Production of squalene from hyper-producing yeasts

Abstract

A method for preparing purified yeast is disclosed, where the squalene source is a yeast that hyperproduces squalene. The squalene is useful for pharmaceutical purposes. For instance, it can be used to prepare an oil-in-water emulsion, and the emulsion is particularly suitable for use as an immmological adjuvant.

1 - 22 . (canceled). 
     
     
         23 . A process for preparing purified squalene for parenteral use in humans, comprising the steps of: (a) growing a yeast culture under conditions such that the yeast comprises at least 2% cell dry weight squalene; (b) purifying squalene from the cultured yeast, such that the squalene is of pharmaceutical grade 
     
     
         24 . The process of  claim 23 , wherein the yeast produces squalene at least 5% cell dry weight. 
     
     
         25 . The process of  claim 23 , wherein the yeast is a  Saccharomyces.    
     
     
         26 . The process of  claim 25 , wherein the yeast is a  Saccharomyces cerevisiae.    
     
     
         27 . The process of  claim 23 , wherein the yeast expresses a truncated 3 hydroxy-3-methylglutaryl-CoA reductase (HMGR) enzyme that has a cytosolic location. 
     
     
         28 . The process of  claim 23 , wherein the yeast is a modified strain that, relative to an unmodified parent strain, hyper expresses HMGR. 
     
     
         29 . The process of  claim 23 , wherein the yeast is a modified strain that, relative to an unmodified parent strain, under expresses zymosterol-24-methyltransferase and/or ergosta-5,7,24(28)-trienol-22-dehydrogenase. 
     
     
         30 . The process of  claim 23 , wherein the yeast expresses a mutant oxidosqualene cyclase. 
     
     
         31 . The process of  claim 23 , wherein the yeast has a disrupted squalene epoxidase. 
     
     
         32 . The process of  claim 23 , wherein the yeast is grown in the presence of factor that increases squalene yield during culture. 
     
     
         33 . The process of  claim 32 , wherein the factor is an allylamine, voriconazole, 6-amino-2-n-pentylthiobenzothiazole, thiamine, or a thiocarbamate. 
     
     
         34 . The process of  claim 23 , wherein the yeast is grown in partial or complete anaerobic conditions prior to squalene purification. 
     
     
         35 . The process of any  claim 23 , wherein the yeast is grown in a controlled medium free from animal-origin products. 
     
     
         36 . A process for preparing an oil-in-water emulsion, comprising the steps of: (a) preparing purified squalene by the process of  claim 23 , and (b) combining the squalene purified in step (a) with an aqueous component to form the oil in water emulsion. 
     
     
         37 . The process of  claim 36 , wherein the oil in water emulsion has oil droplets with a sub-micron diameter. 
     
     
         38 . The process of  claim 36 , wherein the oil in water emulsion is a microfluidised emulsion. 
     
     
         39 . The process of  claim 36 , wherein the oil in water emulsion comprises squalene and polysorbate 80. 
     
     
         40 . A process for preparing an immunogenic composition, comprising a step of preparing an oil in water emulsion by the process of  claim 36 , and mixing the oil-in-water emulsion with an immunogen. 
     
     
         41 . A process for preparing a kit for preparing an immunogenic composition, comprising preparing an oil in water emulsion by the process of  claim 36 , packaging the emulsion in a first container, and combining the first container in kit form with a second container, wherein the second container contains an immunogen. 
     
     
         42 . A process for raising an immune response in a mammal, comprising administering to the mammal an immunogenic composition prepared by the process of  claim 40 .