Formulation of sugar solutions for continuous ultracentrifugation for virus purification
AssigneesBaxter International Inc., Baxter Healthcare S.A.
InventorsManfred Reiter, Leopold Grillberger, Wolfgang Mundt, Artur Mitterer, Horst Schafhauser
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Abstract
The present invention provides a method for purification of a virus or virus antigen comprising providing a virus preparation and centrifugation of said virus preparation in a gradient of a sugar established by the addition of two or more buffered sugar layers of different concentration. The method leads to higher yields and reduces unwanted aggregation of the virus or virus antigen by increasing the volume of the peak pool.
Claims31
A method for purification of an inactivated virus or fragmented virus, the method comprising
- (a) providing a gradient-forming solution containing a sugar gradient by centrifuging in a continuous ultracentrifuge comprising an ultracentrifugation rotor at least
- (i) a first buffered sugar solution comprising a first physiological buffer and
- (ii) a second buffered sugar solution having a higher density than the first buffered sugar solution and comprising
- a second physiological buffer, which is the same or different from the first physiological buffer,
- wherein the concentration of sugar in the first buffered sugar solution has a sucrose equivalent between 35% to 50% (w/w%) and the concentration of sugar in the second buffered sugar solution has a sucrose equivalent between 50% to 65% (w/w%), and the volume ratio of the first buffered sugar solution to the second buffered sugar solution is greater than or equal to 3:1;
- (b) adding an inactivated virus or fragmented virus preparation to the sugar gradient;
- (c) centrifuging the inactivated virus or fragmented virus preparation and sugar gradient to obtain an inactivated virus or fragmented virus peak pool with a sucrose equivalent between 30% and 54% (w/w%) sucrose;
- and
- (d) extracting the inactivated virus or fragmented virus peak pool to obtain the inactivated virus or fragmented virus.
A method for purification of an inactivated virus or fragmented virus comprising
- (a) providing a gradient-forming solution containing a sugar gradient by centrifuging in a continuous ultracentrifuge comprising an ultracentrifugation rotor at least
- (i) a first buffered sugar solution having a density of 1.15 kg/l to 1.23 kg/l and
- (ii) at least a second buffered sugar solution having a density of 1.23 kg/l to 1.32 kg/l and which is higher than the density of the first buffered solution,
- wherein, and the volume ratio of the first buffered sugar solution to the second buffered sugar solution is greater than or equal to 3:1;
- (b) adding an inactivated virus or fragmented virus preparation to the sugar gradient;
- (c) centrifuging the inactivated virus or fragmented virus preparation and sugar gradient to obtain a peak inactivated virus or fragmented virus pool;
- and
- (d) extracting the peak inactivated virus or fragmented virus pool to obtain the inactivated virus or fragmented virus.
Dependencies are inferred from the claim text.