100 to about 150 mL / min, about 125 to about 175 mL / min, about 150 to about 200 mL / min, about 175 to about 250 mL / min, about 225 to about 300 mL / min, about 250 to about 325 mL / min, about 275 to about 350 mL / min, about 300 to about 375 mL / min, about 325 to about 400 mL / min, about 350 to about 425 mL / min, about 375 to about 450 mL / min, about 400 to about 475 mL / min, or about 425 to about 500 mL / min. In some cases, the flow rate for the clarification process may be about 70, 80, 90, 100, 110, 120, 130, 150, 175, 200, 225, 250, 275, 280, 290, 300, or 320 mL / min. The flow rate is expressed in LMH (L / m 2 / hours) may be used. In some cases, the flow rate for the clarification process may be from about 100 to about 750 LMH, e.g., from about 100 to about 175 LMH, from about 125 to about 200 LMH, from about 150 to about 225 LMH, from about 175 to about 250 LMH, from about 200 to about 275 LMH, from about 225 to about 300 LMH, from about 250 to about 325 LMH, from about 275 to about 350 LMH, from about 300 to about 375 LMH, from about 325 to about 400 LMH, from about 350 to about 425 LMH, from about 375 to about 450 LMH, from about 400 to about 475 LMH, from about 425 to about 500 LMH, from about 450 to about 525 LMH, from about 475 to about 550 LMH, from about 500 to about 575 LMH, or from about 525 to about 600 LMH. In some cases, the flow rate for the clarification process may be about 150 LMH. In some cases, the flow rate for the clarification process may be about 490 LMH. In some cases, the flush rate may be selected separately for filters with different pore sizes, e.g., a higher flow rate may be used for filters with larger pore sizes, while a lower flow rate may be used for filters with smaller pore sizes. In some cases, the flush buffer may have similar components as the lysate, e.g., similar ions and ion concentrations, and similar pH, or different components than the lysate. In some cases, the clarification process may be performed at room temperature or any other suitable temperature, e.g., about 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 28°C, 30°C, 32°C, or 37°C.

[0101] After the filtration step, the filtered lysate may be further treated with an enzyme to degrade cell debris, e.g., a nuclease, e.g., Benzonase®, to eliminate contaminants, e.g., nucleic acids, to generate a depth filtration filtrate. In some cases, the filtered lysate may be incubated with a buffer containing Benzonase® for a sufficient period of time. In some cases, the filtered lysate may be incubated with a buffer containing at least about 10 U / ml, at least about 20 U / ml, at least about 40 U / ml, at least about 50 U / ml, at least about 60 U / ml, at least about 80 U / ml, at least about 100 U / ml, at least about 120 U / ml, at least about 130 U / ml, at least about 140 U / ml, at least about 150 U / ml, at least about 160 U / ml, at least about 170 U / ml, at least about 180 U / ml, at least about 200 U / ml, at least about 250 U / ml, at least about 300 U / ml, at least about 500 U / ml, or at least about 1000 U / ml of Benzonase®. In some cases, treatment of the filtered lysate with an enzyme to degrade cell debris may be carried out at a pH of at least about 8.0, at least about 8.1, at least about 8.2, at least about 8.3, at least about 8.4, at least about 8.5, at least about 8.6, at least about 8.7, at least about 8.8, at least about 8.9, at least about 9.0, at least about 9.1, at least about 9.2, at least about 9.3, at least about 9.4, at least about 9.5, at least about 9.6, at least about 9.7, at least about 9.8, or at least about 10.0. In some cases, treatment of the filtered lysate with an enzyme to degrade cell debris may be carried out at a pH of about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9.0, about 9.1, about 9.2, about 9.3, about 9.4, about 9.5, about 9.6, about 9.7, about 9.8, or about 10.0.In some cases, treatment of the filtered lysate with an enzyme to degrade cell debris may be performed at a pH of about 7.5 to about 9.5, about 7.5 to about 10.0, about 8.0 to about 10.0, about 8.5 to about 10.0, about 8.5 to about 10.5, about 9.0 to about 10.5, or about 9.0 to about 11.0. In some cases, the filtered lysate may be treated with an enzyme to degrade cell debris for at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 6 hours, at least about 8 hours, at least about 10 hours, at least about 12 hours, at least about 14 hours, at least about 16 hours, at least about 18 hours, at least about 20 hours, at least about 22 hours, at least about 24 hours, at least about 26 hours, at least about 28 hours, at least about 30 hours, at least about 34 hours, at least about 36 hours, or at least about 48 hours. In some cases, the filtered lysate may be treated with an enzyme to degrade cell debris for about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 22 hours, about 24 hours, about 26 hours, about 28 hours, about 30 hours, about 34 hours, about 36 hours, or about 48 hours. In some cases, the filtered lysate may be treated with an enzyme to degrade cell debris for at most about 3 hours, at most about 5 hours, at most about 7 hours, at most about 9 hours, at most about 11 hours, at most about 13 hours, at most about 15 hours, at most about 17 hours, at most about 19 hours, at most about 21 hours, at most about 23 hours, at most about 25 hours, at most about 27 hours, at most about 29 hours, at most about 31 hours, at most about 35 hours, at most about 40 hours, or at most about 50 hours. In some cases, the filtered lysate may be treated with Benzonase® at room temperature for about 18 hours. In some cases, the enzyme treatment is carried out by removing monocations, such as Na. + For example, the enzyme treatment may be carried out in the presence of sodium chloride (NaCl) in solution. In some cases, the enzyme treatment may be carried out in the presence of a high concentration of monocations, for example Na +For example, the enzyme treatment may be carried out in the presence of sodium chloride (NaCl) in the solution, e.g., at least about 100 mM, at least about 120 mM, at least about 150 mM, at least about 180 mM, at least about 200 mM, at least about 250 mM, at least about 300 mM, at least about 350 mM, at least about 400 mM, at least about 450 mM, at least about 500 mM, at least about 550 mM, at least about 600 mM, at least about 700 mM, at least about 800 mM, at least about 900 mM, or at least about 1000 mM. + , for example, in the presence of sodium chloride (NaCl) in solution, for example at about 100 mM, about 120 mM, about 150 mM, about 180 mM, about 200 mM, about 250 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM, about 500 mM, about 550 mM, about 600 mM, about 700 mM, about 800 mM, about 900 mM, or about 1000 mM.

[0102] In some cases, according to some embodiments of the method provided herein, both unfiltered and filtered viral lysates can be stored at low temperature before the next step.In some cases, filtered lysates can be stored at low temperature before being treated with enzymes to degrade cell debris.For example, filtered lysates can be stored at about 2 to 8°C for a certain period of time, for example, 6 hours, 12 hours, 24 hours, 2 days, 3 days, or 6 days.

[0103] purification

[0104] In some cases, the methods provided herein may include performing tangential flow filtration (TFF) of the filtered lysate to produce a purified preparation of the recombinant oncolytic virus. In some cases, the tangential flow filtration may be followed by diafiltration to further purify the virus. In some cases, more than one round of TFF or diafiltration may be performed. In some cases, two rounds of diafiltration may be performed. In some cases, two, three, four, five or more rounds of TFF or diafiltration may be performed. In other cases, other techniques, such as ultrafiltration or size exclusion chromatography (e.g., using Sephadex® G25 or Sephadex® G10 or equivalent materials), ion exchange chromatography, affinity chromatography, size exclusion chromatography or "reverse phase" chromatography may be used in the subject method for purifying the virus as an alternative or in combination with the TFF process described herein.

[0105] Tangential flow filtration (TFF), also known as cross-flow filtration, may refer to a filtration process in which a feed stream passes parallel to the filter membrane surface while a portion (permeate) passes through the membrane, while the remainder (retentate) can be recycled to the feed reservoir. As provided herein, TFF may be performed to concentrate the filtered lysate. In some cases, after TFF, the filtered lysate may be concentrated at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 12-fold, at least 15-fold, at least 18-fold, at least 20-fold, or at least 30-fold. In some cases, after TFF, the filtered lysate may be concentrated about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 12-fold, about 15-fold, about 18-fold, about 20-fold, or about 30-fold. In some cases, after TFF, the filtered lysate may be concentrated about 10-fold.

