Lipid sterilization method
The present invention relates to a method for sterilisation of phospholipid suspensions, useful in the preparation of ultrasound contrast agent precursors comprising phospholipid-stabilised perfluorobutane microbubbles. The method provides sterility assurance, without undue thermal degradation of the phospholipid. The method is also amenable to commercial scale manufacture. Also provided are methods of preparing kits and ultrasound contrast agents incorporating the sterilisation method of the invention.
1. A method of sterilization of a suspension, which comprises:
(i) mixing a hydrogenated egg phosphatidylserine together with propylene glycol in an aqueous biocompatible carrier using a mixer of a jacketed vessel to give an aqueous hydrogenated egg phosphatidylserine suspension, wherein the suspension contacts an inner wall of the jacketed vessel and the mixer is provided within the jacketed vessel and is configured to mix the contents of the jacketed vessel;
(ii) autoclaving the aqueous hydrogenated egg phosphatidylserine suspension from step (i), wherein said autoclaving enables reaching F 0 values >15 in all parts of the sterilization system, and wherein heating, in addition to sensible heat from heating the jacket of said vessel, comprises the addition of steam to the headspace of the vessel of step (i) to obtain a hot suspension; and
(iii) cooling the hot suspension to 15 to 30° C. to give the sterile hydrogenated egg phosphatidylserine suspension, wherein the mass ratio of phospholipid to propylene glycol is 1:1.5 to 1:2.5.
2. The method of claim 1 , wherein the addition of steam to the headspace is performed multiple times during the autoclave cycle.
3. The method of claim 1 , further comprising the application of vacuum to the headspace of the vessel of step (i), before the addition of steam to said headspace.
4. The method of claim 1 , wherein the F 0 values reacted are between 15 and 25.
5. The method of claim 1 , wherein the content of phosphatidylserine (PS), relative to the sum of PS and related substances, in the sterile hydrogenated egg phosphatidylserine suspension of step (iii) is at least 68%.
6. The method of claim 5 , wherein the concentration of phosphatidic acid (PA), relative to the sum of PS and related substances, in the sterile p hydrogenated egg phosphatidylserine suspension of step (iii), is less than 15%.
7. The method of claim 6 , wherein the combined concentration of phosphatidylserine (PS) and phosphatidic acid (PA) in the sterile hydrogenated egg phosphatidylserine suspension of step (iii) is 5 mg/mL.
8. The method of claim 7 , wherein the mass ratio of the hydrogenated egg phosphatidylserine to propylene glycol is 1:2.
9. The method of claim 1 , wherein clean steam is used in step (ii).
10. The method of claim 1 , wherein a volume of the aqueous hydrogenated egg phosphatidylserine suspension of step (i) is in the range 20 to 80 L.