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US 2026/0085294 A1Published application
Process for producing a purified rhabdovirus from cell culture
AssigneeBoehringer Ingelheim International GmbH
InventorsDethardt MUELLER, Alan Pardo GARCIA, Tanja Anneliese GREIN, Friedrich KAESS, Judy NG, Katharina PESTA, Sabrina SCHNEIDER, Jordan TURNBULL
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Abstract
The present invention relates to the field of upstream and downstream processing and provides a process for producing a purified rhabdovirus from a cell culture, preferably a purified oncolytic rhabdovirus and in particular a vesicular stomatitis virus, including pharmaceutical compositions comprising the rhabdovirus.
Claims26
A method of producing a recombinant vesicular stomatitis virus (rVSV) in a suspension cell culture comprising the steps of:
- (i) infecting host cells with rVSV in said cell culture under conditions that allow replication of the rVSV until at least one of the conditions selected from the following, is met:
- (a) at least 30 hours post infection, (a) a total cell concentration of 2-3×106 cells per milliliter in cell culture supernatant with viability of >90%, (b) infectious virus content in final cell culture supernatant of about 2×109 TCID50/mL, and (c) content of virus genomes in cell culture supernatant of about 3-3.5×1010 TCID50/mL, and
- (ii) harvesting rVSV from the cell culture of step (i) by:
- (a) adding directly to the post-infection cell culture a viral release agent, wherein said viral release agent is selected from the group consisting of dextran sulfate, a solid salt, and an aqueous salt solution, wherein the recovered infectious rVSV harvest concentration, as measured by TCID50/mL calculated according to the Spearman-Karber method, obtained from the cell culture treated with the viral release agent compared to the untreated cell culture is increased by at least a factor of 5 and/or is at an effective viral concentration of at least 1×109 TCID50/mL, followed by clarifying the cell culture, and recovery of the rVSV harvest in the supernatant,
- OR
- (b) subjecting the post-infection cell culture to a filtration step, followed by rinsing of the filter with an aqueous solution containing a viral release agent selected from the group consisting of dextran sulfate, and a salt, wherein the recovered infectious rVSV harvest concentration, as measured by TCID50/mL calculated according to the Spearman-Karber method, obtained from the cell culture treated with the viral release agent compared to the untreated cell culture is increased by at least a factor of 5 and/or is at an effective viral concentration of at least 1×109 TCID50/ml, and recovery of the rVSV harvest in the supernatant.
3 more elements
A process for purifying a rVSV from a suspension cell culture infected with the rVSV, comprising the steps of:
- (a) adding directly to a post-infection cell culture a viral release agent, wherein said viral release agent is selected from the group consisting of dextran sulfate, a solid salt, and an aqueous salt solution, wherein the recovered infectious rVSV harvest concentration, as measured by TCID50/mL calculated according to the Spearman-Karber method, obtained from the cell culture treated with the viral release agent compared to the untreated cell culture is increased by at least a factor of 5 and/or is at an effective viral concentration of at least 1×109 TCID50/ml, followed by clarifying the cell culture, and recovery of the rVSV harvest in the supernatant,
- OR
- (b) subjecting the post-infection cell culture to a filtration step, followed by rinsing of the filter with an aqueous solution containing a viral release agent selected from the group consisting of dextran sulfate, and a salt, wherein the recovered infectious rVSV harvest concentration, as measured by TCID50/mL calculated according to the Spearman-Karber method, obtained from the cell culture treated with the viral release agent compared to the untreated cell culture is increased by at least a factor of 5 and/or is at an effective viral concentration of at least 1×109 TCID50/ml, and recovery of the rVSV harvest in the supernatant
- wherein the post-infection step in (a) and (b) is defined by one and/or all of the conditions selected from the following (i) at least 30 hours post infection, (ii) a total cell concentration of 2-3×106 cells per milliliter in cell culture supernatant with viability of >90%, (iii) infectious virus content in final cell culture supernatant of about 2×109 TCID50/mL, and (iv) content of virus genomes in cell culture supernatant of about 3-3.5×1010 TCID50/mL
1 more element
Dependencies are inferred from the claim text.