WO2020106975A1 - Manufacture of virus - Google Patents

Manufacture of virus

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Publication number
WO2020106975A1
WO2020106975A1 PCT/US2019/062643 US2019062643W WO2020106975A1 WO 2020106975 A1 WO2020106975 A1 WO 2020106975A1 US 2019062643 W US2019062643 W US 2019062643W WO 2020106975 A1 WO2020106975 A1 WO 2020106975A1
Authority
WO
WIPO (PCT)
Prior art keywords
virus
culture
recombinant oncolytic
host cells
bioreactor
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2019/062643
Other languages
English (en)
French (fr)
Inventor
Paul Andrew Wright
Katy WACK
Mingrui Zhang
Jackie Vasseur
Susan Nicole HOLLEY
August Allen RENSHAW
Guangyu Zhu
Michelle Catherine VENDEL
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Western Oncolytics Ltd
Original Assignee
Western Oncolytics Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Western Oncolytics Ltd filed Critical Western Oncolytics Ltd
Priority to EP19887930.6A priority Critical patent/EP3884044A4/en
Priority to CN201980089784.0A priority patent/CN113330111A/zh
Priority to CA3120129A priority patent/CA3120129A1/en
Priority to JP2021528362A priority patent/JP7625519B2/ja
Priority to KR1020217018523A priority patent/KR20210093965A/ko
Priority to AU2019384164A priority patent/AU2019384164A1/en
Publication of WO2020106975A1 publication Critical patent/WO2020106975A1/en
Priority to US17/324,756 priority patent/US20210277363A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C12N7/02—Recovery or purification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
    • C12N2710/24151—Methods of production or purification of viral material

Definitions

  • Viruses can find use in many areas, e.g., therapeutics, e.g., oncolytic therapeutics.
  • Oncolytic viruses hold great promise for the treatment of many types of cancer.
  • recombinant oncolytic viruses generated through genetic engineering of naturally occurring viruses prove to be effective tools for eradicating cancer cells.
  • therapeutic applications of oncolytic viruses have requirements of high standard in many aspects. For example, clinical applicable viruses may need to be of high purity. Also high titer of viruses may be needed in order to be therapeutically effective.
  • how to scale up viral production for therapeutic applications also remains a great challenge. Therefore, an improved method of manufacturing viruses is desirable.
  • the present disclosure provides a method of manufacture, comprising: 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 a recombinant oncolytic virus per host cell, wherein said bioreactor comprises a surface area of about 0.2 m 2 to about 500 m 2 .
  • PFU plaque forming units
  • the present disclosure provides a method of manufacture, comprising: (i) growing a culture comprising a plurality of virus infected host cells in a bioreactor; and (ii) harvesting from said culture a population of recombinant oncolytic virus, wherein said harvesting comprises lysing said culture to produce a lysate comprising said population of recombinant oncolytic virus, and (iii) clarifying said lysate to produce a population of purified recombinant oncolytic virus, wherein said population of purified recombinant oncolytic virus comprises at least about 50% to about 90% of said population recombinant oncolytic virus in said lysate, and wherein said bioreactor comprises a surface area of about 0.2 m 2 to about 500 m 2 .
  • the present disclosure provides a method of manufacture, comprising: harvesting from a culture comprising a plurality of virus infected host cells, grown in a bioreactor, a population of a recombinant oncolytic virus, wherein said population comprises about 1.5 x 10 11 to about 5 x 10 13 PFU of said recombinant oncolytic virus, and wherein said bioreactor comprises a surface area of about 0.2 m 2 to about 500 m 2 .
  • the present disclosure provides a method of manufacture, comprising: harvesting from a culture comprising a plurality of virus infected host cells, grown in a bioreactor, a population of a recombinant oncolytic virus, wherein said population comprises a viral titer of about 1.5 x 10 8 to about 2 x 10 10 PFU per mL of said culture, wherein bioreactor comprises a surface area of about 0.2 m 2 to about 500 m 2 .
  • the present disclosure provides a method of manufacture, comprising: (i) growing a culture comprising a plurality of virus infected host cells, in a bioreactor; and (ii) harvesting from said culture a population of recombinant oncolytic virus, wherein said bioreactor comprises a surface area of about 0.2 m 2 to about 500 m 2 , and wherein said harvesting is carried out in an alkaline condition.
  • the present disclosure provides a method of manufacture, comprising growing a culture comprising a plurality of virus infected host cells in a bioreactor to produce a population of recombinant oncolytic virus, wherein said plurality of virus infected host cells have been infected with a virus at a multiplicity of infection (m.o.i.) of about 0.0001 to about 0.01 PFU/cell, and wherein said bioreactor comprises a surface area of about 0.2 m 2 to about 500 m 2 .
  • m.o.i. multiplicity of infection
  • the present disclosure provides a method of manufacture, comprising: (i) growing a culture comprising a plurality of virus infected host cells in a bioreactor, wherein said plurality of virus infected host cells have been infected with a virus at a multiplicity of infection (m.o.i.) of about 0.0001 to about 0.01, and (ii) harvesting from said culture a population of a recombinant oncolytic virus, wherein said population comprises about 1.5 x 10 11 to about 5 x 10 13 PFU of said recombinant oncolytic virus.
  • m.o.i. multiplicity of infection
  • said bioreactor comprises a fixed-bed.
  • said fixed-bed comprises microcarriers.
  • said fixed bed comprises a volume of about 0.01 L to about 25 L.
  • said fixed bed comprises a compaction density of at least about 80 g/L to about 144 g/L.
  • said fixed bed comprises a compaction density of at least about 96 g/L.
  • said plurality of virus infected host cells are seeded at a density of at least about 1,000 cells/cm 2 to about 150,000 cells/ cm 2 in said bioreactor.
  • said culture comprising said virus-infected host cells is grown for about 24 hours to about 96 hours. In some embodiments, said culture comprising said virus-infected host cells is grown for about 48 hours to about 72 hours. In some embodiments, said culture comprising said virus-infected host cells comprises a dissolved oxygen tension (DOT) level of at least about 20% to about 100%. In some embodiments, said culture comprising said virus-infected host cells is maintained at a temperature of about 32 °C to about 38 °C. In some embodiments, said culture comprising said virus-infected host cells is maintained at a pH of about 7.0 to about 7.5. In some embodiments, said plurality of virus infected cells have been infected with a virus at a multiplicity of infection (m.o.i.) of about 0.0005 to about 0.2.
  • m.o.i. multiplicity of infection
  • the method further comprises lysing said plurality of virus infected host cells by incubating in an alkaline buffer. In some embodiments, the method further comprises lysing said plurality of virus infected host cells by incubating with an enzyme to degrade cell debris. In some embodiments, the method further comprises lysing said plurality of virus infected host cells by incubating with an enzyme to degrade cell debris and an alkaline buffer. In some embodiments, said harvesting comprises lysing said plurality of virus infected host cells by incubating in an alkaline buffer. In some embodiments, said harvesting comprises lysing said plurality of virus infected host cells by incubating with an enzyme to degrade cell debris.
  • said harvesting comprises lysing said plurality of virus infected host cells by incubating with an enzyme to degrade cell debris and an alkaline buffer.
  • a concentration of said enzyme, during said incubating is from about 50 IU/mL to about 200 IU/mL.
  • said incubating is for about 2 hours to about 8 hours.
  • said incubating is at a temperature of about 22 °C to about 28 °C.
  • said enzyme to degrade cell debris comprises Benzonase®.
  • said alkaline buffer comprises a pH of above 8.
  • said alkaline buffer comprises a pH of above 8.0 to about 10.0.
  • said alkaline buffer comprises a pH of about 9.5.
  • said alkaline buffer comprises a Tris buffer.
  • the method further comprises monitoring pH during said incubating.
  • the method further comprises conducting a filtration to generate a filtered lysate.
  • the method further comprises treating said filtered lysate with an enzyme to degrade cell debris.
  • said treating is in an alkaline condition hi some embodiments, said treating is for about 10 hours to about 24 hours.
  • said treating is followed by a tangential flow filtration of said filtered lysate to generate a purified preparation of said recombinant oncolytic virus.
  • said tangential flow filtration comprises concentrating said filtered lysate followed by at least a first and a second round of diafiltration.
  • said 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, said first round of diafiltration comprises about 10 volumes of said buffer exchange. In some embodiments, said 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, said buffer comprising 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, said second round of diafiltration comprises a buffer exchange with a buffer comprising a pH of above 7.5.
  • said second round of diafiltration comprises about 6 volumes of said buffer exchange with said buffer comprising a pH of above 7.5.
  • said buffer comprising a pH of above 7.5 comprises a Tris concentration of about 10 mM to about 30 mM.
  • said buffer comprising a pH of above 7.5 further comprises at least about 5% to at least about 10% sucrose, by volume.
  • said purified preparation of said recombinant oncolytic virus comprises from about 5 ng to about 100 ng of host cell DNA in a unit dose of said recombinant oncolytic virus. In some embodiments, said purified preparation of said recombinant oncolytic virus comprises from about 0.1 pg to about 10 pg of host cell protein in a unit dose of said recombinant oncolytic virus. In some embodiments, said unit dose comprises about 1 x 10 9 to about 1 xlO 13 PFU of said recombinant oncolytic virus.
  • said purified preparation of said 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 5 x 10 9 PFU of said recombinant oncolytic virus. In some embodiments, said purified preparation of said recombinant oncolytic virus comprises up to about 80 ng, about 40 ng, about 20 ng, about 10 ng, or about 8 ng of host cell DNA per 1 x 10 9 PFU of said recombinant oncolytic virus.
  • said purified preparation of said recombinant oncolytic virus comprises up to about 7.5 pg, about 7 pg, about 6 pg, about 5 pg, or about 4 pg of host cell protein per 5 x 10 9 PFU of said
  • recombinant oncolytic virus comprises up to about 1.5 pg, about 1.4 pg, about 1.2 pg, about 1 pg, or about 0.8 pg of host cell protein per 1 x 10 9 PFU of said recombinant oncolytic virus.