[0106] In some cases, the TFF filter membrane may have a pore size of about 300 kDa, about 350 kDa, about 400 kDa, about 450 kDa, about 500 kDa, about 550 kDa, about 600 kDa, about 650 kDa, about 700 kDa, about 750 kDa, about 800 kDa, about 850 kDa, about 900 kDa, or about 1000 kDa. In some cases, the TFF filter membrane may have a pore size of at most about 300 kDa, at most about 350 kDa, at most about 400 kDa, at most about 450 kDa, at most about 500 kDa, at most about 550 kDa, at most about 600 kDa, at most about 650 kDa, at most about 700 kDa, at most about 750 kDa, at most about 800 kDa, at most about 850 kDa, at most about 900 kDa, or at most about 1000 kDa.

[0107] The appropriate TFF filter and filtration parameters may be selected and adjusted depending on a number of parameters, including, but not limited to, the type of virus to be produced, the type of host cell, the cell culture medium, and the filter buffer. In some cases, the TFF process may be carried out at any suitable temperature, for example, at about 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., 28° C., 30° C., 32° C., or 37° C. In some cases, the load for the TFF is at least about 10 L / m 2 , at least about 20 L / m 2 , at least about 30 L / m 2 , at least about 40 L / m 2 , at least about 50 L / m 2 , at least about 60 L / m 2 , at least about 70 L / m 2 , at least about 80 L / m 2 , at least about 100 L / m 2 , at least about 120 L / m 2 , at least about 150 L / m 2 , or at least about 200 L / m 2 In some cases, the load for the TFF may be about 10 L / m 2 , about 20L / m 2 , about 30L / m 2 , about 40L / m2 , about 50L / m 2 , about 60L / m 2 , about 70L / m 2 , about 80L / m 2 , about 100L / m 2 , about 120L / m 2 , about 150L / m 2 , or about 200 L / m 2 In some cases, the TFF process may be performed for about 2000 to about 6000 s -1 , for example, from about 2000 to about 3500s -1 , from about 2500 to about 4000s -1 , from about 3000 to about 4500s -1 , from about 3000 to about 5000s -1 , from about 3500 to about 5000s -1 , from about 3500 to about 5500s -1 , from about 4000 to about 6000s -1 , or about 4500 to about 6000s -1 In some cases, the TFF process may be carried out at a shear rate of about 2000 s -1 , about 2500s -1 , about 3000s -1 , about 3500, about 3500s -1 , about 4000s -1 , about 4500s -1 , about 5000s -1 , about 5500s -1 , about 6000s -1 In some cases, during the TFF process, the lysate may be flowed at a rate of about 0.25 to about 2.5 L / min, e.g., about 0.25 to about 0.75 L / min, about 0.5 to about 1 L / min, about 0.75 to about 1.25 L / min, about 1 to about 1.5 L / min, about 1.25 to about 1.75 L / min, about 1.5 to about 2.0 L / min, about 1.75 to about 2.25 L / min, or about 2 to about 2.5 L / min. In some cases, the flow rate and shear rate may be selected in combination, e.g., the lysate may be flowed at a rate of about 1.5 L / min, 5000 s -1 or about 0.8 L / min for 3000 s -1The shear rate may be controlled throughout the TFF process. In some cases, the permeate flux rate may be controlled between 5 and about 50 LMH, for example, between 5 and about 15 LMH, between 10 and about 20 LMH, between 15 and about 25 LMH, between 20 and about 30 LMH, between 25 and about 35 LMH, between 30 and about 40 LMH, between 35 and about 45 LMH, or between 40 and about 50 LMH. In some cases, the permeate flux rate may be controlled between about 15 and about 20 LMH. In some cases, the permeate flux rate may be controlled between about 10 and about 30 LMH. In some cases, the permeate flux rate may be controlled between about 15 and about 30 LMH. In some cases, the permeate flux rate may be controlled at about 15 LMH. In some cases, the permeate flux rate may be controlled at about 20 LMH.

[0108] In some cases, the method may further include diafiltration of the filtered lysate or purified lysate. In some cases, diafiltration may be performed to remove salts or other contaminants from the lysate to produce a purified virus preparation. As provided herein, diafiltration can be a fractionation process that washes small molecules through a membrane and leaves larger molecules in the retentate, ultimately leaving the concentration unchanged.

[0109] Either continuous or discontinuous diafiltration may be used in the methods provided herein. In continuous diafiltration, diafiltration buffer may be added to the feed reservoir at the same rate that filtrate can be produced. In this way, the volume in the sample reservoir can be kept constant, but small molecules (e.g., salts) that can freely pass through the membrane can be washed out. As a result, each additional volume of buffer exchange may further reduce the salt concentration. As provided herein, the "volume of buffer exchange" may be equal to the volume of the starting solution before diafiltration solution can be added. In discontinuous diafiltration, the starting solution may be first diluted and then concentrated back to the starting volume. This process may then be repeated until the small molecules (e.g., salts) remaining in the reservoir reach a desired concentration. In some cases, for the initial concentration process, the diafiltration process may be performed using the same TFF system. In some cases, the diafiltration process may be performed using a different system. As provided herein, diafiltration may include adding at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 14, at least 15, at least 16, at least 18, at least 20, at least 21, at least 22, at least 23, at least 25, or at least 30 volumes of buffer exchange. In some cases, diafiltration provided herein may include 6 volumes of buffer exchange. In some cases, diafiltration provided herein may include 10 volumes of buffer exchange. In some cases, diafiltration provided herein may include 20 volumes of buffer exchange. In some cases, the method provided herein may include 2 rounds of diafiltration, with 1 round with 10 volumes of buffer exchange, followed by 2 rounds with 6 volumes of buffer exchange, or 1 round with 20 volumes of buffer exchange, followed by 2 rounds with 10 volumes of buffer exchange.In some cases, the first round of buffer exchange can be performed with a buffer having the same components as the lysate, while the second round of buffer exchange can be performed with a buffer used to filter out ions contained in the lysate, such as a buffer containing sucrose. In some cases, the buffer exchange can be performed with a buffer that comprises a pH of about 7.8. In some cases, the buffer exchange can be performed with a buffer that comprises a pH of about 7.0. In some cases, the buffer exchange can be performed with a buffer that comprises a pH of about 7.0, about 7.1, about 7.2, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 9.0, or about 10.0. In some cases, the buffer exchange may be performed using a buffer that comprises a pH of at least about 7.0, at least about 7.1, at least about 7.2, at least about 7.4, at least about 7.5, at least about 7.6, at least about 7.7, at least about 7.8, at least about 7.9, at least about 8.0, at least about 9.0, or at least about 10.0. In some cases, the buffer exchange may be performed using a buffer that comprises a pH of about 7 or about 7.8. In some cases, the buffer exchange may be performed using a Tris buffer. The Tris buffer may comprise about 5 mM, about 10 mM, about 12 mM, about 15 mM, about 20 mM, about 22 mM, about 24 mM, about 25 mM, about 26 mM, about 28 mM, about 30 mM, about 35 mM, or about 40 mM Tris. In some cases, the buffer exchange may be performed using a buffer that comprises about 30 mM or about 20 mM Tris. In some cases, the buffer exchange may be performed using a buffer. Water may be the solvent of the buffer. The buffer may contain uncharged molecules such as saccharides, such as sucrose or glucose, histidine, sorbitol. In some cases, the buffer may contain ionic components such as MgCl2 or NaCl, or other stabilizing molecules. In some cases, the buffer for diafiltration may contain about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, about 10%, about 10.5%, about 11%, about 11.5%, about 12%, about 13%, about 14%, about 15%, about 20%, or about 30% sucrose.In some cases, buffer exchange may be performed with a buffer containing about 10% w / v or about 8.5% w / v sucrose. In some cases, additional rounds of buffer exchange may be performed to achieve the desired concentration. For example, the target concentration may be 0.5x, 1x, 1.25x, 1.5x, 1.75x, 2x, 2.25x, 2.5x, 2.75x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 11x, 12x, 13x, 14x, 15x, 16x, 17x, 18x, 19x, or up to about 20x.

[0110] In some cases, additional clarification or filtration steps may be performed depending on the need for purity, concentration, or titer of the virus product. In some cases, for example, a final filtration step may be performed, and the TFF retentate may be further filtered through a filter using a final formulation buffer. The formulation buffer may include ionic components, for example, as required for the final virus product. Any suitable flow parameters may be selected as described above. A relatively small pore size filter, for example 1.2 μm, may be used in the final step. Filters with various pore sizes may be utilized in any embodiment of the present disclosure. In some embodiments, the filter may have a pore size of about 0.1 μm, 0.2 μm, 0.3 μm, 0.4 μm, 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1.0 μm, 1.1 μm, 1.2 μm, 1.3 μm, 1.4 μm, 1.5 μm, 1.6 μm, 1.7 μm, 1.8 μm, 1.9 μm, 2.0 μm, 2.5 μm, 3.0 μm, 4.0 μm, or up to about 5.0 μm.

