Depletion of host cell components from live vector vaccines
Inventors
Jordan, Ingo • Bernhardt, Holger • Hartmann, Stefan
Assignees
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Publication Number
US-9273289-B2
Publication Date
2016-03-01
Expiration Date
Abstract
It is desirable to produce live vaccines, which are highly attenuated and which do only contain minimal or no animal-derived components. The production of highly attenuated live viruses can be better achieved when using specifically designed cell lines as producer substrate as opposed to using less defined primary cells. However, live viruses, thus produced comprise undesirable components from the cell lines and cell culture. The present invention relates to methods of production and purification of live enveloped viruses, which are suitable for vaccination.
Core Innovation
The invention provides a virus purification method that incubates a virus producing cell with at least one chaotropic salt prior to cell lysis and then separates the virus from at least part of the non-viral substances in the virus producing cell or its cell culture medium. The chaotropic salt is selected from NaBr and KCl, with specified minimum concentration thresholds prior to lysis.
The method includes performing cell lysis after the pre-lysis incubation in a chaotropic context, followed by separation of the virus from non-viral substances. In representative embodiments, the separation step is performed by filtration using a siliceous substance, including diatomaceous earth, acid washed diatomaceous earth, acid etched diatomaceous earth, or diatomaceous earth treated with a silane.
In additional embodiments, pre-lysis incubation may include at least one chaotropic salt together with one or more additives selected from dextrane sulphate, polyphosphoric acid, polyvinylpyrrolidon, NaI, chitosan, and detergents. The described framework is applied in the context of producing live, highly attenuated enveloped viruses from continuous cell lines, with improved pfU:DNA ratios and downstream compatibility.
Claims Coverage
The independent claim identifies a three-step virus purification method with one inventive feature set centered on pre-lysis incubation with specified chaotropic salts, followed by cell lysis and separation from non-viral substances. Dependent claim coverage narrows the separation step to filtration over siliceous substances and adds optional pre-lysis additives and concentration constraints.
Chaotropic salt pre-lysis incubation for virus purification
A method of virus purification comprising incubating a virus producing cell with at least one chaotropic salt selected from NaBr and KCl prior to cell lysis.
Virus separation from non-viral substances after lysis
A method of virus purification comprising lysing the virus producing cell after the incubating and separating the virus from at least part of the non-viral substances comprised in the virus producing cell or its cell culture medium.
Filtration through a siliceous substance for separation
The method where separation is performed by filtration using a siliceous substance.
Diatomaceous earth siliceous filter selection
The siliceous substance is selected from diatomaceous earth, acid washed diatomaceous earth, acid etched diatomaceous earth, or diatomaceous earth treated with a silane.
Additional pre-lysis additives alongside chaotropic salts
The method including incubating the virus-producing cells with dextrane sulphate, polyphosphoric acid, polyvinylpyrrolidon, NaI, chitosan, and/or detergents before cell lysis.
Overall, the claim coverage centers on pre-lysis incubation of a virus producing cell with specified chaotropic salts, followed by lysis and separation from non-viral substances. Dependent claims further specify separation by filtration over siliceous substances and add optional pre-lysis additives.
Stated Advantages
Improved pfU:DNA ratios.
Scalable downstream compatibility, including improved performance enabling further downstream steps.
Reduced host-cell derived contaminants via selective DNA depletion as part of the purification concept.
Documented Applications
Purification associated with producing live, highly attenuated enveloped viruses and/or vectored vaccines from continuous cell lines.
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