  • said purified preparation of said recombinant oncolytic virus comprises up to about 100 ng, about 60 ng, about 50 ng, about 30 ng, or about 25 ng of serum albumin that is used in said culture per 5 x 10 9 PFU of said recombinant oncolytic virus.
  • said host cell is selected from a group consisting of a HeLa cell, 293 cells, and Vero cells.
  • said recombinant oncolytic virus comprises a recombinant oncolytic vaccinia virus.
  • the present disclosure provides a composition comprising a population of recombinant oncolytic virus produced by the method as described herein. [0017] In one aspect, the present 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.
  • the bioreactor comprises a fixed bed, wherein said fixed bed comprises said culture of host cells.
  • said culture of host cells is propagated in microcarriers within said fixed bed.
  • the bioreactor comprises said culture of host cells, wherein said culture comprises a host cell density of about 1,000 cells/ cm 2 to about 10,000 cells/ cm 2 of said fixed bed.
  • said fixed bed comprises a volume of about 0.01 L to about 25 L.
  • said fixed bed comprises a compaction density of at least about 80 g/L to about 144 g/L.
  • said fixed bed comprises a surface area of about 0.2 m 2 to about 500 m 2 .
  • the present disclosure provides a bioreactor comprising about 1.5 x 10 11 to about 5 x 10 13 PFU of a recombinant oncolytic virus.
  • the bioreactor comprises a fixed bed, wherein said fixed bed comprises a culture comprising said recombinant oncolytic virus.
  • said fixed bed comprises a volume of about 0.01 L to about 25 L.
  • said fixed bed comprises a compaction density of at least about 80 g/L to about 144 g/L.
  • said fixed bed comprises a surface area of about 0.2 m 2 to about 500 m 2 .
  • said recombinant oncolytic virus comprises a recombinant oncolytic vaccinia virus.
  • the present disclosure provides a method for producing a recombinant oncolytic virus, the method comprising: (i) contacting a culture of host cells in a bioreactor with a vaccinia virus at a multiplicity of infection of about 0.001 PFU/cell; (ii) growing said culture for a period of about 72 hours to generate a further culture comprising a plurality of virus infected host cells; (iii) harvesting from said further culture comprising said plurality of virus infected host cells a population of a recombinant oncolytic virus, wherein said population comprises about 1.5 x 10 11 to about 5 x 10 13 PFU of said recombinant oncolytic virus, wherein said bioreactor comprises a surface area of about 0.2 m 2 to about 500 m 2 , and wherein said harvesting is carried out in an alkaline condition.
  • said bioreactor comprises a fixed-bed.
  • said fixed-bed comprises microcarriers.
  • said fixed bed comprises a volume of about 0.01 L to about 25 L.
  • said fixed bed comprises a compaction density of at least about 80 g/L to about 144 g/L.
  • said fixed bed comprises a compaction density of at least about 96 g/L.
  • said host cells are seeded at a density of at least about 1,000 cells/ cm 2 to about 150,000 cells/ cm 2 in said bioreactor.
  • said further culture comprises a dissolved oxygen tension (DOT) level of at least about 20% to about 100%.
  • said further culture comprises a dissolved oxygen tension (DOT) level of about 50%.
  • said further culture is maintained at a temperature of about 32 °C to about 38 °C.
  • said further culture is maintained at a temperature of about 36 °C. In some embodiments, said further culture is maintained at a pH of about 7.0 to about 7.5. In some embodiments, said further culture is maintained at a pH of about 7.2. In some embodiments, said harvesting comprises lysing said plurality of virus infected host cells by incubating with an enzyme to degrade cell debris and an alkaline buffer. In some embodiments, a concentration of said enzyme to degrade cell debris, during said incubating, is from about 50 IU/mL to about 200 IU/mL. In some embodiments, said incubating is at a temperature of about 22 °C to about 28 °C.
  • said incubating is 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, said incubating is at a temperature of about 25 °C. In some embodiments, said incubating is at a temperature of about 27 °C. In some embodiments, said enzyme to degrade cell debris comprises Benzonase®.
  • said cell enzyme to degrade cell debris comprises Benzonase® and wherein said incubating is in the presence of said Benzonase® at a concentration of about 150 IU/mL, at a temperature of about 27 °C, for a period of about 5 hours.
  • said alkaline buffer comprises a pH of above 8.
  • said alkaline buffer comprises a pH of above 8.0 to about 10.0.
  • said alkaline buffer comprises a pH of about 9.5.
  • said alkaline buffer comprises a Tris buffer.
  • the method further comprises monitoring pH during said incubating.
  • the method further comprises conducting a filtration to generate a filtered lysate.
  • the method further comprises treating said filtered lysate with a cell lysing enzyme.
  • said treating is in an alkaline condition.
  • said treating is for about 10 hours to about 24 hours.
  • said treating is for about 18 hours at a temperature of about 4 °C.
  • said treating is followed by a tangential flow filtration of said filtered lysate to generate a purified preparation of said recombinant oncolytic virus.
  • said tangential flow filtration comprises concentrating said filtered lysate followed by at least a first and a second round of diafiltration.
  • a second round of concentration can be performed.
  • said first round of diafiltration comprises a buffer exchange with a buffer comprising a pH of about 9.0 to about 9.5.
  • said first round of diafiltration comprises about 20 volumes of said buffer exchange.
  • said buffer comprising a pH of about 9.0 to about 9.5 comprises a Tris concentration of about 40 mM to about 100 mM.
  • said second round of diafiltration comprises a buffer exchange with a buffer comprising a pH of about 7.0 to about 8.0.
  • said second round of diafiltration comprises about 10 volumes of said buffer exchange.
  • said buffer comprising a pH of about 7.0 to about 8.0 comprises a Tris
  • said buffer comprising a pH of about 7.0 to about 8.0 further comprises at least about 5% to at least about 20% sucrose, by volume.
  • said purified preparation of said recombinant oncolytic virus comprises from about 5 ng to about 100 ng of host cell DNA in a unit dose of said recombinant oncolytic virus. In some embodiments, said purified preparation of said recombinant oncolytic virus comprises from about 0.1 pg to about 10 pg of host cell protein in a unit dose of said recombinant oncolytic virus.
  • said unit dose comprises about 1 x 10 9 to about 1 x 10 13 PFU of said recombinant oncolytic virus.
  • said purified preparation of said 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 5 x 10 9 PFU of said recombinant oncolytic virus.
  • said purified preparation of said recombinant oncolytic virus comprises up to about 2000 ng, about 1000 ng, about 500 ng, about 250 ng, or about 200 ng of host cell DNA per 1 x 10 9 PFU of said recombinant oncolytic virus.
  • said purified preparation of said recombinant oncolytic virus comprises up to about 7.5 pg, about 7 pg, about 6 pg, about 5 pg, or about 4 pg of host cell protein per 5 x 10 9 PFU of said recombinant oncolytic virus. In some embodiments, said purified preparation of said
  • recombinant oncolytic virus comprises up to about 37.5 pg, about 35 pg, about 30 pg, about 25 pg, or about 20 pg of host cell protein per 1 x 10 9 PFU of said recombinant oncolytic virus.
  • said purified preparation of said recombinant oncolytic virus comprises up to about 100 ng, about 60 ng, about 50 ng, about 30 ng, or about 25 ng of serum albumin that is used in said culture per 5 x 10 9 PFU of said recombinant oncolytic virus.
  • the present disclosure provides a method for producing a recombinant oncolytic virus, the method comprising: (i) contacting a culture of host cells in a bioreactor with a vaccinia virus at a multiplicity of infection of about 0.001 PFU/cell; (ii) growing said culture for a period of about 72 hours, at a temperature of about 36 °C, and a dissolved oxygen tension level of about 50%, to generate a further culture comprising a plurality of virus infected host cells; (iii) harvesting from said further culture comprising said plurality of virus infected host cells a population of a recombinant oncolytic virus, and wherein said harvesting is carried out in presence of a buffer comprising a pH of about 9.5.
  • the present disclosure provides a method for producing a recombinant oncolytic virus, the method comprising: (i) contacting a culture of host cells in a bioreactor with a vaccinia virus at a multiplicity of infection of about 0.003 PFU/cell; (ii) growing said culture for a period of about 48 hours, at a temperature of about 35 °C, and a dissolved oxygen tension level of about 45%, to generate a further culture comprising a plurality of virus infected host cells; (iii) harvesting from said further culture comprising said plurality of virus infected host cells a population of a recombinant oncolytic virus, wherein said bioreactor comprises a surface area of about 0.2 m 2 to about 500 m 2 , and wherein said harvesting is carried out in presence of a buffer comprising a pH of about 8.5.
  • the present disclosure provides a method for producing a recombinant oncolytic virus, the method comprising: (i) contacting a culture of host cells in a bioreactor with a vaccinia virus at a multiplicity of infection of about 0.002 PFU/cell; (ii) growing said culture for a period of about 24 hours, at a temperature of about 32 °C, and a dissolved oxygen tension level of about 50%, to generate a further culture comprising a plurality of virus infected host cells; (iii) harvesting from said further culture comprising said plurality of virus infected host cells a population of a recombinant oncolytic virus, wherein said bioreactor comprises a surface area of about 0.2 m 2 to about 500 m 2 , and wherein said harvesting is carried out in presence of a buffer comprising a pH of about 8.0.
  • the present disclosure provides a method for producing a recombinant oncolytic virus, the method comprising: (i) contacting a culture of host cells in a bioreactor with a vaccinia virus at a multiplicity of infection of about 0.002 PFU/cell; (ii) growing said culture for a period of about 96 hours, at a temperature of about 37 °C, and a dissolved oxygen tension level of about 50%, to generate a further culture comprising a plurality of virus infected host cells; (iii) harvesting from said further culture comprising said plurality of virus infected host cells a population of a recombinant oncolytic virus, wherein said bioreactor comprises a surface area of about 0.2 m 2 to about 500 m 2 , and wherein said harvesting is carried out in presence of a buffer comprising a pH of about 7.8.