[0111] EXAMPLES

[0112] The following examples are offered by way of illustration and not by way of limitation.

[0113] Example 1 Upstream Process (I)

[0114] In this example, an exemplary protocol for the upstream process for the production of recombinant vaccinia virus, from the culture of virus-infected host cells, to harvesting and cell lysis, is described, as illustrated in the flow chart of FIG.

[0115] 1. Host Hela cells are cultured in iCELLis nanobioreactors (Pall Corporation, Port Washington, NY) for 5 days at 37° C. The pH of the culture medium is maintained at 7.1, and the dissolved oxygen tension (DOT) level of the culture medium is 50%;

[0116] 2. On day 6, the culture medium was replaced with the culture medium containing the recombinant vaccinia virus at a multiplicity of infection (moi) of 0.02. After infection, the virus-infected HeLa cells were continued to be cultured at 37°C, pH 7.2, 50% DOT for 3 days;

[0117] 3. On day 9, the bioreactor is pre-washed and then harvested, and the culture medium is replaced with a wash buffer containing 90 mM Tris and having a pH of 7.5 for 0.5 h at 25°C;

[0118] 4. After pre-washing, the wash buffer is replaced with lysis medium containing 90 mM Tris, 2 mM MgCl2, and 150 iU / mL Benzonase®, pH 9.5. Virus-infected cells are incubated with the lysis medium at 25°C for 4-6 hours, after which the lysis medium containing the cell lysate is collected for further processing.

[0119] Example 2 Upstream Process (II)

[0120] In this example, an exemplary protocol for the upstream process for the production of recombinant vaccinia virus, from the culture of virus-infected host cells, to harvesting and cell lysis, is described, as illustrated in the flow chart of FIG.

[0121] 1. Host Hela cells are cultured in iCELLis nanobioreactors (Pall Corporation, Port Washington, NY) for 7 days at 37° C. The pH of the culture medium is maintained at 7.2, and the dissolved oxygen tension (DOT) level of the culture medium is 50%;

[0122] 2. On day 8, the culture medium was replaced with the culture medium containing the recombinant vaccinia virus at a multiplicity of infection (moi) of 0.02. After infection, the virus-infected HeLa cells were continued to be cultured at 37°C, pH 7.2, 50% DOT for 3 days;

[0123] 3. On day 11, the bioreactor is pre-washed and then harvested, and the culture medium is replaced with wash buffer containing 75 mM Tris and having a pH of 7.3 for 0.5 h at 25°C;

[0124] 4. After pre-washing, the wash buffer is replaced with lysis medium containing 75 mM Tris, 2 mM MgCl2, and 150 iU / mL Benzonase®, with a pH of 9.5. Virus-infected cells are incubated with the lysis medium at 25° C. for 4 hours, after which the lysis medium containing the cell lysate is collected for further processing.

[0125] Example 3 Upstream Process (III)

[0126] In this example, an exemplary protocol for the upstream process for the production of recombinant vaccinia virus, from the culture of virus-infected host cells, to harvesting and cell lysis, is described, as illustrated in the flow chart of FIG.

[0127] 1. Host Hela cells are cultured in iCELLis nanobioreactors (Pall Corporation, Port Washington, NY) for 7 days at 37° C. The pH of the culture medium is maintained at 7.2, and the dissolved oxygen tension (DOT) level of the culture medium is 50%;

[0128] 2. On day 8, the culture medium was replaced with the culture medium containing the recombinant vaccinia virus at a multiplicity of infection (moi) of 0.002. After infection, the virus-infected HeLa cells were continued to be cultured at 36°C, pH 7.2, 50% DOT for 3 days;

[0129] 3. On day 11, the bioreactor is pre-washed and then harvested, and the culture medium is replaced with wash buffer containing 75 mM Tris, pH 7.3, for 10 min at 26°C;

[0130] 4. After pre-washing, the wash buffer is replaced with lysis medium containing 75 mM Tris, 2 mM MgCl2, and 150 iU / mL Benzonase®, with a pH of 9.5. Virus-infected cells are incubated with the lysis medium at 25° C. for 4 hours, after which the lysis medium containing the cell lysate is collected for further processing.

[0131] Example 4 Downstream Process (I)

[0132] In this example, an exemplary protocol for downstream processing to produce an exemplary vaccinia virus is described, as diagrammed in FIG.

[0133] The virus-infected host HeLa cells are cultured in the iCELLis nanobioreactor for 3 days before the virus is harvested.

[0134] 1. Collection of control samples: As a control, sample 10 carriers from the reactor bed and hydrate the samples with 15 mM Tris, pH 8.0 (0.05 ml / cm 2 ) lysis buffer for 30 minutes (volume of lysis buffer: 7 ml).

[0135] 2. Pre-wash the culture medium by draining it, stop the pump and remove all medium following the procedure:

[0136] (a) Leave the medium flowing out of the loop and allow it to flow in a counterclockwise direction until the medium is unprimed;

[0137] (b) Stop the medium;

[0138] (c) Discard the medium in the recirculation bottle (open and pour out);

[0139] (d) Stop adjusting;

[0140] (e) Close all clamps;

[0141] (f) Open the flow path (two clamps) from the sample bottle and open the gas exhaust clamp;

[0142] (g) Use a large syringe (or a hand pump) in the gas outlet line to pressure pump the medium into a sampling bottle.

[0143] 3. Wash the floor.

[0144] (a) Unclamp the recirculation loop;

[0145] (b) adding 800 ml of a 25° C. wash buffer containing 90 mM Tris and having a pH of 7.5±0.2 through the sample bottle at a linear flow rate of 1 cm / sec at 25° C. for 0.5 hours;

[0146] (c) After incubation, the wash buffer is discarded.

[0147] 4. During cleaning, collect samples and measure pH offline. Adjust pH if necessary.

[0148] 5. Immediately prior to removing the wash buffer from the bioreactor, collect another sample and read the offline pH to ensure that the iCELLis online pH is reading accurately.

[0149] 6. Set the bioreactor pH control to 9.5±0.2.

[0150] 7. Set the bioreactor temperature control to 25°C.

[0151] Add 600 ml of lysis buffer containing 8.90 mM Tris and 150 U Benzonase® / ml, having a pH of 9.0±0.2, and add 2.4 ml of 0.5 M MgCl2.

[0152] 9. Wait for the pH to drop and stabilize.

[0153] 10. Take an offline pH measurement and adjust the iCELLis offset if necessary:

[0154] The pH control is turned on (set point 9.0±0.2) and the linear flow rate is increased to 1.25 cm / sec for 4.0 hours at 25° C.

[0155] 11. The dissolution time begins when the bioreactor indicates that the pH has reached 9.0. Take another offline pH reading to verify that the reading is accurate.

[0156] 12. Two hours after dissolution has begun, samples are taken.

[0157] 13. Four hours after lysis begins, drain the bioreactor.

[0158] 14. Measure pH and adjust if necessary (if pH is too high, use 1M HCL). Record if adjusted.

[0159] Clarification

[0160] 1. Set up the filtration system as shown in FIG.

[0161] 2. Prime the Sartopure PP3 (5.0 μm / 1.2 μm) filter by flushing the filter with 750 mL of Priming Buffer (75 mM Tris, pH 9, 2 mM MgCl2) by using a peristaltic pump at 250 ml / min:

[0162] (a) Open the air ports of both filters, flush until buffer is out, then close until fully primed;

[0163] (b) Pump until all the priming buffer is gone and reopen the air port (no more liquid should come out).

[0164] 3. Filter the entire lysate by pumping it through the filter using a peristaltic pump at 250 ml / min.

[0165] Start pumping the viral lysate slowly until liquid comes out of the side air port, then remove the cap and wipe with ethanol;

[0166] (d) Pump the entire lysate (approximately 600 ml) through the filter at a rate of 250 ml / min.

[0167] (e) Finally, pump through (open) the air port until only air comes out;

[0168] (f) Separate the container and collect a sample.

[0169] 4. Flush the filter with an equal volume of flush buffer (75 mM Tris, pH 9, 2 mM MgCl2) into a separate vessel at 250 ml / min and sample the flush buffer.

[0170] 5. Pool the filtered lysate and flush buffer and collect a separate sample.

[0171] 6. Immediately adjust the pH of the pooled lysates from step 4 to 9 using a small amount of 1 M HCl.

