Method for separating virus-like particles from a cell suspension
Inventors
Wetzel, David • JENZELEWSKI, Volker • Piontek, Michael
Assignees
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Publication Number
US-11053478-B2
Publication Date
2021-07-06
Expiration Date
Abstract
A method for separating virus-like particles from a cell suspension of host cells. The virus-like particles having at least one envelope protein embedded in a lipid double membrane including at least a portion corresponding to a small envelope protein of a virus of the family Hepadnaviridae. The host cells are disrupted to obtain a first suspension. A supernatant containing the virus-like particles is separated from the first suspension. Then, an adsorbent is added to the supernatant and separated off. Then, the virus-like particles are desorbed from the adsorbent by adding a desorption buffer. A soluble calcium salt is added to a supernatant separated from the second suspension to form a precipitate, the precipitate formed is separated off and transferred to a third suspension. The virus-like particles are separated from the third suspension and purified.
Core Innovation
The invention provides a method for separating virus-like particles from a cell suspension of host cells containing virus-like particles, wherein the virus-like particles have at least one envelope protein embedded in a lipid double membrane. The protein comprises at least a portion corresponding to a small envelope protein of a virus of the family Hepadnaviridae. The method begins by disrupting the host cells to obtain a first suspension containing the virus-like particles and separating a supernatant containing the virus-like particles from the first suspension.
The method further adsorbs the virus-like particles onto an adsorbent by adding the adsorbent to the separated supernatant, then separates off the adsorbent with the adsorbed virus-like particles. The virus-like particles are desorbed from the separated-off adsorbent using a desorption buffer that contains deoxycholic acid and/or at least one soluble salt of deoxycholic acid. The supernatant containing the virus-like particles is separated from the second suspension formed thereafter.
After separating the supernatant containing the virus-like particles from the second suspension, the method adds a soluble calcium salt to the separated-off supernatant to form a precipitate containing a calcium salt of deoxycholic acid together with the virus-like particles. The precipitate is separated off, transferred to a third suspension, and the virus-like particles are separated from the third suspension and purified.
Claims Coverage
The claim coverage indicates two inventive features in the separation and purification workflow.
Adsorbent adsorption and deoxycholic acid-based desorption
A method including disrupting host cells to obtain a first suspension containing virus-like particles, separating a supernatant, adding an adsorbent so the virus-like particles are adsorbed, separating off the adsorbent with the adsorbed virus-like particles, and desorbing the virus-like particles using a desorption buffer containing deoxycholic acid and/or at least one soluble salt of deoxycholic acid.
Calcium salt co-precipitation of deoxycholic acid with virus-like particles
Adding a soluble calcium salt to the separated-off supernatant to form a precipitate containing a calcium salt of deoxycholic acid together with the virus-like particles, separating off the precipitate, transferring it to a third suspension, and separating and purifying the virus-like particles from the third suspension.
The inventive features center on adsorbent capture and deoxycholic acid-based desorption, followed by calcium salt addition to form a precipitate with the virus-like particles and subsequent separation and purification.
Stated Advantages
Cost reduction by dispensing with cesium chloride density gradient ultracentrifugation and size exclusion chromatography.
Documented Applications
Purification and separation of virus-like particles having at least one Hepadnaviridae small envelope protein embedded in a lipid double membrane, including s-HBsAg and s-dHBsAg embodiments.
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