  • the present disclosure provides a method for producing a recombinant oncolytic virus, the method comprising: (i) contacting a culture of host cells in a bioreactor with a vaccinia virus at a multiplicity of infection of about 0.005 PFU/cell; (ii) growing said culture for a period of about 96 hours, at a temperature of about 37 °C, and a dissolved oxygen tension level of about 50%, to generate a further culture comprising a plurality of virus infected host cells; (iii) harvesting from said further culture comprising said plurality of virus infected host cells a population of a recombinant oncolytic virus , wherein said bioreactor comprises a surface area of about 0.2 m 2 to about 500 m 2 , and wherein said harvesting is carried out in presence of a buffer comprising a pH of about 9.5.
  • the present disclosure provides a method for producing a recombinant oncolytic virus, the method comprising: (i) contacting a culture of host cells in a bioreactor with a vaccinia virus at a multiplicity of infection of about 0.001 PFU/cell; (ii) growing said culture for a period of about 72 hours, at a temperature of about 36 °C, and a dissolved oxygen tension level of about 50%, to generate a further culture comprising a plurality of virus infected host cells; (iii) harvesting from said further culture comprising said plurality of virus infected host cells a population of a recombinant oncolytic virus comprising at least about 10 12 PFU of said recombinant oncolytic virus, and wherein said harvesting is carried out in presence of a buffer comprising a pH of about 9.5.
  • the present disclosure provides a method for producing a recombinant oncolytic virus, the method comprising: (i) contacting a culture of host cells in a bioreactor with a vaccinia virus at a multiplicity of infection of about 0.003 PFU/cell; (ii) growing said culture for a period of about 48 hours, at a temperature of about 35 °C, and a dissolved oxygen tension level of about 45%, to generate a further culture comprising a plurality of virus infected host cells; (iii) harvesting from said further culture comprising said plurality of virus infected host cells a population of a recombinant oncolytic virus comprising at least about 10 12 PFU of said recombinant oncolytic virus, wherein said bioreactor comprises a surface area of about 0.2 m 2 to about 500 m 2 , and wherein said harvesting is carried out in presence of a buffer comprising a pH of about 8.5.
  • the present disclosure provides a method for producing a recombinant oncolytic virus, the method comprising: (i) contacting a culture of host cells in a bioreactor with a vaccinia virus at a multiplicity of infection of about 0.002 PFU/cell; (ii) growing said culture for a period of about 24 hours, at a temperature of about 32 °C, and a dissolved oxygen tension level of about 50%, to generate a further culture comprising a plurality of virus infected host cells; (iii) harvesting from said further culture comprising said plurality of virus infected host cells a population of a recombinant oncolytic virus comprising at least about 10 12 PFU of said recombinant oncolytic virus, wherein said bioreactor comprises a surface area of about 0.2 m 2 to about 500 m 2 , and wherein said harvesting is carried out in presence of a buffer comprising a pH of about 8 0 [0032] In another aspect, the present
  • the present disclosure provides a method for producing a recombinant oncolytic virus, the method comprising: (i) contacting a culture of host cells in a bioreactor with a vaccinia virus at a multiplicity of infection of about 0.005 PFU/cell; (ii) growing said culture for a period of about 96 hours, at a temperature of about 37 °C, and a dissolved oxygen tension level of about 50%, to generate a further culture comprising a plurality of virus infected host cells; (iii) harvesting from said further culture comprising said plurality of virus infected host cells a population of a recombinant oncolytic virus comprising at least about 1012 PFU of said recombinant oncolytic virus, wherein said bioreactor comprises a surface area of about 0.2 m 2 to about 500 m 2 , and wherein said harvesting is carried out in presence of a buffer comprising a pH of about 9.5.
  • the method further comprises purifying said population of recombinant oncolytic virus harvested in step (iii) to produce a purified preparation of said recombinant oncolytic virus.
  • said purified preparation of said recombinant oncolytic virus comprises at least about 2 x 10 11 to about 7 x 10 11 PFU of virus.
  • FIG. l is a flow chart of an exemplary virus manufacturing process.
  • FIG. 2 is a flow chart of an exemplary virus manufacturing process.
  • FIG. 3 is a flow chart of an exemplary virus manufacturing process.
  • FIG. 4 is a flow chart of an exemplary virus manufacturing process.
  • FIG. 5 is a flow chart of an exemplary virus manufacturing process.
  • FIG. 6 is a flow chart of an exemplary downstream process for virus manufacturing.
  • FIG. 7 is a flow chart of an exemplary downstream process for virus manufacturing.
  • FIG. 8 is a flow chart of an exemplary downstream process for virus manufacturing.
  • FIG. 9 is a flow chart of an exemplary downstream process for virus manufacturing.
  • FIG. 10 shows a schematic of an exemplary setup for clarification process.
  • FIG. 11 shows a schematic of an exemplary setup for TFF and diafiltration process.
  • FIG. 12 shows a schematic of an exemplary setup for final filtration process.
  • the term“about” or“approximately” can mean within an acceptable error range for the 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” can mean within 1 or more than 1 standard deviation, per the practice in the given value. Where particular values are described in the application and claims, unless otherwise stated the term“about” should be assumed to mean an acceptable error range for the particular value, such as ⁇ 10% of the value modified by the term“about”.
  • the terms“individual”,“patient”, or“subject” can be used interchangeably. None of the terms require or are limited to situation characterized by the supervision (e.g. constant or intermittent) of a health care worker (e.g. a doctor, a registered nurse, a nurse practitioner, a physician’s assistant, an orderly, or a hospice worker). In some embodiments, patients, subjects, or individuals can be under the supervision of a health care worker.
  • a health care worker e.g. a doctor, a registered nurse, a nurse practitioner, a physician’s assistant, an orderly, or a hospice worker.
  • patients, subjects, or individuals can be under the supervision of a health care worker.
  • mutation as used herein, can refer to a deletion, an insertion of a heterologous nucleic acid, an inversion or a substitution, including an open reading frame ablating mutations as commonly understood in the art.
  • gene can refer to a segment of nucleic acid that encodes an individual protein or RNA (also referred to as a "coding sequence” or “coding region”), optionally together with associated regulatory regions such as promoters, operators, terminators and the like, which may be located upstream or downstream of the coding sequence.
  • recombinant virus and “modified virus”, as used interchangeably herein, can refer to a virus comprising one or more mutations in its genome, including but not limited to deletions, insertions of heterologous nucleic acids, inversions, substitutions or combinations thereof.
  • the term“oncolytic”, as used herein, can refer to killing of cancer or tumor cells by an agent, such as an oncolytic vaccinia virus, e.g., through the direct lysis of said cells, by stimulating immune response towards said cells, apoptosis, expression of toxic proteins, autophagy and shut-down of protein synthesis, induction of anti-tumoral immunity, or any combinations thereof.
  • the direct lysis of the cancer or tumor cells infected by the agent, such as an oncolytic vaccinia virus can be a result of replication of the virus within said cells.
  • the term“oncolytic,” refers to killing of cancer or tumor cells without lysis of said cells.
  • the term“subject” can refer to an animal, including, but not limited to, a primate (e.g., human), cow, sheep, goat, horse, dog, cat, rabbit, rat, or mouse.
  • a primate e.g., human
  • cow, sheep, goat, horse, dog, cat, rabbit, rat, or mouse e.g., cow, sheep, goat, horse, dog, cat, rabbit, rat, or mouse.
  • patient are used interchangeably herein in reference, for example, to a mammalian subject, such as a human subject.
  • the terms“treat”,“treating”, and“treatment” can be meant to include alleviating or abrogating a disorder, disease, or condition; or one or more of the symptoms associated with the disorder, disease, or condition; or alleviating or eradicating the cause(s) of the disorder, disease, or condition itself. Desirable effects of treatment can include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishing any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state and remission or improved prognosis.
  • One aspect of the present disclosure provides methods and systems for manufacturing viruses.
  • the methods and systems provided herein can be efficient and productive in manufacturing viruses.
  • the methods and systems as described herein can produce viruses at a high titer per host cell.
  • the methods and systems as described herein can be applied to produce viruses at an industrial scale, e.g., with a high yield per production cycle, e.g., from infection of host cell, culture of virus-infected cells, to harvest of viruses and clarification and purification of the viruses.
  • the method provided herein can produce virus preparation of high purity, with limited amount to none of protein or nucleic acid contamination.
  • the methods and systems provided herein can be applied to produce recombinant viruses.
  • the methods and systems provided herein can be applied to produce oncolytic viruses.
  • a method of manufacturing virus as provided herein can comprise growing a cell culture comprising virus-infected host cells in a bioreactor. In some cases, methods and systems as provided herein can make use of fixed-bed bioreactor to culture virus-infected host cells.
  • a method of manufacturing virus can comprise infecting (inoculating) host cells to generate a cell culture comprising virus-infected host cells.
  • a method of manufacturing virus can comprise 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 can be performed in an alkaline condition.
  • a method can comprise conducting clarification and purification of cell lysate obtained from the harvesting to generate a purified virus preparation.
  • the clarification and purification can comprise filtration of the cell lysate.
  • the clarification and purification can comprise treating the cell lysate with an enzyme to degrade cell debris followed by filtration.
  • a method can also comprise conducting filtration to generate a concentrated virus preparation of high purity.
  • the filtration can comprise tangential flow filtration.
  • a method of manufacturing virus as provided herein can comprise (a) infecting
  • the clarification and purification can comprise: (i) treating the cell lysate with an enzyme to degrade cell debris; and (ii) conducting filtration of the treated cell lysate to generate a concentrated virus preparation of high purity.
  • a method as provided herein can comprise growing a cell culture to produce viruses, e.g., oncolytic viruses, e.g., recombinant oncolytic viruses, at a titer of 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 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
  • viruses e
  • 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
  • the method can comprise growing a cell culture to produce viruses, e.g., oncolytic viruses, e.g., recombinant oncolytic viruses, at a titer of about 50 PFU/cell to about 350 PFU/cell.