[0172] 7. Store the pooled filtrate at pH 9.0 at 2-8° C. (over the weekend if necessary): if storing over the weekend, take 6×250 μl samples before proceeding to the next step.

[0173] 8. Further dissolve to produce depth filtrate: add Benzonase® 150 iU / ml (720 μl) and enough 2M NaCl (90 mL) to reach 500 mM NaCl and pH 9.0 and incubate at room temperature overnight for 18±4 hours.

[0174] TFF and diafiltration

[0175] 1. Set up a sterile 750 kD mPES (modified polyethersulfone), 790 cm2 hollow fiber filter (Spectrum) in a KrosFlo® TFF system.

[0176] 2. Pre-flush the TFF system with pre-flush buffer (75 mM Tris, pH 9, 2 mM MgCl2) until everything is primed. Flush out the 60-70 ml hold-up volume in the system and discard the pre-flush buffer.

[0177] 3. Place the depth filtration filtrate on the balance and connect the tubing to the inlet and outlet lines of the TFF system.

[0178] 4. Recirculate the sample for 10 minutes until the shear reaches approximately 4000-5000 s -1 Ensure that it is.

[0179] 5. Run the depth filtration filtrate through the filter membrane (aim to keep the shear rate at 5000 sec-1 and TMP (transmembrane pressure) around or below 1.2 psid) and slowly open the permeate line until the cross flow is below 30 LMH (L / m2 / hr). Run until the sample is concentrated about 10 times. The final volume should be about 100-120 ml in the reservoir bottle.

[0180] 7. Open the VFB line and start the first round of diafiltration (virus formulation buffer "VFB", pH 7) by maintaining a constant weight in the sample container (shear rate 5000 sec -1 , aiming for a maximum of 25-30 LMH, approximately 960 ml / min), and perform a 10-fold buffer exchange with VFB at pH 7.

[0181] 8. Open the VFB line and begin the final round of diafiltration (20 mM Tris, pH 7.8, 8.5% sucrose) by maintaining a constant weight in the sample container (shear rate 5000 sec-1, again aiming for a maximum of 25 LMH) and perform a 6-volume buffer exchange with 20 mM Tris, 8.5% sucrose, pH 8.

[0182] 9. Perform a final concentration step until only 15-30 mL is left in the reservoir, then flush the system until the retentate volume is added to the reservoir (60-70 ml, giving a final total of 75-100 ml).

[0183] 10. Store the final retentate in approximately 5 ml aliquots in 50 ml conical tubes at -80°C.

[0184] Example 5 Downstream Process (II)

[0185] In this example, an exemplary protocol for downstream processing to produce an exemplary vaccinia virus is described, as illustrated in Figure 8. Listed below are records obtained from running the protocol experiments.

[0186] Virus-infected host HeLa cells are cultured in the iCELLis nanobioreactor for 3 days before virus is harvested.

[0187] 1. Collection of control samples: As a control, 10 carriers were sampled from the reactor bed and the samples were diluted with 15 mM Tris, pH 8.0 (0.05 ml / cm 2 ) lysis buffer for 30 minutes (volume of lysis buffer: 7 ml).

[0188] 2. Pre-wash the culture medium by draining it, stop the pump and remove all medium following the procedure:

[0189] (a) Keep the medium out of the loop flow and allow the medium to flow in a counterclockwise direction until the prime is complete;

[0190] (b) Stop the medium;

[0191] (c) Discard the medium in the recirculation bottle (open and pour out);

[0192] (d) Stop adjusting;

[0193] (e) Close all clamps;

[0194] (f) Open the flow path (two clamps) from the sample bottle and open the gas exhaust clamp;

[0195] (g) Use a large syringe (or a hand pump) in the gas outlet line to pressure pump the medium into a sampling bottle.

[0196] 3. Wash the floor.

[0197] (a) Unclamp the recirculation loop;

[0198] (b) adding 800 ml of 25° C. wash buffer containing 75 mM Tris and having a pH of 7.3±0.2 through the sample bottle at a linear flow rate of 1 cm / sec at 25° C. for 0.5 hours;

[0199] (c) After incubation, the wash buffer is discarded.

[0200] 4. During cleaning, collect samples and measure pH offline. Adjust pH if necessary.

[0201] 5. Immediately prior to removing the wash buffer from the bioreactor, collect another sample and read the offline pH to ensure that the iCELLis online pH is reading accurately.

[0202] 6. Set the bioreactor pH control to 9.5±0.2.

[0203] 7. Set the bioreactor temperature control to 25°C.

[0204] Add 600 ml of lysis buffer containing 8.75 mM Tris and 150 U Benzonase® / ml, with a pH of 9.5±0.2, and add 2.4 ml of 0.5 M MgCl2.

[0205] 9. Wait for the pH to drop and stabilize.

[0206] 10. Take an offline pH measurement and adjust the iCELLis offset if necessary:

[0207] The pH control is turned on (set point 9.5±0.2) and the linear flow rate is increased to 1.25 cm / sec for 4.0 hours at 25° C.

[0208] 11. The dissolution time begins when the bioreactor indicates that the pH has reached 9.5. Take another offline pH reading to verify that the reading is accurate.

[0209] 12. Two hours after dissolution has begun, samples are taken.

[0210] 13. Four hours after lysis begins, drain the bioreactor.

[0211] 14. Measure pH and adjust if necessary (if pH is too high, use 1M HCL). Record if adjusted.

[0212] Clarification

[0213] 1. Set up a filter clarification system as depicted in FIG. 10.

[0214] 2. Prime the autoclaved pre-assembled Sartopure PP3 (5.0 μm / 1.2 μm) filters by flushing the filters with 750 mL of Priming Buffer (75 mM Tris, pH 9, 2 mM MgCl2) by using a peristaltic pump at 250 ml / min:

[0215] (a) Open the air ports of both filters, flush until buffer is out, then close until fully primed;

[0216] (b) Pump until all the priming buffer is gone and reopen the air port (no more liquid should come out).

[0217] 3. Filter the entire lysate by pumping it through the filter using a peristaltic pump at 250 ml / min.

[0218] (c) Start pumping the viral lysate slowly until liquid comes out of the side air port, then remove the cap and wipe with ethanol;

[0219] (d) Pump the entire lysate (approximately 600 ml) through the filter at a rate of 250 ml / min.

[0220] (e) Finally, pump through (open) the air port until only air comes out;

[0221] (f) Separate the container and collect a sample.

[0222] 4. Flush the filter with an equal volume of flush buffer (75 mM Tris, pH 9, 2 mM MgCl2) into a separate vessel at 250 ml / min and sample the flush buffer.

[0223] 5. Pool the filtered lysate and flush buffer and collect a separate sample.

[0224] 6. Immediately adjust the pH of the pooled lysates from step 4 to 9 using a small amount of 1 M HCl.

[0225] 7. Store the pooled filtrate at pH 9.0 at 2-8°C (for about 8 hours, about 10 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 22 hours, about 24 hours, about 26 hours, about 28 hours, about 30 hours, about 36 hours, about 40 hours, about 45 hours, about 50 hours, about 70 hours, or about 72 hours). If storing for an extended period of time, for example about 36 hours, take 6 x 250 μl of sample before proceeding to the next step.

[0226] 8. Further lyse to produce depth filtrate: add Benzonase® 150 U / ml (720 μl) and enough 2 M NaCl (90 mL) to reach 150 mM NaCl and pH 9.0 and incubate at room temperature overnight for 18±4 hours.

[0227] TFF and diafiltration

[0228] 1. Set up a sterile 0.1 μm hollow fiber filter (GE) RTP HF cartridge TFF system as diagrammed in FIG. 11.

[0229] 2. Pre-flush the TFF system with 1 L of Pre-flush Buffer (75 mM Tris, 2 mM MgCl2, 150 mM NaCl, pH 9.00).

[0230] 3.TFF-1 enrichment:

[0231] (a) Fill the retentate reservoir with approximately 45 mL of filtered lysate.

[0232] (b) Start the pump at approximately 100 mL / min to fill the tubing with the permeate valve closed.

[0233] (c) When the retentate reservoir reaches approximately 5 mL, add an additional 40 mL volume of filtered lysate to the reservoir. Increase the feed flow rate to the specified flow rate of 1.472 L / min and close the permeate line for 5-10 min to recirculate.

[0234] (d) After membrane conditioning, the permeate valve is opened slowly to prevent spikes in flux rate above 15 LMH.

[0235] (e) The filtered lysate is concentrated to 1 / 20 of its original volume (20x concentration) and the permeate flux is controlled with a target of 15 LMH by applying a control valve to the permeate line; the temperature is not controlled.

