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US 11,453,854 B2Granted
Lyophilization methods that provide stably dehydrated protozoans for use as potent live vaccines
AssigneeZoetis Services LLC
InventorsRim Daoussi, Frederick H. Weber
Life of this patent
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Abstract
An improved stabilization buffer for use in the lyophilization of Neospora and other protozoans is disclosed. Also disclosed is an improved lyophilization method, which includes controlling ice nucleation during the freezing step.
Claims19
A method of lyophilizing a protozoan by controlling ice nucleation by pressurizing and depressurizing with an inert gas or air, wherein the method comprises the steps of:
- (A) Loading a mixture of cultured cells and a stabilizing buffer into a dryer chamber which is at approximately 5° C. and about 1 Bar, the cultured cells including protozoa infected cells;
- (B) Lowering the temperature of the chamber to between −1° C. and −15° C., and holding for approximately 5 minutes;
- (C) Pressurizing the chamber to between 1.2 to 2 Bar, and holding for approximately 45 minutes, while maintaining the temperature of the chamber at between −1° C. and −15° C.;
- (D) Rapidly depressurizing the chamber to about 1 Bar, while holding the temperature at between −1° C. and −15° C.;
- (E) Holding the pressure in the chamber at about 1 Bar, lowering the temperature to as low as −50° C., and holding for approximately 45 minutes;
- (F) Holding the pressure in the chamber at about 1 Bar, the temperature at as low as −50° C., for a further approximate 90 minutes;
- (G) Holding the temperature in the chamber at as low as −50° C., decreasing the chamber pressure to about 0.1 mBar, and holding for approximately 30 minutes;
- (H) Holding the temperature in the chamber at as low as −50° C., decreasing the chamber pressure to about 0.01 mBar, and holding for approximately 60 minutes;
- (I) Holding the pressure in the chamber at about 0.01 mBar, raising the temperature in the chamber to as high as −35° C., and holding for approximately 60 minutes;
- (J) Holding the pressure in the chamber at about 0.01 mBar, and the temperature at as high as −35° C., and holding for up to an additional 3500 minutes;
- (K) Decreasing the chamber pressure to about 0.005 mBar;
- raising the temperature to as high as 20° C., and holding for up to an additional 360 minutes;
- (L) Holding the pressure in the chamber at about 0.005 mBar;
- and the temperature at as high as 20° C., for up to an additional 900 minutes.
11 more elements
Dependencies are inferred from the claim text.