  • viruses e.g., oncolytic viruses, e.g., recombinant oncolytic viruses
  • the method can comprise growing a cell culture to produce viruses at titer of 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, 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 P
  • a method as provided herein can comprise harvesting a population of viruses, e.g., oncolytic viruses, e.g., recombinant oncolytic viruses, from a culture that comprises about 1.5 x 10 11 to about 5 x 10 13 PFU of the virus.
  • a method can comprise harvesting viruses from a culture that comprises at least about 1 x 10 11 PFU, at least about 1.5 x 10 11 PFU, at least about 2 x 10 11 PFU, at least about 2.5 x 10 11 PFU, at least about 3 x 10 11 PFU, at least about 4 x
  • 10 11 PFU at least about 5 x 10 11 PFU, at least about 7.5 x 10 11 PFU, at least about 1 x 10 12 PFU, at least about 1.5 x 10 12 PFU, at least about 2 x 10 12 PFU, at least about 2.5 x 10 12 PFU, at least about 3 x 10 12 PFU, at least about 4 x 10 12 PFU, at least about 5 x 10 12 PFU, at least about 7.5 x
  • 10 12 PFU at least about 1 x 10 13 PFU, at least about 1.5 x 10 13 PFU, at least about 2 x 10 13 PFU, at least about 2.5 x 10 13 PFU, at least about 3 x 10 13 PFU, at least about 4 x 10 13 PFU, at least about 5 x 10 13 PFU, at least about 7.5 x 10 13 PFU, or at least about 1 x 10 14 PFU of the viruses.
  • a method can comprise harvesting viruses from a culture that can comprise about 1 x 10 11 to about 1 x 10 14 PFU, about 5 x 10 11 to about 1 x 10 14 PFU, about 1 x 10 12 to about 1 x 10 14 PFU, about 5 x 10 12 to about 1 x 10 14 PFU, about 1 x 10 13 to about 1 x 10 14 PFU, about 5 x
  • 10 13 to about 1 x 10 14 PFU about 1 x 10 11 to about 5 x 10 13 PFU, about 2 x 10 11 to about 5 x 10 13 PFU, about 5 x 10 11 to about 5 x 10 13 PFU, about 1 x 10 12 to about 5 x 10 13 PFU, about 2 x 10 12 to about 5 x 10 13 PFU, about 5 x 10 12 to about 5 x 10 13 PFU, or about 1 x 10 13 to about 5 x 10 13 PFU of the viruses.
  • a method as provided herein can comprise harvesting a population of viruses, e.g., oncolytic viruses, e.g., recombinant oncolytic viruses, from a culture that comprises the viruses at a viral titer of about 1.5 x 10 8 to about 2 x 10 10 PFU per mL of said culture, such as about 1.5 x
  • viruses e.g., oncolytic viruses, e.g., recombinant oncolytic viruses
  • a method as provided herein can comprise harvesting from a culture a population of viruses, e.g., oncolytic viruses, e.g., recombinant oncolytic viruses, to produce a lysate comprising the population of viruses, and clarifying the lysate to produce a population of purified viruses, the population of purified viruses can comprise at least about 50% to about 90% of the population of viruses in the lysate.,.
  • viruses e.g., oncolytic viruses, e.g., recombinant oncolytic viruses
  • the population of purified viruses can 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 in the cell culture, e.g., oncolytic viruses, e.g., recombinant oncolytic viruses.
  • the population of purified viruses can comprise 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 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 population of viruses in the cell culture, e.g., oncolytic viruses, e.g., recombinant oncolytic viruses.
  • oncolytic viruses e.g., recombinant oncolytic viruses.
  • a method as provided herein can comprise purifying the harvested virus lysate to generate a purified preparation of the virus.
  • a purified preparation of the virus generated according to the methods provided herein can comprise 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 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
  • a purified preparation of the virus generated according to the methods provided herein can comprise about 2 ng, about 3 ng, about 4 ng, about 5 ng, about 6 ng, about 7 ng, about 8 ng, about 9 ng, about 10 ng, about 11 ng, about 12 ng, about 13 ng, about 14 ng, about 15 ng, about 16 ng, about 17 ng, about 18 ng, about 19 ng, about 20 ng, about 21 ng, about 22 ng, about 23 ng, about 24 ng, about 25 ng, about 28 ng, about 30 ng, about 50 ng, about 82 mg, about 100 ng, about 200 ng, about 500 ng, about 750 ng, about 1 pg, or about 2 pg of host cell DNA in a unit dose of the virus.
  • the host cell DNA in the purified preparation of the virus can be measured by extraction and purification of DNAs using a silica-based column, or extraction of DNAs by standard methods. In some cases, the host cell DNA in the purified preparation of the virus can be measured after processing with QuickExtractTM or Qiagen ® kits by quantitative real-time PCR with probes specific to the host cell genome.
  • a unit dose of the virus can comprise about 0.5 x 10 9 , about 1 x 10 9 , about 2 x 10 9 , about 5 x 10 9 , about 7.5 x 10 9 , about 1 x 10 10 , about 2 x 10 10 , about 5 x 10 10 , about 7.5 x 10 10 , about 1 x 10 11 , about 2 x 10 11 , about 5 x 10 11 , about 7.5 x 10 11 , about 1 x 10 12 , about 2 x 10 12 , about 5 x 10 12 , about 7.5 x 10 12 , about 1 x 10 13 , about 2 x 10 13 , about 5 x 10 13 , about 7.5 x 10 13 , about 1 x 10 14 , about 2 x 10 14 , about 5 x 10 14 , about 7.5 x 10 14 , about 1 x 10 15 , or about 1 x 10 16 PFU of the virus.
  • a unit dose of the virus can comprise about 1 x 10 9 to about 1 x 10 13 PFU of the virus, e.g ., oncolytic virus, e.g. , recombination oncolytic virus. In some cases, a unit dose of the virus can comprise about 1 x 10 9 to about 1 x 10 13 viruses, such as 5 xlO 9 viruses. In some examples, the unit dose of the virus can include intracellular mature virus (IMV), intracellular enveloped virus (IEV), cell-associated enveloped virus (CEV) and extracellular enveloped virus (EEV).
  • IMV intracellular mature virus
  • IEV intracellular enveloped virus
  • CEV cell-associated enveloped virus
  • EEV extracellular enveloped virus
  • a purified preparation of the virus generated according to the methods provided herein can comprise at most about 5 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 dose of 5 x 10 9 of the virus.
  • a purified preparation of the virus generated according to the methods provided herein can comprise 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 gg, about 1.5 gg, about 2 gg, about 3 gg, or about 5 gg of host cell DNA in dose of 5 x 10 9 of the virus.
  • a purified preparation of the virus generated according to the methods provided herein can comprise 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 gg, about 0.3 gg, about 0.4 gg, or about 1 gg of host cell DNA in dose of 1 x 10 9 of the virus.
  • a purified preparation of the virus generated according to the methods provided herein can comprise about 51 ng of host cell DNA in dose of 1 x 10 9 of the virus.
  • a purified preparation of the virus generated according to the methods provided herein can comprise at most about 0.1 gg, at most about 0.2 gg, at most about 0.3 gg, at most about 0.4 gg, at most about 0.5 gg, at most about 0.6 gg, at most about 0.7 gg, at most about 0.8 gg, at most about 0.9 gg, at most about 1 gg, at most about 1.1 gg, at most about 1.2 gg, at most about 1.3 gg, at most about 1.4 gg, at most about 1.5 gg, at most about 2 gg, at most about 5 gg, at most about 7.5 gg, at most about 10 gg, at most about 20 gg, at most about 30 gg, at most about 50 gg, or at most about 100 gg of host cell protein in a unit dose of the virus.
  • a purified preparation of the virus generated according to the methods provided herein can comprise about 0.1 gg, about 0.2 gg, about 0.3 gg, about 0.4 gg, about 0.5 gg, about 0.6 gg, about 0.7 gg, about 0.8 gg, about 0.9 gg, about 1 gg, about 1.1 gg, about 1.2 gg, about 1.3 gg, about 1.4 gg, about 1.5 gg, about 2 gg, about 5 gg, about 7.5 gg, about 10 gg, about 20 gg, or about 30 gg of host cell protein in a unit dose of the virus.
  • a purified preparation of the virus generated according to the methods provided herein can comprise from about 0.1 gg to about 7.5 gg of host cell protein in a unit dose of said recombinant oncolytic virus.
  • the host cell protein content is determined by ELISA (enzyme-linked immunosorbent assay) targeting one or more host cell proteins.
  • a purified preparation of the virus generated according to the methods provided herein can comprise at most about 0.1 gg, at most about 0.5 gg, at most about 1 gg, at most about 1.5 gg, at most about 2 gg, at most about 2.5 gg, at most about 3 gg, at most about 3.5 gg, at most about 4 gg, at most about 4.5 gg, at most about 5 gg, at most about 5.5 gg, at most about 6 gg, at most about 6.5 gg, at most about 7 gg, at most about 7.5 gg, at most about 10 gg, or at most about 20 gg of host cell protein in dose of 5 x 10 9 of the virus.
  • a purified preparation of the virus generated according to the methods provided herein can comprise about 0.1 pg, about 0.5 pg, about 1 pg, about 1.5 pg, about 2 pg, about 2.5 pg, about 3 pg, about 3.5 pg, about 4 pg, about 4.5 pg, about 5 pg, about 5.5 pg, about 6 pg, about 6.5 pg, about 7 pg, about 7.5 pg, about 10 pg, or about 20 pg of host cell protein in dose of 5 x 10 9 of the virus.
  • a purified preparation of the virus generated according to the methods provided herein can comprise about 0.02 pg, about 0.1 pg, about 0.2 pg, about 0.3 pg, about 0.4 pg, about 0.5 pg, about 0.6 pg, about 0.7 pg, about 0.8 pg, about 0.9 pg, about 1 pg, about 1.1 pg, about 1.2 pg, about 1.3 pg, about 1.4 pg, about 1.5 pg, about 2 pg, or about 4 pg of host cell protein in dose of 5 x 10 9 of the virus.