[0236] 4. TFF-1 Diafiltration 1: Perform the first diafiltration using 75 mM Tris, 2 mM MgCl, 150 mM NaCl buffer 20 DV, pH 9.00; permeate flux 20 liters / m2 / hr (LMH); shear rate controlled at 3000-5000 s-1; temperature not controlled.

[0237] 5. TFF-1 Diafiltration 2: Perform a second diafiltration using 10 DV of 20 mM Tris, 8.5% w / v sucrose buffer, pH 7.80; permeate flux 30 LMH; shear rate controlled at 3000-5000 s-1; TMP not controlled. After diafiltration, collect the retentate from the TFF system (tubing and HF cartridge) in the retentate reservoir and reduce the pump flow rate while collecting the product.

[0238] Final filtration

[0239] As depicted in FIG. 12, the TFF retentate is passed through a 1.2 μm filter and pumped into the drug substance bottle at a pump flow rate of 150 LMH.

[0240] Example 6 Virus Production (I)

[0241] This example describes an example of virus production according to an embodiment of the present disclosure and an exemplary yield of an exemplary vaccinia virus from a manufacturing process.

[0242] Host HeLa cells were infected with an exemplary vaccinia virus at an moi of 0.02 and incubated for 4 min. 2 The virus was cultured in a microcarrier fixed-bed-based bioreactor with a surface area of ​​100 μg / ml. Virus was harvested 72 h post-infection and subjected to clarification, benzonase treatment, TFF for 20-fold concentration, DF-1 and DF-2, then the obtained purified virus preparation was kept overnight at 2-8 °C, followed by a final filtration. Table 1 shows the measured values ​​of virus titer, the amount of host cell protein (HCP), host cell DNA (hcDNA) and BSA in the virus preparation obtained at the end of each manufacturing step. Table 1. Productivity and purity measurements [Table 1]

[0243] Example 7 Virus Production (II)

[0244] This example describes an example of virus production according to an embodiment of the present disclosure and an exemplary yield of an exemplary vaccinia virus from a manufacturing process.

[0245] Host HeLa cells were infected with an exemplary vaccinia virus at an moi of 0.001 and incubated for 4 min. 2The virus was cultured in a microcarrier fixed-bed-based bioreactor with a surface area of ​​100 μg / ml. Virus was harvested 72 h post-infection and subjected to clarification, Benzonase® treatment, TFF for 20-fold concentration, DF-1 and DF-2, then the obtained purified virus preparation was kept overnight at 2-8 °C, followed by a final filtration. Table 2 shows the measured virus titers, the amount of host cell proteins (HCPs), host cell DNA (hcDNA) and BSA in the virus preparation obtained at the end of each manufacturing step. Table 2. Productivity and purity measurements [Table 2]

[0246] Example 8 Comparison of virus recovery

[0247] This example describes an example of virus production according to an embodiment of the present disclosure and an exemplary yield of an exemplary vaccinia virus from the manufacturing process compared to production using conventional hypo-osmotic harvest methods.

[0248] In each series of experiments, two batches of Hela cells were infected with an exemplary modified vaccinia virus at an MOI of 0.02 and grown in a microcarrier fixed-bed-based bioreactor for 48 hours. One batch of virus-infected Hela cells was harvested using a hypotonic lysis buffer, and the other batch was harvested using an exemplary high pH buffer according to the present disclosure. Table 3 below summarizes the average yields in the harvest step. Table 3. Viral yields for different recovery methods [Table 3]

[0249] Although preferred embodiments of the present disclosure have been shown and described herein, such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will normally occur to those skilled in the art without departing from the present disclosure. It should be understood that various alternatives to the embodiments of the present disclosure may be used in the practice of the present disclosure. The following claims define the scope of the present disclosure, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby. The present invention provides, for example, the following items. (Item 1) growing a culture comprising a plurality of virus-infected host cells in a bioreactor to produce at least about 50 plaque-forming units (PFU) to about 350 PFU of the recombinant oncolytic virus per host cell, said bioreactor having a capacity of about 0.2 m 2 Approximately 500m from 2 A manufacturing method including a surface area of. (Item 2) (i) growing a culture comprising a plurality of virus-infected host cells in a bioreactor; and (ii) recovering a population of recombinant oncolytic viruses from said culture, said recovering comprising lysing said culture to produce a lysate comprising said population of recombinant oncolytic viruses; and (iii) clarifying said lysate to produce a purified population of recombinant oncolytic viruses, said population of purified recombinant oncolytic viruses comprising at least about 50% to about 90% of said population of recombinant oncolytic viruses in said lysate, wherein said bioreactor is about 0.2 m 2 Approximately 500m from 2 A manufacturing method including a surface area of. (Item 3) Recovering a population of recombinant oncolytic viruses from a culture comprising a plurality of virus-infected host cells grown in a bioreactor, said population being about 1.5×10 11 From about 5 × 10 13 PFU of the recombinant oncolytic virus, and the bioreactor is about 0.2 m 2 Approximately 500m from 2 A manufacturing method including a surface area of. (Item 4) 1. Recovering a population of recombinant oncolytic viruses from a culture comprising a plurality of virus-infected host cells grown in a bioreactor, said population being approximately 1.5×10 virus / mL of said culture. 8 From about 2 × 10 10 The bioreactor contained a viral titer of 1000 PFU and was approximately 0.2 m 2 Approximately 500m from 2 A manufacturing method including a surface area of. (Item 5) (i) growing a culture comprising a plurality of virus-infected host cells in a bioreactor; and (ii) recovering a population of recombinant oncolytic viruses from said culture, wherein said bioreactor has a volume of about 0.2 m 2 Approximately 500m from 2 wherein said recovering is carried out under alkaline conditions. (Item 6) 2. Growing a culture comprising a plurality of virus-infected host cells in a bioreactor to produce a population of recombinant oncolytic viruses, wherein the plurality of virus-infected host cells are infected with the viruses at a multiplicity of infection (moi) of about 0.0001 to about 0.01 PFU / cell, and wherein the bioreactor is about 0.2 m 2 Approximately 500m from 2 A manufacturing method including a surface area of. (Item 7) (i) growing in a bioreactor a culture comprising a plurality of virus-infected host cells, said plurality of virus-infected host cells being infected with a virus at a multiplicity of infection (moi) of about 0.0001 to about 0.01; and (ii) recovering from said culture a population of recombinant oncolytic viruses, said population being about 1.5×10 11 From about 5 × 10 13 A method of producing PFU of said recombinant oncolytic virus. (Item 8) 8. The method according to any one of items 1 to 7, wherein the bioreactor comprises a fixed bed. (Item 9) 7. The method of claim 6, wherein the fixed bed comprises microcarriers. (Item 10) 9. The method according to any one of items 1 to 8, wherein the fixed bed comprises a volume of about 0.01 L to about 25 L. (Item 11) 11. The method according to any one of the preceding claims, wherein the fixed bed comprises a packed density of at least about 80 g / L to about 144 g / L. (Item 12) 12. The process according to any one of the preceding claims, wherein the fixed bed comprises a packed density of at least about 96 g / L. (Item 13) The plurality of virus-infected host cells is at least about 1,000 cells / cm in the bioreactor. 2 to approximately 150,000 cells / cm 2 13. The method according to any one of items 1 to 12, wherein the cells are seeded at a density of (Item 14) 14. The method of any one of items 1 to 13, wherein the culture comprising the virus-infected host cells is grown for about 24 hours to about 96 hours. (Item 15) 15. The method of any one of items 1 to 14, wherein the culture comprising the virus-infected host cells is grown for about 48 hours to about 72 hours. (Item 16) 16. The method of any one of items 1 to 15, wherein the culture comprising the virally-infected host cells comprises a dissolved oxygen tension (DOT) level of at least about 20% to about 100%. (Item 17) 17. The method of any one of items 1 to 16, wherein the culture comprising the virus-infected host cells is maintained at a temperature of about 32° C. to about 38° C. (Item 18) 18. The method of any one of items 1 to 17, wherein the culture comprising the virus-infected host cells is maintained at a pH of about 7.0 to about 7.5. (Item 19) 19. The method of any one of paragraphs 1 to 5 and 8 to 18, wherein a plurality of said virus-infected cells are infected with the virus at a multiplicity of infection (moi) of about 0.0005 to about 0.2. (Item 20) 19. The method of any one of items 1 and 6 to 18, further comprising lysing the plurality of virus-infected host cells by incubation in an alkaline buffer. (Item 21) 19. The method of any one of items 1 and 6 to 18, further comprising lysing the plurality of virus-infected host cells by incubating with an enzyme to degrade cellular debris. (Item 22) 19. The method of any one of items 1 and 6 to 18, further comprising lysing the plurality of virus-infected host cells by incubating with an enzyme and an alkaline buffer to degrade cellular debris. (Item 23) 20. The method of any one of items 2 to 5 and 8 to 19, wherein the recovering comprises lysing the plurality of virus-infected host cells by incubation in an alkaline buffer. (Item 24) 20. The method of any one of items 2 to 5 and 8 to 19, wherein the recovering comprises lysing the plurality of virus-infected host cells by incubation with an enzyme to degrade cellular debris. (Item 25) 20. The method of any one of items 2 to 5 and 8 to 19, wherein said recovering comprises lysing said plurality of virus-infected host cells by incubation with an enzyme and an alkaline buffer to degrade cellular debris. (Item 26) 26. The method of any one of items 21, 22, 24, and 25, wherein the concentration of the enzyme during the incubation is from about 50 IU / mL to about 200 IU / mL. (Item 27) 27. The method of any one of items 20 to 26, wherein the incubating is performed for about 2 hours to about 8 hours. (Item 28) 28. The method of any one of items 20 to 27, wherein the incubating is performed at a temperature of about 22° C. to about 28° C. (Item 29) 29. The method according to any one of items 21 to 22 and 24 to 28, wherein said enzyme for degrading cell debris comprises benzonase. (Item 30) 30. The method according to any one of items 20, 22 to 24, and 25 to 29, wherein the alkaline buffer comprises a pH greater than 8. (Item 31) 31. The method of claim 30, wherein the alkaline buffer comprises a pH of greater than 8.0 to about 10.0. (Item 32) 32. The method of claim 31, wherein the alkaline buffer comprises a pH of about 9.5. (Item 33) 33. The method of any one of items 20 to 32, wherein the alkaline buffer comprises a Tris buffer. (Item 34) 34. The method of any one of items 20 to 33, further comprising monitoring the pH during the incubation. (Item 35) 35. The method of any one of items 20 to 34, further comprising filtering to produce a filtered lysate. (Item 36) 36. The method of claim 35, further comprising treating the filtered lysate with an enzyme to degrade cell debris. (Item 37) 37. The method of claim 36, wherein the treating is carried out under alkaline conditions. (Item 38) Item 38. The method of item 37, wherein the treating is carried out for about 10 hours to about 24 hours. (Item 39) 39. The method of any one of items 36 to 38, wherein the treating is followed by tangential flow filtration of the filtered lysate to produce a purified preparation of the recombinant oncolytic virus. (Item 40) 40. The method of claim 39, wherein the tangential flow filtration comprises concentrating the filtered lysate, followed by at least one and two rounds of diafiltration. (Item 41) 41. The method of claim 40, wherein the first round of diafiltration comprises buffer exchange with a buffer comprising a pH of about 9.0 to about 9.5. (Item 42) 42. The method of claim 41, wherein the first round of diafiltration comprises about 20 volumes of the buffer exchange. (Item 43) 43. The method according to item 41 or 42, wherein the buffer comprising a pH of about 9.0 to about 9.5 comprises a Tris concentration of about 40 mM to about 80 mM. (Item 44) 44. The method of claim 43, wherein the buffer comprises a pH of about 9.0 and a Tris concentration of about 75 mM. (Item 45) 45. The method of any one of items 40 to 44, wherein the second round of diafiltration comprises buffer exchange with a buffer having a pH greater than 7.5. (Item 46) 46. ​​The method of claim 45, wherein the second round of diafiltration comprises about a 10-fold volume exchange of the buffer with the buffer comprising a pH greater than 7.5. (Item 47) 47. The method according to item 45 or 46, wherein the buffer comprising a pH greater than 7.5 comprises a Tris concentration of about 10 mM to about 30 mM. (Item 48) 48. The method of claim 47, wherein the buffer having a pH greater than 7.5 further comprises at least about 5% to at least about 10% by volume of sucrose. (Item 49) 49. The method of any one of items 39 to 48, wherein the tangential flow filtration is carried out at a flow rate comprised between about 15 LMH and about 50 LMH. (Item 50) 50. The method of any one of items 39 to 49, wherein the purified preparation of the recombinant oncolytic virus comprises from about 5 ng to about 100 ng of host cell DNA in a unit dose of the recombinant oncolytic virus. (Item 51) 51. The method of any one of items 39 to 50, wherein the purified preparation of the recombinant oncolytic virus contains from about 0.1 μg to about 10 μg of host cell protein in a unit dose of the recombinant oncolytic virus. (Item 52) The unit dose is about 10 9 About 10 13 52. The method of claim 50 or 51, comprising a PFU of said recombinant oncolytic virus. (Item 53) The purified preparation of the recombinant oncolytic virus is 5×10 9 50. The method of any one of items 39 to 49, comprising up to about 400 ng, about 200 ng, about 100 ng, about 50 ng, or about 40 ng of host cell DNA per PFU. (Item 54) The purified preparation of the recombinant oncolytic virus is 5×10 9 54. The method of any one of items 39 to 49 and 53, comprising up to about 7.5 μg, about 7 μg, about 6 μg, about 5 μg, or about 4 μg of host cell DNA per PFU. (Item 55) The purified preparation of the recombinant oncolytic virus is 5×10 9 55. The method of any one of items 39 to 49 and 54, comprising up to about 100 ng, about 60 ng, about 50 ng, about 30 ng, or about 25 ng of serum albumin per PFU used in the culture. (Item 56) 56. The method of any one of items 1 to 55, wherein the host cell is selected from the group consisting of a HeLa cell, a 293 cell, and a Vero cell. (Item 57) 57. The method of any one of items 1 to 56, wherein the recombinant oncolytic virus comprises a recombinant oncolytic vaccinia virus. (Item 58) 58. A composition comprising a population of recombinant oncolytic viruses produced by the method of any one of items 1 to 57. (Item 59) A bioreactor comprising a culture of host cells containing at least about 50 PFU to about 350 PFU of the recombinant oncolytic virus per host cell. (Item 60) 60. The bioreactor of item 59, comprising a fixed bed, said fixed bed comprising said culture of host cells. (Item 61) 61. The bioreactor of item 60, wherein the culture of host cells is grown in microcarriers within the fixed bed. (Item 62) said culture of host cells, said culture having about 1,000 cells / cm 2 Approximately 10,000 cells / cm from the fixed bed 2 62. The bioreactor according to item 60 or 61, comprising a host cell density of the fixed bed. (Item 63) 63. The bioreactor of any one of items 59 to 62, wherein the fixed bed comprises a volume of about 0.01 L to about 25 L. (Item 64) 64. The bioreactor of any one of items 59 to 63, wherein the fixed bed comprises a packed density of at least about 80 g / L to about 144 g / L. (Item 65) The fixed bed is about 0.2 m 2 Approximately 500m from 2 64. The bioreactor according to any one of items 59 to 63, comprising a surface area of (Item 66) Approximately 1.5×10 11 From about 5 × 10 13 A bioreactor containing PFU of recombinant oncolytic virus. (Item 67) 67. The bioreactor of item 66, comprising a fixed bed, said fixed bed containing a culture comprising said recombinant oncolytic virus. (Item 68) 67. The bioreactor of any one of items 59 to 65 and 66, wherein the fixed bed comprises a volume of about 0.01 L to about 25 L. (Item 69) 70. The bioreactor of item 68, wherein the fixed bed comprises a packed density of at least about 80 g / L to about 144 g / L. (Item 70) The fixed bed is about 0.2 m 2 Approximately 500m from 2 70. The bioreactor according to item 68 or 69, comprising a surface area of (Item 71) 71. The bioreactor of any one of items 59 to 70, wherein the recombinant oncolytic virus comprises a recombinant oncolytic vaccinia virus. (Item 72) 1. A method for producing a recombinant oncolytic virus, comprising: (i) contacting a culture of host cells in a bioreactor with vaccinia virus at a multiplicity of infection of about 0.001 PFU / cell; (ii) growing the culture for about 72 hours to generate a further culture comprising a plurality of virus-infected host cells; (iii) recovering a population of recombinant oncolytic virus from the further culture comprising the plurality of virus-infected host cells, wherein the population is about 1.5×10 11 From about 5 × 10 13 PFU of the recombinant oncolytic virus, and the bioreactor is about 0.2 m 2 Approximately 500m from 2 wherein said recovering is performed under alkaline conditions. (Item 73) 73. The method of claim 72, wherein the bioreactor comprises a fixed bed. (Item 74) 