  • Methods and systems provided herein can be applicable to manufacturing a variety of different viruses, e.g., oncolytic viruses, e.g., recombinant oncolytic viruses.
  • the methods and systems provided herein can be applicable to manufacturing vaccinia viruses, e.g., recombinant vaccinia viruses.
  • Exemplary oncolytic viruses that the methods and system provided herein are applicable to include measles virus, poliovirus, poxvirus, vaccinia virus, an adenovirus, an adeno associated virus, herpes simplex virus, vesicular stomatitis virus, reovirus, Newcastle disease virus, Seneca virus, lentivirus, mengovirus, and myxoma virus.
  • the oncolytic virus can be a vaccinia virus.
  • the methods and systems provided herein can be applicable to different strain variants of vaccinia viruses, such as, Lister, Dryvax, EM63, ACAM2000, Modified Vaccinia Ankara, LC16m8, CV-1, Western Reserve, Copenhagen, Connaught
  • vaccinia viruses such as, Lister, Dryvax, EM63, ACAM2000, Modified Vaccinia Ankara, LC16m8, CV-1, Western Reserve, Copenhagen, Connaught
  • Viruses that the methods provided herein are applicable to can comprise genetic modifications, such as, deletion or mutation of one or more viral endogenous genes, or introduction of an exogenous virus.
  • the virus can be a recombinant vaccinia virus.
  • the recombinant vaccinia virus can comprise 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.
  • a deletion of a viral gene may include a partial or a complete deletion of the viral gene.
  • “partial deletion” or“mutation” can refer to an in situ partial deletion or mutation of an endogenous viral gene, respectively. Alternatively, they can refer to replacing the endogenous viral gene with an otherwise identical exogenous nucleic acid that lacks a portion of the gene (“partial deletion”) or has one or more nucleotide change in the gene (“mutation”).
  • the virus that can be manufactured by the methods disclosed herein can be vaccinia virus comprising one or more genetic modifications.
  • the vaccinia virus can comprise TK (thymidine kinase) viral gene mutation, such that the vaccinia virus is thymidine kinase negative, C12L (IL18 binding protein) viral gene deletion, TRIF gene insertion, HPGD gene insertion, and reduced surface glycosylation.
  • the vaccinia virus can comprise at least one of the following engineered modifications: (i) C12L (IL18 binding protein) viral gene deletion; (ii) TRIF gene insertion; or (iii) HPGD gene insertion.
  • the vaccinia virus can comprise a reduced surface glycosylation.
  • the reduced surface glycosylation can be reduced sialylation of the surface of the virus.
  • the vaccinia virus can have a reduced glycosylation level as compared to a modified vaccinia virus.
  • the vaccinia virus can be treated with an agent that reduces the amount of glycosylation (e.g., sialylation) during or after the manufacture process as described herein but prior to administration to a host.
  • the vaccinia virus can further comprise one or more modifications selected from the group of a A34R Lysl51 to Glu mutation; complete or partial deletion of B5R; mutation/deletion of A36R and/or mutation/deletion of A56R.
  • the vaccinia virus can be a deglycosylated VV which comprises a complete or partial deletion of B5R.
  • any suitable host cells can be chosen for manufacturing viruses according to the present disclosure.
  • the host cells used in the methods provided herein can be adherent cells.
  • the host cells can be cultured in suspension.
  • the host cells can be subcultured from an established cell line, such as, but not limited to, human embryonic kidney (HEK) cells such as HEK 293 cells, African green monkey kidney (AGMK) cells such as Vero cells, Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells, and Hela cells.
  • the host cells can be Hela cells.
  • the hots cells can be genetically modified cells, for instance, Hela cells that can be genetically modified by
  • the host cells can be primary cells obtained from an organism, e.g., human.
  • the host cells can be immortalized cells that can be passaged (or subcultured) indefinitely.
  • the method provided herein can comprise directly infecting the host cells with the virus, e.g., oncolytic virus, e.g., recombinant oncolytic virus.
  • the host cells can be brought into direct contact with the virus to become infected.
  • the viruses can be added into the culture medium of the host cells for inoculation.
  • the host cells can be infected by the virus at a multiplicity of infection (m.o.i.) 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.
  • m.o.i. multiplicity of infection
  • multiplicity of infection can be calculated as plaque forming units of the virus used for infection divided by the number of host cells.
  • the host cells can be infected by the virus at a m.o.i. of 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 0.035, 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.
  • the host cells can be infected by the virus at a m.o.i. 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 .
  • the host cells can be infected by the virus at an m.o.i. of about 0.001 to about 0.02 PFU/cell.
  • Methods provided herein can comprise growing a culture comprising a plurality of virus- infected host cells in a bioreactor. Methods provided herein can produce viruses at high titer as described above.
  • the bioreactor as used in the methods provided herein can be an integrated mixing system, offering even distribution of the culture medium. In some cases, the bioreactor can be capable of culturing cells at high density, leading to high production yield. In some cases, the bioreactor can have linear scalability, e.g., the configuration of the bioreactor can be adapted to both bench scale and industrial scale production depending on the need.
  • the bioreactor can comprise a fixed-bed reactor.
  • Methods and systems as provided herein can comprise a fixed bed reactor that comprise a vessel filled or packed with carrier materials that can be used as support for the immobilization of cells.
  • the fixed bed reactor can also comprise a conditioning vessel that contains the culture medium.
  • the vessel with carrier materials and the conditioning vessel can be coupled via a circulation system.
  • the circulation system can be a circulation loop.
  • the circulation system can contain oxygen enriched medium that can be pumped from the conditioning vessel through the fixed-bed and back. In some cases, oxygen can be supplied directly into the fixed-bed.
  • the cells In a fixed bed reactor, the cells can be retained in the fixed-bed.
  • the medium in the conditioning vessel can be exchanged either batch wise or continuously, so that the exhausted product-containing medium can be replaced with fresh medium. In some cases, the medium can be exchanged continuously.
  • the fixed bed reactor can be an axial flow-based fixed bed system, in which an axial pumping of the media through the fixed bed can be used to provide the cells with fresh media and oxygen. In some cases, the fixed bed reactor can be a radial flow-based fixed bed system, in which a radial pumping of the media through the fixed bed, e.g., the media can be pumped through the fixed bed from a center point radially toward the surrounding the fixed bed.
  • the fixed bed can comprise microcarriers. The microcarriers can be made of appropriate materials.
  • the microcarrier can be made of PET (polyethylene terephthalate).
  • the microcarrier can be made of synthetic materials, natural materials, or both. Synthetic materials include, but not limited to, glass (e.g., natron, borosilicate), DEAE-dextran, polypropylene, polyurethane, ceramic, acrylamide, polyhydroxyethylmethacrylate, polystyrene, and
  • Natural materials include, but not limited to, collagen, cellulose, gelatin, fibrin, chitin and its derivatives, chitosan, alginate, polysaccharide, and polyglycan.
  • the cell culture comprising virus-infected host cells can be grown in a fixed-bed bioreactor.
  • the fixed-bed bioreactor can comprise microcarriers that can provide relatively large surface-to-volume ration with sufficient oxygen and nutrition exposure to the cells.
  • the fixed-bed bioreactor can have a low draining time, e.g., time needed to drain up culture medium in the bioreactor to a maximum extent. In some cases, the draining time can be less than about 3 hours, less than about 2 hours, less than about 1 hours, less than about 30 min, less than about 20 min, less than about 15 min, less than about 14 min, less than about 13 min, less than about 12 min, less than about 11 min, or less than about 10 min.
  • the draining time can be about 80 min, about 50 min, about 30 min, about 12.5 min, about 12 min, about 11.5 min, about 11 min, or about 10.5 min.
  • the fixed-bed bioreactor can have a low residual volume, e.g., the residual volume of culture medium when the medium can be drained to the maximum extent.
  • the bioreactor can 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.
  • the bioreactor can 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.
  • the method can comprise growing the culture in a fixed bed that comprises a volume of about 0.01 L to about 25 L.
  • the fixed bed can comprise a volume of at least about 0.005 L, at least about 0.01 L, at least about 0.05 L, at least about 0.1 L, at least about 0.5 L, at least about 1 L, at least about 2 L, at least about 3 L, at least about 4 L, at least about 5 L, at least about 6 L, at least about 7 L, at least about 8 L, at least about 9 L, at least about 10 L, at least about 12 L, at least about 14 L, at least about 15 L, at least about 16 L, at least about 18 L, at least about 20 L, at least about 21 L, at least about 22 L, at least about 23 L, at least about 24 L, at least about 25 L, at least about 26 L, at least about 27 L, at least about 28 L, at least about 29 L, at least about 30 L, at least about 35 L, at least about 40 L, at least about
  • the fixed bed can comprise a volume of about 0.005 L, about 0.01 L, about 0.05 L, about 0.1 L, about 0.5 L, about 1 L, about 2 L, about 3 L, about 4 L, about 5 L, about 6 L, about 7 L, about 8 L, about 9 L, about 10 L, about
  • the fixed bed can comprise a volume of about 1 L. In some cases, the fixed bed can comprise a volume of about 70 L.
  • the fixed bed can comprise a volume of about 0.005 L to about 25 L, about 0.01 L to about 25 L, about 0.05 L to about 25 L, about 0.1 L to about 25 L, about 0.15 L to about 25 L, about 0.2 L to about 25 L, about 0.5 L to about 25 L, about 1 L to about 25 L, about 5 L to about 25 L, about 10 L to about 25 L, about 15 L to about 25 L, about 20 L to about 25 L, about 0.005 L to about 15 L, about 0.05 L to about 15 L, about 0.5 L to about 15 L, about 5 L to about 15 L, about 10 L to about 15 L, about 0.005 L to about 50 L, about 0.5 L to about 50 L, about 5 L to about 50 L, about 10 L to about 50 L, about 15 L to about 50 L, about 25 L to about 50 L, about 30 L to about 50 L, about 1 L to about 100 L, about 5 L to about 100 L, about 10 L to about 100 L, about 20 L to about 100 L, about 50 L
  • the method can comprise growing the culture in a fixed bed that comprises a compaction density of at least about 80 g/L to about 144 g/L.