74. The method of claim 73, wherein the fixed bed comprises microcarriers. (Item 75) 75. The method of claim 73 or 74, wherein the fixed bed comprises a volume of about 0.01 L to about 25 L. (Item 76) 76. The method of any one of items 73 to 75, wherein the fixed bed comprises a packed density of at least about 80 g / L to about 144 g / L. (Item 77) 77. The method of any one of items 73 to 76, wherein the fixed bed comprises a packed density of at least about 96 g / L. (Item 78) The host cells are grown in the bioreactor at a density of at least about 1,000 cells / cm. 2 to approximately 150,000 cells / cm 2 78. The method according to any one of items 72 to 77, wherein the cells are seeded at a density of (Item 79) 79. The method of any one of items 72 to 78, wherein the further culture comprises a dissolved oxygen tension (DOT) level of at least about 20% to about 100%. (Item 80) 80. The method of claim 79, wherein the further culture comprises a dissolved oxygen tension (DOT) level of about 50%. (Item 81) 81. The method of any one of paragraphs 72 to 80, wherein the further culture is maintained at a temperature of about 32° C. to about 38° C. (Item 82) 82. The method of claim 81, wherein the further culture is maintained at a temperature of about 36° C. (Item 83) 83. The method of any one of items 72 to 82, wherein the further culture is maintained at a pH of about 7.0 to about 7.5. (Item 84) 84. The method of claim 83, wherein the further culture is maintained at a pH of about 7.2. (Item 85) 85. The method of any one of items 72 to 84, wherein said recovering comprises lysing the plurality of virus-infected host cells by incubation with an enzyme and an alkaline buffer to degrade cellular debris. (Item 86) 86. The method of claim 85, wherein the concentration of the enzyme for degrading cell debris during the incubation is from about 50 IU / mL to about 200 IU / mL. (Item 87) 87. The method of claim 86, wherein said incubating is performed for about 5 hours. (Item 88) 88. The method of claim 86 or 87, wherein the incubating is performed at a temperature of about 22° C. to about 28° C. (Item 89) 89. The method of claim 88, wherein the incubating is performed at a temperature of about 27° C. (Item 90) 90. The method of any one of items 85 to 89, wherein said enzyme for degrading cell debris comprises benzonase. (Item 91) 91. The method of claim 90, wherein the enzyme for degrading cell debris comprises benzonase, and the incubating is performed in the presence of benzonase at a concentration of about 150 IU / mL at a temperature of about 27° C. for about 5 hours. (Item 92) 92. The method according to any one of items 85 to 91, wherein the alkaline buffer comprises a pH greater than 8. (Item 93) 93. The method of claim 92, wherein the alkaline buffer comprises a pH of greater than 8.0 to about 10.0. (Item 94) 94. The method of claim 93, wherein the alkaline buffer has a pH of about 9.5. (Item 95) 95. The method of any one of items 85 to 94, wherein the alkaline buffer comprises a Tris buffer. (Item 96) 96. The method of any one of items 85 to 95, further comprising monitoring the pH during said incubation. (Item 97) 97. The method of any one of items 85 to 96, further comprising filtering to produce a filtered lysate. (Item 98) 98. The method of claim 97, further comprising treating the filtered lysate with a cell lytic enzyme. (Item 99) 99. The method of claim 98, wherein the treating is carried out under alkaline conditions. (Item 100) Item 100. The method of item 99, wherein the treating is carried out for about 10 hours to about 24 hours. (Item 101) Item 100. The method of item 99, wherein said treating is carried out at a temperature of about 4°C for about 18 hours. (Item 102) 102. The method of any one of items 98 to 101, wherein the treating is followed by tangential flow filtration of the filtered lysate to produce a purified preparation of the recombinant oncolytic virus. (Item 103) The tangential flow filtration includes concentrating the filtered lysate. 103. The method of claim 102, further comprising the step of: (Item 104) 104. The method of claim 103, wherein the first round of diafiltration comprises buffer exchange with a buffer having a pH of about 9.0 to about 9.5. (Item 105) 105. The method of claim 104, wherein the first round of diafiltration comprises about 20 volumes of the buffer exchange. (Item 106) 106. The method according to claim 104 or 105, wherein the buffer solution having a pH of about 9.0 to about 9.5 comprises a Tris concentration of about 40 mM to about 100 mM. (Item 107) 107. The method of any one of items 103 to 106, wherein the second round of diafiltration comprises buffer exchange with a buffer comprising a pH of about 7.0 to about 8.0. (Item 108) 108. The method of claim 107, wherein the second round of diafiltration comprises about 10 volumes of the buffer exchange. (Item 109) 109. The method according to claim 107 or 108, wherein the buffer solution having a pH of about 7.0 to about 8.0 comprises a Tris concentration of about 15 mM to about 30 mM. (Item 110) 110. The method according to any one of items 107 to 109, wherein the buffer comprising a pH of about 7.0 to about 8.0 further comprises at least about 5% to at least about 20% by volume of sucrose. (Item 111) 111. The method of any one of items 102 to 110, wherein the purified preparation of the recombinant oncolytic virus contains from about 5 ng to about 100 ng of host cell DNA in a unit dose of the recombinant oncolytic virus. (Item 112) 112. The method of any one of items 102 to 111, wherein the purified preparation of the recombinant oncolytic virus contains from about 0.1 μg to about 10 μg of host cell protein in a unit dose of the recombinant oncolytic virus. (Item 113) The unit dose is about 10 9 About 10 13 113. The method of claim 111 or 112, comprising a PFU of said recombinant oncolytic virus. (Item 114) The purified preparation of the recombinant oncolytic virus is 5×10 9 114. The method of any one of items 102 to 113, comprising up to about 400 ng, about 200 ng, about 100 ng, about 50 ng, or about 40 ng of host cell DNA per PFU. (Item 115) The purified preparation of the recombinant oncolytic virus is 5×10 9 114. The method of any one of items 102 to 113, comprising up to about 7.5 μg, about 7 μg, about 6 μg, about 5 μg, or about 4 μg of host cell DNA per PFU. (Item 116) The purified preparation of the recombinant oncolytic virus is 5×10 9 116. The method of any one of items 102 to 113 and 115, comprising using up to about 100 ng, about 60 ng, about 50 ng, about 30 ng, or about 25 ng of serum albumin per PFU in the culture. (Item 117) A method for producing a recombinant oncolytic virus, comprising: (i) contacting a culture of host cells with vaccinia virus in a bioreactor at a multiplicity of infection of about 0.001 PFU / cell; (ii) growing the culture at a temperature of about 36°C and a dissolved oxygen tension level of about 50% for about 72 hours to generate a further culture comprising a plurality of virus-infected host cells; and (iii) recovering a population of recombinant oncolytic virus from the further culture comprising the plurality of virus-infected host cells, wherein the recovering is performed in the presence of a buffer having a pH of about 9.5. (Item 118) 1. A method for producing a recombinant oncolytic virus, comprising: (i) contacting a culture of host cells with vaccinia virus in a bioreactor at a multiplicity of infection of about 0.003 PFU / cell; (ii) growing the culture at a temperature of about 35° C. and a dissolved oxygen tension level of about 45% for about 48 hours to generate a further culture comprising a plurality of virus-infected host cells; and (iii) recovering a population of recombinant oncolytic virus from the further culture comprising the plurality of virus-infected host cells, wherein the bioreactor is about 0.2 m 2 Approximately 500m from 2 and said recovering is performed in the presence of a buffer comprising a pH of about 8.5. (Item 119) 1. A method for producing a recombinant oncolytic virus, comprising: (i) contacting a culture of host cells with vaccinia virus in a bioreactor at a multiplicity of infection of about 0.002 PFU / cell; (ii) growing the culture at a temperature of about 32° C. and a dissolved oxygen tension level of about 50% for about 24 hours to generate a further culture comprising a plurality of virus-infected host cells; and (iii) recovering a population of recombinant oncolytic virus from the further culture comprising the plurality of virus-infected host cells, wherein the bioreactor is about 0.2 m 2 Approximately 500m from 2 and said recovering is performed in the presence of a buffer comprising a pH of about 8.0. (Item 120) 1. A method for producing a recombinant oncolytic virus, comprising: (i) contacting a culture of host cells with vaccinia virus in a bioreactor at a multiplicity of infection of about 0.002 PFU / cell; (ii) growing the culture at a temperature of about 37° C. and a dissolved oxygen tension level of about 50% for about 96 hours to generate a further culture comprising a plurality of virus-infected host cells; and (iii) recovering a population of recombinant oncolytic virus from the further culture comprising the plurality of virus-infected host cells, wherein the bioreactor is about 0.2 m 2 Approximately 500m from 2 and said recovering is performed in the presence of a buffer comprising a pH of about 7.8. (Item 121) 1. A method for producing a recombinant oncolytic virus, comprising: (i) contacting a culture of host cells with vaccinia virus in a bioreactor at a multiplicity of infection of about 0.005 PFU / cell; (ii) growing the culture at a temperature of about 37° C. and a dissolved oxygen tension level of about 50% for about 96 hours to generate a further culture comprising a plurality of virus-infected host cells; and (iii) recovering a population of recombinant oncolytic virus from the further culture comprising the plurality of virus-infected host cells, wherein the bioreactor is about 0.2 m 2 Approximately 500m from 2 and said recovering is performed in the presence of a buffer comprising a pH of about 9.5. (Item 122) A method for producing a recombinant oncolytic virus, comprising: (i) contacting a culture of host cells with a vaccinia virus in a bioreactor at a multiplicity of infection of about 0.001 PFU / cell; (ii) growing the culture at a temperature of about 36° C. and a dissolved oxygen tension level of about 50% for about 72 hours to generate a further culture comprising a plurality of virus-infected host cells; (iii) recovering at least about 10 virus-infected host cells from the further culture comprising the plurality of virus-infected host cells. 12 and recovering a population of recombinant oncolytic viruses comprising PFU of said recombinant oncolytic viruses, said recovering being performed in the presence of a buffer comprising a pH of about 9.5. (Item 123) 1. A method for producing a recombinant oncolytic virus, comprising: (i) contacting a culture of host cells with a vaccinia virus in a bioreactor at a multiplicity of infection of about 0.003 PFU / cell; (ii) growing the culture at a temperature of about 35° C. and a dissolved oxygen tension level of about 45% for about 48 hours to generate a further culture comprising a plurality of virus-infected host cells; (iii) extracting at least about 10 virus-infected host cells from the further culture comprising the plurality of virus-infected host cells. 12 and harvesting a population of recombinant oncolytic viruses comprising PFU of said recombinant oncolytic viruses, said bioreactor being about 0.2 m 2 Approximately 500m from 2 and said recovering is performed in the presence of a buffer comprising a pH of about 8.5. (Item 124) A method for producing a recombinant oncolytic virus, comprising: (i) contacting a culture of host cells with a vaccinia virus in a bioreactor at a multiplicity of infection of about 0.002 PFU / cell; (ii) growing the culture at a temperature of about 32° C. and a dissolved oxygen tension level of about 50% for about 24 hours to generate a further culture comprising a plurality of virus-infected host cells; (iii) recovering at least about 10 virus-infected host cells from the further culture comprising the plurality of virus-infected host cells. 12 and harvesting a population of recombinant oncolytic viruses comprising PFU of said recombinant oncolytic viruses, said bioreactor being about 0.2 m 2 Approximately 500m from 2 and said recovering is performed in the presence of a buffer comprising a pH of about 8.0. (Item 125) A method for producing a recombinant oncolytic virus, comprising: (i) contacting a culture of host cells with a vaccinia virus in a bioreactor at a multiplicity of infection of about 0.002 PFU / cell; (ii) growing the culture at a temperature of about 37° C. and a dissolved oxygen tension level of about 50% for about 96 hours to generate a further culture comprising a plurality of virus-infected host cells; (iii) recovering at least about 10 virus-infected host cells from the further culture comprising the plurality of virus-infected host cells. 12 and harvesting a population of recombinant oncolytic viruses comprising PFU of said recombinant oncolytic viruses, said bioreactor being about 0.2 m 2 Approximately 500m from 2 and said recovering is performed in the presence of a buffer comprising a pH of about 7.8. (Item 126) 1. A method for producing a recombinant oncolytic virus, comprising: (i) contacting a culture of host cells with a vaccinia virus in a bioreactor at a multiplicity of infection of about 0.005 PFU / cell; (ii) growing the culture at a temperature of about 37° C. and a dissolved oxygen tension level of about 50% for about 96 hours to generate a further culture comprising a plurality of virus-infected host cells; (iii) recovering at least about 10 virus-infected host cells from the further culture comprising the plurality of virus-infected host cells. 12 and harvesting a population of recombinant oncolytic viruses comprising PFU of said recombinant oncolytic viruses, said bioreactor being about 0.2 m 2 Approximately 500m from 2 and said recovering is performed in the presence of a buffer comprising a pH of about 9.5. (Item 127) 127. The method of any one of items 117 to 126, further comprising purifying the population of recombinant oncolytic viruses recovered in step (iii) to produce a purified preparation of the recombinant oncolytic viruses. (Item 128) The purified preparation of the recombinant oncolytic virus is at least about 2×10 11 From about 7 × 10 11 128. The method of claim 127, comprising a PFU of the virus.