  • the fixed bed can comprise a compaction 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
  • the fixed bed can comprise a compaction density of at least about 96 g/L. In some cases, the fixed bed can comprise a compaction 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.
  • the method can comprise seeding host cells in the bioreactor for growing for a certain period before virus inoculation. In other cases, the method can comprise seeding the virus infected host cells in the bioreactor for growing the culture. In some cases, the host cells prior to virus inoculation can be seeded at a density of at least about 1,000 cells/cm 2 to about 50,000 cells/cm 2 in the bioreactor. In some cases, the host cells prior to virus inoculation can be seeded at a density of at least about 10,000 cells/cm 2 to about 400,000 cells/cm 2 .
  • the host cells prior to virus inoculation can 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
  • the host cells can be seeded at a density of about 2,500 cells/cm 2 .
  • the host cells prior to virus inoculation can be seeded at a density of about 1,500 cells/cm 2 , about 1,600 cells/cm 2 , about 1,800 cells/cm 2 , about 2,200 cells/cm 2 , about 2,200 cells/cm 2 , about 2,400 cells/cm 2 , about 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 , about 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/
  • the method as described herein can comprise growing the host cells in the bioreactor for a determined period and then infecting the host cells at a determined density in the bioreactor.
  • the host cells can 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.
  • the host cells can be grown in the bioreactor for about 5, 6, 7, 8, 9, or 10 days.
  • the present disclosure provides a bioreactor comprising a culture of host cells comprising a large number of viruses.
  • the bioreactor can be a bioreactor that can be used to culture virus-infected host cells according to the methods provided herein.
  • An exemplary bioreactor can comprise a culture of a host cells comprising at least about 50 PFU to about 350 PFU of a recombinant oncolytic virus per host cell.
  • Another exemplary bioreactor can comprise a culture of a host cells comprising at least about 1.5 x 10 11 to about 5 x 10 13 PFU of a recombinant oncolytic virus.
  • the bioreactor can comprise a fixed bed, and the fixed bed can comprise the culture of host cells.
  • the culture of host cells can be propagated in microcarriers within the fixed bed.
  • the culture can comprise a host cell density of about 2,000 cells/cm 2 to about 10,000 cells/cm 2 of the fixed bed.
  • the fixed bed can comprise a volume of about 0.01 L to about 25 L.
  • the fixed bed can comprise a compaction density of at least about 80 g/L to about 144 g/L.
  • the fixed bed can comprise a surface area of about 0.2 m 2 to about 500 m 2 , such as about 0.2 m 2 to about 0.5 m 2 , about 0.4 m 2 to about 1 m 2 , about 0.8 m 2 to about 1.5 m 2 , about 1 m 2 to about 5 m 2 , about 4 m 2 to about 6 m 2 , about 5 m 2 to about 20 m 2 , about 20 m 2 to about 50 m 2 , about 50 m 2 to about 100 m 2 , about 100 m 2 to about 200 m 2 , about 200 m 2 to about 400 m 2 , about 250 m 2 to about 500 m 2 .
  • the fixed bed can comprise a surface area of about 4 m 2 . In some cases, the fixed bed can comprise a surface area of about 500 m 2 .
  • the virus production scale can depend on the surface area of the fixed bed used in the method as described herein. For example, if a fixed bed having a surface area of about 4 m 2 is used to culture virus-infected cells, 4 xlO 11 to 1 x 10 12 PFU of virus can be produced using the method provided herein. In some cases, if a fixed bed having a surface area of about 500 m 2 is used to culture virus-infected cell, 1 x 10 13 to 2.5 x 10 14 PFU of virus can be produced using the method provided herein.
  • the method as provided herein can comprise growing a culture for a certain period of time, during which the viruses can replicate.
  • the method as provided herein can comprise growing a culture for a sufficient period of time for reaching a desired viral titer in the culture.
  • the culture can be grown for about 24 hours to about 96 hours.
  • the culture can 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
  • the culture can be grown for about 72 hours. In some cases, the culture can 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.
  • the culture can 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 case, the culture can be grown for about 3 days.
  • the method as provided herein can comprise growing a culture at a dissolved oxygen tension (DOT) level of at least about 20% to about 100%.
  • the culture can comprise 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%.
  • DOT dissolved oxygen tension
  • the culture can 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%.
  • the culture can comprise a DOT level of about 50%.
  • the culture can comprise 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 case, the culture can comprise a DOT level of about 50%.
  • the method as provided herein can comprise growing a culture comprising the virus- infected host cells at a temperature of about 32 °C to about 38 °C.
  • the method as provided herein can comprise growing a culture comprising virus-infected host cells at a maintained temperature.
  • the maintained temperature can 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.
  • the temperature of the culture can vary from time to time, e.g., at a first maintained temperature for a first period of time, and at a second maintained temperature for a second period of time, depending on the life cycle of the viruses, the growth stage of the host cells, or both.
  • the maintained temperature can be controlled very precisely, for instance, with a variation 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 at which the temperature of the culture can be maintained can be adjusted depending on a number of factors, such as, the life cycle of the viruses, the temperature sensitivity of the virus and the host cells, and the culture medium. In some cases, the culture can be maintained at about 37°C.
  • the method provided herein can comprise growing a culture comprising virus-infected host cells at a pH of about 7.0 to about 7.5.
  • the pH of the culture can be maintained at a certain level, such as 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.
  • the maintained pH can be at least about 8.0, or at most about 7.0, depending on the type of the host cells and the viruses.
  • the pH of the culture can be maintained about 7.2.
  • the method can comprise growing a culture at a pH maintained with a variation 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.
  • the pH of the culture comprising virus-infected host cells can be adjusted by exchanging the medium with fresh medium that can be well pH-adjusted.
  • the pH of the culture can be maintained by pH buffer.
  • the pH can be adjusted by CO2 gas control or by supplementing acid or alkaline into the culture medium, for instance, adding HC1 to reduce the pH, or adding NaOH to increase the pH, to a desired level.
  • pH can also be modified by adding CO2.
  • Some aspects of the present disclosure provide a method of harvesting viruses, e.g., oncolytic viruses, e.g., recombinant oncolytic viruses, from a culture comprising a plurality of virus-infected host cells.
  • Harvesting viruses can comprise lysis of the virus-infected host cells, thereby releasing the viruses into the lysate, and collecting the lysate.
  • Harvesting viruses can also comprise pre-harvest wash of the bioreactor. In some cases, the pre-harvest wash can be conducted in order to remove contaminants (e.g., proteins, nucleic acids, exhaust products, and other molecules in the culture medium) from the lysate.
  • the bioreactor e.g., the carriers
  • the wash solution can be a buffer solution, such as Tris buffer solution.
  • the wash solution can comprise 75 mM Tris and a pH of 7.3.
  • the washing step can be performed at any appropriate temperature for any appropriate duration.
  • the parameters of the washing step such as, the wash solution (e.g., ionic concentration, pH), temperature, washing duration, can be well adjusted so that the host cells can be rarely lysed at this step.
  • the carriers can be washed by 75mM Tris solution (pH 7.3, 25 °C) for 30 min.
  • the present disclosure provides a pH shift method for harvesting viruses from the culture.
  • Harvesting can comprise incubating the virus-infected host cells in an alkaline buffer.
  • harvesting also can comprise incubating the virus-infected host cells with an enzyme to degrade cell debris.
  • harvesting can comprise incubating the virus-infected host cells with an enzyme to degrade cell debris and an alkaline buffer.
  • the alkaline buffer can comprise a pH of at least about 8.
  • the alkaline buffer can 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.
  • the alkaline buffer can 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.
  • the alkaline buffer can comprise Tris.
  • the alkaline buffer can comprise a pH of about 9.5.
  • the alkaline buffer can comprise a pH of about 9.6.
  • the alkaline buffer can have a determined pH 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.
  • the alkaline buffer can comprise 75mM Tris, 2mM MgCh and pH of 9.5.
  • the alkaline buffer can comprise 90mM Tris, 2mM MgCb and pH of 9.5.
  • the alkaline buffer can comprise 75mM Tris, 2mM MgCh and pH of 9.6.
  • the alkaline buffer can comprise 75mM Tris, 2.5mM MgCh and pH of 9.6.
  • high pH of the lysis buffer can contribute to better lysis of the host cells, the endosomes of the host cells, or both, so that the viruses that can be “trapped” therein can be more easily exposed, thereby leading to a higher yield of viruses.
  • an enzyme can be used to degrade cell debris in the method provided herein.
  • the enzyme can comprise nuclease, e.g., endonuclease for clearing free nucleic acids in the cell lysate.
  • the enzyme can comprise proteases, e.g., TrypLE or trypsin.
  • a nuclease can refer to an enzyme capable of cleaving the phosphodi ester bonds between monomers of nucleic acids. Nucleases can cause single and double stranded breaks in their target molecules. Different nucleases, such as exonuclease and endonuclease can be used in the methods provided herein, which act on different loci of a nucleic acid target. Exonucleases can digest nucleic acids from the ends, while endonucleases can act on regions in the middle of target molecules.
  • the nucleases as used herein can be naturally-occurring nucleases, or recombinant (engineered) nucleases, whose amino acid sequences deviate from naturally-occurring nucleases.
  • genetically engineered Serratia nuclease e.g., Benzonase®
  • the method can comprise incubating the virus-infected host cells with an enzyme to degrade cell debris that can be about 50 IU/mL to about 200 IU/mL.
  • the enzyme to degrade cell debris can 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.
  • the enzyme to degrade cell debris can 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.
  • the enzyme to degrade cell debris can 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.
  • the virus harvesting can comprise incubating the virus-infected host cells with Benzonase® at about 150 IU/mL
  • the incubation of the virus-infected host cells in a cell lysis buffer for virus harvesting can last for an appropriate amount of time. In some cases, the incubation can be for about 2 hours to about 8 hours. In some cases, the incubation can 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, the incubation can be for at least about 2 hours to at least about 8 hours.