Claims

1. A method of production includes growing a culture comprising a plurality of virus-infected host cells in a bioreactor to produce a population of recombinant oncolytic viruses, the plurality of virus-infected host cells being infected with the viruses at a multiplicity of infection (moi) of about 0.0001 to about 0.01 PFU / cell, and the bioreactor being configured to grow at a multiplicity of infection (moi) of about 0.2 ml. ２ About 500m from ２ the bioreactor comprises a surface area of ​​about 100 μg / l×10 9 PFU of host cell protein and less than 300 ng / l×10 9 PFU of host cell DNA, the bioreactor comprises a fixed bed, the culture comprising the virus-infected host cells comprises a dissolved oxygen tension (DOT) level of about 50%, and the population of recombinant oncolytic virus comprises less than 10 μg / l×10 9 PFU of host cell protein and less than 300 ng / l×10 9 PFU of host cell DNA.

2. The method of claim 1 , wherein the fixed bed comprises microcarriers.

3. 3. The method of claim 1, wherein the fixed bed comprises a volume of about 0.01 L to about 25 L, or about 20 L to about 100 L.

4. 10. The method of claim 1, wherein the fixed bed comprises a packed density of at least about 80 g / L to about 144 g / L.

5. The plurality of virus-infected host cells is at least about 1,000 cells / cm in the bioreactor. ２ to about 150,000 cells / cm ２ 5. The method according to claim 1 , wherein the seeds are seeded at a density of 0.1-0.2g / g.

6. 6. The method of any one of claims 1 to 5, wherein the culture comprising the virus-infected host cells is grown for about 24 hours to about 96 hours.

7. 7. The method of any one of claims 1 to 6, wherein the culture containing the virally-infected host cells is maintained at a temperature of about 32°C to about 38°C.

8. 8. The method of any one of claims 1 to 7, wherein the culture containing the virally-infected host cells is maintained at a pH of about 7.0 to about 7.

5.

9. 9. The method of any one of claims 1 to 8, further comprising lysing the plurality of virus-infected host cells by incubation in an alkaline buffer.

10. 10. The method of any one of claims 1 to 9, further comprising lysing the plurality of virus-infected host cells by incubation with an enzyme to degrade cellular debris.

11. 11. The method of claim 10, wherein the concentration of the enzyme during said incubating is from about 50 IU / mL to about 200 IU / mL.

12. 12. The method of any one of claims 9 to 11, wherein the incubating is performed for about 2 hours to about 8 hours.

13. 13. The method of any one of claims 9 to 12, wherein the incubating is performed at a temperature of about 22°C to about 28°C.

14. 14. The method of any one of claims 10 to 13, wherein the enzyme for degrading cell debris comprises Benzonase®.

15. 15. The method of any one of claims 9 to 14, wherein the alkaline buffer comprises a Tris buffer.

16. 16. The method of any one of claims 1 to 15, wherein the host cell is selected from the group consisting of Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells, HeLa cells, HEK293 cells, and Vero cells.

17. 17. The method of any one of claims 1 to 16, wherein the recombinant oncolytic virus comprises a recombinant oncolytic poxvirus.

18. The method of any one of claims 1 to 17, further comprising filtration to produce a filtered lysate.

19. 20. The method of claim 18, further comprising performing one or more tangential flow filtration steps of the filtered lysate to produce a purified preparation of the recombinant oncolytic virus.

20. 20. The method of claim 19, further comprising 1.2 μm filtration after the one or more tangential flow filtration steps.

21. 20. The method of claim 19, wherein said population of purified recombinant oncolytic viruses comprises at least about 50% to about 90% of said population of recombinant oncolytic viruses in said lysate.

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