  • the incubation can 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, the incubation can be for about 2 hours to about 8 hours. In some cases, the incubation can 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.
  • the virus harvesting can comprise incubating the virus-infected host cells in a cell lysis buffer for about 4 hours.
  • the incubation of the virus-infected host cells in a cell lysis buffer for virus harvesting can be at any appropriate temperature, for instance, about 22 °C to about 28 °C.
  • the temperature for the incubation for virus harvesting can be at about 22 °C, at about 23 °C, at about 24 °C, at about 25 °C, at about 26 °C, at about 27 °C, at about 28 °C, at about 29 °C, at about 30 °C, at about 31 °C, or at about 32 °C.
  • the temperature can be maintained at a certain degree.
  • the maintained temperature can be controlled very precisely, for instance, with a variation 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 virus harvesting can comprise incubating the virus-infected host cells at about 25 °C.
  • the method provided herein can further comprise conducting a clarification step to generate a filtered lysate.
  • the step of clarification can be performed to eliminate or reduce contaminants and purify the virus.
  • the step of clarification as provided herein can comprise filtration and nuclease treatment.
  • the step of clarification can comprise purification of the virus by centrifugation.
  • the step of clarification can comprise filtration.
  • the lysate harvested by the method provided herein can be subject to filtration through a filter to generate a filtered lysate.
  • a filter can have a pore size that retains particles of a size larger than the pore size and passes particles of a size smaller than the pore size. Any appropriate filter can be chosen to conduct the filtration step for the purpose of isolating and purifying the virus.
  • the filter can have an average filter size that can be at least about 0.15 pm, at least about 0.18 pm, at least about 0.2 pm, at least about 0.22 pm, at least about 0.25 pm, at least about 0.28 pm, at least about 0.3 pm, at least about 0.35 pm, at least about 0.4 pm, at least about 0.45 pm, at least about 0.5 pm, at least about 0.55 pm, at least about 0.6 pm, at least about 0.65 pm, at least about 0.7 pm, at least about 0.75 pm, at least about 0.8 pm, at least about 0.85 pm, at least about 0.9 pm, at least about 0.95 pm, or at least about 1 pm.
  • the filter can have an average filter size that can be at most about 0.18 pm, at most about 0.2 pm, at most about 0.22 pm, at most about 0.25 pm, at most about 0.28 pm, at most about 0.3 pm, at most about 0.35 pm, at most about 0.4 pm, at most about 0.45 pm, at most about 0.5 pm, at most about 0.55 pm, at most about 0.6 pm, at most about 0.65 pm, at most about 0.7 pm, at most about 0.75 pm, at most about 0.8 pm, at most about 0.85 pm, at most about 0.9 pm, at most about 0.95 pm, at most about 1 pm, or at most about 1.5 pm.
  • filtration can be performed with more than one filter, e.g., 2, 3, 4, 5, 6, or more filters.
  • the more than one filter can have the same pore size or different sizes, for instance, in some examples, the lysate can be filtered by a filter with average size of about 5 pm followed by a filter with average size of about 1.2 pm. In some examples, the lysate can be filtered by a filter with average size of about 5 pm followed by a filter with average size of about 2.4 pm, and then about a filter about 1 pm.
  • the clarification process can be carried out by flushing the lysate through a filter with the aid of a flush buffer.
  • the flow rate for the clarification process can be about 50 to about 500 mL/ min, such as 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.
  • the flow rate for the clarification process can be about 70, 80, 90, 100, 110, 120, 130, 150, 175, 200, 225, 250, 275, 280, 290, 300, or 320 mL/min.
  • Flow rate can sometimes be described in LMH (L/m 2 /h).
  • the flow rate for the clarification process can be about 100 to about 750 LMH, such as about 100 to about 175 LMH, about 125 to about 200 LMH, about 150 to about 225 LMH, about 175 to about 250 LMH, about 200 to about 275 LMH, about 225 to about 300 LMH, about 250 to about 325 LMH, about 275 to about 350 LMH, about 300 to about 375 LMH, about 325 to about 400 LMH, about 350 to about 425 LMH, about 375 to about 450 LMH, about 400 to about 475 LMH, about 425 to about 500 LMH, about 450 to about 525 LMH, about 475 to about 550 LMH, about 500 to about 575 LMH, or about 525 to about 600 LMH.
  • the flow rate for the clarification process can be about 150 LMH. In some cases, the flow rate for the clarification process can be about 490 LMH. In some cases, the flush rate can be chosen differently for filters of different pore sizes, for example, a higher flow rate can be used for filter of a larger pore size whereas a lower flow rate can be used for filter of a smaller pore size. In some cases, the flush buffer can have similar constituents as the lysate, for example, similar ions and ionic concentrations, and similar pH, or different constituents from the lysate.
  • the clarification process can be carried out at room temperature, or any other appropriate temperature, for instance, at about 20 °C, 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 28 °C, 30 °C, 32 °C, or 37 °C.
  • the filtered lysate can be further treated with an enzyme to degrade cell debris, e.g., a nuclease, e.g., Benzonase®, to eliminate contaminants, e.g., nucleic acids, and to generate a depth filtrate.
  • a nuclease e.g., Benzonase®
  • the filtered lysate can be incubated with a buffer containing Benzonase® for a sufficient amount of time.
  • the filtered lysate can 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 Benzonase®.
  • 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
  • the treatment of the filtered lysate by the enzyme to degrade cell debris can be conducted at 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
  • the treatment of the filtered lysate by the enzyme to degrade cell debris can be conducted at pH of about 8.0, about 8.1, about 8.2, about
  • the treatment of the filtered lysate by the enzyme to degrade cell debris can be conducted at 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.
  • the filtered lysate can be treated by the 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.
  • the filtered lysate can be treated by the 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.
  • the filtered lysate can be treated by the 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.
  • the filtered lysate can be treated with Benzonase® for about 18 hours at room temperature.
  • the enzyme treatment can be conducted in the presence of a monocation, e.g., Na + , e.g., sodium chloride (NaCl) in the solution.
  • a monocation e.g., Na + , e.g., sodium chloride (NaCl)
  • the enzyme treatment can be conducted in the presence of high concentration of monocation, e.g., Na + , e.g., sodium chloride (NaCl), in the solution, for instance, 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
  • the enzyme treatment can be conducted in the presence of high concentration of monocation, e.g., Na + , e.g., sodium chloride (NaCl), in the solution, for instance, 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.
  • monocation e.g., Na + , sodium chloride (NaCl)
  • NaCl sodium chloride
  • the virus lysate can be stored at a low temperature before the next step.
  • the filtered lysate can be saved at low temperature before the treatment by enzyme to degrade cell debris.
  • the filtered lysate can be stored at about 2 to 8 °C for a certain period of time, e.g., 6 hours, 12 hours, 24 hours, 2 days, 3 days, or 6 days.
  • the methods provided herein can comprise conducting tangential flow filtration (TFF) of the filtered lysate to generate a purified preparation of said recombinant oncolytic virus.
  • tangential flow filtration can be followed by diafiltration to further purify the virus.
  • more than one round of TFF or diafiltration can be conducted.
  • 2 rounds of diafiltration can be conducted.
  • 2, 3, 4, 5, or more rounds of TFF or diafiltration can be conducted.
  • other techniques can be used in the subject methods for purifying the viruses as an alternative of or in combination with the TFF process as described herein, for instance, ultrafiltration or size-exclusion
  • Tangential Flow Filtration also known as crossflow filtration
  • TFF can refer to a filtration process where the feed stream passes parallel to the filter membrane face as one portion passes through the membrane (permeate) while the remainder (retentate) can be recirculated back to the feed reservoir.
  • TFF can be performed to concentrate the filtered lysate.
  • the filtered lysate can be concentrated by at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 12 times, at least 15 times, at least 18 times, at least 20 time, or at least 30 times.
  • the filtered lysate can be concentrated by about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 12 times, about 15 times, about 18 times, about 20 time, or about 30 times. In some cases, after TFF, the filtered lysate can be concentrated by about 10 times.
  • a TFF filter membrane having a pore size that can be 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.
  • a TFF filter membrane having a pore size that can be 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.
  • TFF filter and filtration parameters can be selected and adjusted depending on a number of parameters, including, but not limited to, the type of virus to be manufactured, the type of host cells, cell culture medium, and filter buffer.
  • the TFF process can be carried out at any appropriate temperature, such as at about 20 °C, 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 28 °C, 30 °C, 32 °C, or 37 °C.
  • the loading for TFF can be 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 .
  • the loading for TFF can be about 10 L/m 2 , about 20 L/m 2 , about 30 L/m 2 , about 40 L/m 2 , about 50 L/m 2 , about 60 L/m 2 , about 70 L/m 2 , about 80 L/m 2 , about 100 L/m 2 , about 120 L/m 2 , about 150 L/m 2 , or about 200 L/m 2 .
  • the TFF process can be carried out at shear rate of about 2000 to about 6000 s 1 , such as about 2000 to about 3500 s 1 , about 2500 to about 4000 s 1 , about 3000 to about 4500 s 1 , about 3000 to about 5000 s 1 , about 3500 to about 5000 s 1 , about 3500 to about 5500 s 1 , about 4000 to about 6000 s 1 , or about 4500 to about 6000 s 1 .
  • the TFF process can be carried out at shear rate of about 2000 s 1 , about 2500 s 1 , about 3000 s 1 , about 3500, about 3500 s 1 , about 4000 s 1 , about 4500 s 1 , about 5000 s 1 , about 5500 s about 6000 s 1 .
  • the lysate can be flowed at a rate of about 0.25 to about 2.5 L/min, such as 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.
  • the flow rate and the shear rate can be chosen in combination, for instance, lysate can be flowed at about 1.5 L/min at a shear rate of 5000 s 1 , or about 0.8 L/min at a shear rate of 3000 s 1 .
  • permeate flux rate can be controlled throughout the TFF process.
  • the permeate flux rate can be controlled at between 5 and about 50 LMH, such as 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.
  • the permeate flux rate can be controlled at between about 15 to about 20 LMH.
  • the permeate flux rate can be controlled at between about 10 to about 30 LMH.
  • the permeate flux rate can be controlled at between about 15 to about 30 LMH.
  • the permeate flux rate can be controlled at between about 15 LMH. In some cases, the permeate flux rate can be controlled at between about 20 LMH.
  • the method can further comprise diafiltration of the filtered lysate or the purified lysate.
  • diafiltration can be performed to remove salts or other contaminants from the lysate to generate a purified virus preparation.
  • diafiltration can be a fractionation process that washes smaller molecules through a membrane and leaves larger molecules in the retentate without ultimately changing concentration.
  • Either continuous diafiltration or discontinuous diafiltration can be used in the method provided herein.
  • the diafiltration buffer can be added to the feed reservoir at the same rate as a filtrate can be generated.
  • the volume in the sample reservoir can remain constant, but the small molecules (e.g. salts) that can freely permeate through the membrane can be washed away.
  • each additional volume of buffer exchange can reduce the salt concentration further.
  • a“volume of buffer exchange” can be equal to the volume of the starting solution before the diafiltration solution can be added.
  • the starting solution can be first diluted and then concentrated back to the starting volume. This process can then be repeated until the desirable concentration of small molecules (e.g.
  • a diafiltration process can be carried out using the same TFF system for the earlier concentration process. In some cases, the diafiltration process can be carried out using a different system.
  • a diafiltration can comprise 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.
  • a diafiltration as provided herein can comprise 6 volumes of buffer exchange. In some cases, a diafiltration as provided herein can comprise 10 volumes of buffer exchange.
  • a diafiltration as provided herein can comprise 20 volumes of buffer exchange.
  • the methods provided herein can comprise 2 rounds of diafiltration, a first round having 10 volumes of buffer exchange followed by a second round having 6 volumes of buffer exchange, or a first round having 20 volumes of buffer exchange followed by a second round having 10 volumes of buffer exchange.
  • the first round of buffer exchange can be carried with a buffer having similar constituents to the lysate, whereas the second round of buffer exchange can be carried out with a buffer used to filter out the ions contained in the lysate, for example a buffer containing sucrose.
  • the buffer exchange can be conducted with a buffer comprising a pH of about 7.8.
  • the buffer exchange can be conducted with a buffer comprising a pH of about 7.0. In some cases, the buffer exchange can be conducted with a buffer comprising 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 can be conducted with a buffer comprising 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.
  • the buffer exchange can be conducted with a buffer comprising a pH of about 7 or about 7.8.
  • the buffer exchange can be conducted with a Tris buffer.
  • the Tris buffer can 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.
  • the buffer exchange can be conducted with a buffer comprising about 30 mM, or about 20 mM Tris.
  • the buffer exchange can be conducted with a buffer. Water can be solvent of the buffer.
  • the buffer can comprise uncharged molecules, like saccharide, such as sucrose or glucose, histidine, sorbitol.
  • the buffer can comprise, in some cases, ionic constituents, like MgCk or NaCl, or other stabilizing molecules.
  • the buffer for diafiltration can comprise 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% weight by volume of sucrose.
  • the buffer exchange can be conducted with a buffer comprising about 10% or about 8.5% weight by volume of sucrose.
  • a target concentration can be 0.5X, IX, 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.
  • additional clarification or filtration steps can be performed depending on the need for purity, concentration, or titer of the virus product.
  • a final filtration step can be performed in which TFF retentate can be further filtered through a filter with the aid of a final formulation buffer.
  • the formulation buffer can comprise ion constituents as needed for the final virus product, for instance. Any appropriate flow parameters can be chosen as described above.
  • a filter of relatively small pore size, for instance, 1.2 pm, can be used for the final step. Filters of a variety of pore sizes can be utilized in any aspect of the present disclosure.
  • a filter can have a pore size of about 0.1 pm, 0.2pm, 0.3 pm, 0.4pm, 0.5pm, 0.6pm, 0.7pm, 0.8pm, 0.9pm, 1.0pm, 1.1pm, 1.2pm, 1.3pm, 1.4pm, 1.5pm, 1.6pm, 1.7pm, 1.8pm, 1.9pm, 2.0pm, 2.5pm, 3.0pm, 4.0pm, or up to about 5.0pm.
  • This example describes an exemplary protocol for upstream processes, as depicted in the flow chart in FIG. 1, for manufacturing a recombinant vaccinia virus from culture of virus- infected host cells, harvesting, to cell lysis.
  • DOT dissolved oxygen tension
  • culture medium is exchange with culture medium containing initial recombinant vaccinia virus at multiplicity of infection (m.o.i.) of 0.02. After infection, the virus- infected Hela cells continued to be cultured at 37°C, pH of 7.2, and DOT of 50% for 3 days;
  • the wash buffer is exchanged with lysis medium containing 90 mM Tris, 2 mM MgC12, and 150 iU/mL Benzonase®, and having pH of 9.5.
  • the virus-infected cells are incubated with the lysis medium for 4-6 hours at 25°C, after which the lysis medium containing the cell lysate is collected for further processing.
  • This example describes an exemplary protocol for upstream processes, as depicted in the flow chart in FIG. 2, for manufacturing a recombinant vaccinia virus from culture of virus- infected host cells, harvesting, to cell lysis.
  • Host Hela cells are cultured in iCELLis nano bioreactor (Pall Corporation, Port
  • DOT dissolved oxygen tension
  • culture medium is exchange with culture medium containing initial recombinant vaccinia virus at multiplicity of infection (m.o.i.) of 0.02. After infection, the virus- infected Hela cells continued to be cultured at 37°C, pH of 7.2, and DOT of 50% for 3 days;
  • the wash buffer is exchanged with lysis medium containing 75 mM Tris, 2 mM MgC12, and 150iU/mL Benzonase®, and having pH of 9.5.
  • the virus-infected cells are incubated with the lysis medium for 4 hours at 25°C, after which the lysis medium containing the cell lysate is collected for further processing.
  • This example describes an exemplary protocol for upstream processes, as depicted in the flow chart in FIG. 3, for manufacturing a recombinant vaccinia virus from culture of virus- infected host cells, harvesting, to cell lysis.
  • DOT dissolved oxygen tension
  • culture medium is exchange with culture medium containing initial recombinant vaccinia virus at multiplicity of infection (m.o.i.) of 0.002.
  • multiplicity of infection m.o.i.
  • the virus-infected Hela cells continued to be cultured at 36°C, pH of 7.2, and DOT of 50% for 3 days;
  • the wash buffer is exchanged with lysis medium containing 75 mM Tris, 2 mM MgCh, and 150iU/mL Benzonase®, and having pH of 9.5.
  • the virus-infected cells are incubated with the lysis medium for 4 hours at 25°C, after which the lysis medium containing the cell lysate is collected for further processing.
  • This example describes an exemplary protocol for downstream process, as depicted in FIG. 6, for producing an exemplary vaccinia virus.
  • Lysis time starts when the bioreactor indicates that pH reaches 9.0. Take another offline pH reading to verify it’s reading correctly.
  • [00181] 8. Begin the final round (20mM Tris, pH 7.8, 8.5% sucrose) of diafiltration by opening the VFB line and maintaining constant weight in the sample vessel (again aim for 5000 sec-1 shear rate, maximum 25 LMH), and do 6 volumes of buffer exchange with 20 mM Tris, pH 8, 8.5% sucrose.
  • This example describes an exemplary protocol for downstream process, as depicted in FIG. 8, for producing an exemplary vaccinia virus.
  • the records listed below are records taken from an experimental run of the protocol.
  • Control sample collection as a control, sample ten carriers from the reactor bed and place the sample in 15mM Tris, pH 8.0 (0.05ml/cm 2 ) lysis buffer for 30 min (volume of lysis buffer: 7ml).
  • Lysis time starts when the bioreactor indicates that pH reaches 9.5. Take another offline pH reading to verify it’s reading correctly.
  • TFF-1 Concentration [00231] (a) Fill retentate reservoir with ⁇ 45mL of filtered lysate
  • TFF-1 Diafiltration 1 conduct the first diafiltration with 20 DV of the buffer 75 mM Tris, 2 mM MgC12, 150 mM NaCl, pH 9.00; control the permeate flux at 20 Liter/m2/h (LMH); shear rate at 3000-5000 s-1; no temperature control.
  • LMH Liter/m2/h
  • TFF-1 Diafiltration 2 conduct the second diafiltration with 10 DV of the buffer
  • This example describes an example of virus production according to an embodiment of the present disclosure and the exemplary yields of an exemplary vaccinia virus from the manufacturing process.
  • Host Hela cells were infected with an exemplary vaccinia virus at m.o.i. of 0.02, and cultured in a microcarrier fixed-bed based bioreactor of 4 m 2 surface area. At 72 hours post infection, the viruses were harvested, and subject to clarification, benzonase treatment, TFF for 20 times concentration, DF-1 and DF-2, and then the resultant purified virus preparation was kept at 2-8 °C overnight followed by final filtering.
  • Table 1 shows measurements of the virus titer, amount of host cell protein (HCP), host cell DNA (hcDNA), and BSA in the virus preparation obtained at the end of each manufacture step.
  • This example describes an example of virus production according to an embodiment of the present disclosure and the exemplary yields of an exemplary vaccinia virus from the manufacture process.
  • Host Hela cells were infected with an exemplary vaccinia virus at m.o.i. of 0.001, and cultured in a microcarrier fixed-bed based bioreactor of 4 m 2 surface area. At 72 hours post infection, the viruses were harvested, and subject to clarification, Benzonase® treatment, TFF for 20 times concentration, DF-1 and DF-2, and then the resultant purified virus preparation was kept at 2-8 °C overnight followed by final filtering.
  • Table 2 shows measurements of the virus titer, amount of host cell protein (HCP), host cell DNA (hcDNA), and BSA in the virus preparation obtained at the end of each manufacture step.

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