USOO9346722B2 (12) United States Patent (10) Patent No.: US 9,346,722 B2 L00ten et al. (45) Date of Patent: May 24, 2016 (54) METHOD FOR REFINING SQUALENE (52) U.S. Cl. PRODUCED BY MICROALGAE CPC ................. C07C 7/08 (2013.01); A23L I/3002 (2013.01); A23L I/337 (2013.01); A61K 8/31 (71) Applicant: ROQUETTE FRERES, Lestrem (FR) (2013.01); A61K 8/975 (2013.01); A61 K3I/01 (2013.01); A61 K36/02 (2013.01); A61O 19/00 (72) Inventors: Philippe Looten, Lomme (FR); Samuel (2013.01); B0ID 3/40 (2013.01); B0ID Patinier, Lille (FR): Michel Perrut, II/0403 (2013.01); BOID LL/0426 (2013.01); Villers les Nancy (FR): Vincent Perrut, C07C5/03 (2013.01); CIIB I/4 (2013.01); Venterol (FR) CI2P5/007 (2013.01); A61 K 2800/10 (2013.01); A61 K 2800/85 (2013.01); A61 K (73) Assignee: ROQUETTE FRERES, Lestrem (FR) 2800/86 (2013.01); Y02E 50/343 (2013.01); Y02P 20/544 (2015.11) (*) Notice: Subject to any disclaimer, the term of this (58) Field of Classification Search patent is extended or adjusted under 35 CPC ......................................................... CO7C 7/08 U.S.C. 154(b) by 0 days. See application file for complete search history. 56 References Cited (21) Appl. No.: 14/394,813 (56) U.S. PATENT DOCUMENTS (22) PCT Filed: Apr. 15, 2013 7,575,767 B2 * 8/2009 May ..................... CO7D 311/72 424,776 (86). PCT No.: PCT/FR2O13/OSO812 2004/00 15033 A1 1/2004 Steiner et al. S371 (c)(1) 2011/0243969 A1 10, 2011 Broeker (2) Date: Oct. 16, 2014 FOREIGN PATENT DOCUMENTS (87) PCT Pub. No.: WO2013/156720 CN 102 100 260 6, 2011 EP O 541999 5, 1993 PCT Pub. Date: Oct. 24, 2013 EP 1818388 8, 2007 WO WO 2005, O75614 8, 2005 (65) Prior Publication Data WO WO 2008. 142175 11, 2008 OTHER PUBLICATIONS US 2015/O140030 A1 May 21, 2015 Luetal (Journal of Chromatography A. 994, pp. 37-43, 2003).* (30) Foreign Application Priority Data Chen, G. et al. "Optimization of nitrogen source for enhanced pro duction of squalene from thraustochytrid Aurantiochyrium sp.” New Apr. 16, 2012 (FR)...................................... 1253496 Biotechnology, Sep. 2010, pp. 382-389, vol.27, No. 4. Apr. 19, 2012 (FR) ...................................... 12 53614 Written Opinion in International Application No. PCT/FR2013/ 050812, Oct. 14, 2013, pp. 1-8. (51) Int. Cl. * cited by examiner CI2P 7/64 (2006.01) C07C 7/08 (2006.01) Primary Examiner — Albert Navarro A2.3L I/30 (2006.01) (74) Attorney, Agent, or Firm — Saliwanchik, Lloyd & A2.3L I/337 (2006.01) Eisenschenk A61O 19/00 (2006.01) (57) ABSTRACT BOLD II/04 (2006.01) CIIB I/O (2006.01) The invention relates to a method for preparing a composition CI2P 5/00 (2006.01) that is very rich in squalene produced by fermentation of A6 IK 8/3 (2006.01) micro-organisms. The method is characterized in that it com A 6LX8/97 (2006.01) prises a purification step selected from the group including: A6 IK3I/01 (2006.01) Supercritical CO2 extraction in a multi-stage counter-current A6 IK36/02 (2006.01) fractionation column with extract reflux, and short-path C07C5/03 (2006.01) 13 Claims, No Drawings US 9,346,722 B2 1. 2 METHOD FOR REFINING SQUALENE tion of the 22 million doses administered since 1997 (MF59 at PRODUCED BY MICROALGAE a rate of 10 mg per dose of FLUADR), without serious postvaccinal reactions. CROSS-REFERENCE TO RELATED Addition of adjuvants, squalene or aluminum salts (used APPLICATION since 1926) is necessary at present for certain vaccines which, being inactivated or Subunit vaccines, do not contain the This application is the U.S. national stage application of signals enabling the immune system to employ the appropri International Patent Application No. PCT/FR2013/050812, ate defense mechanisms. filed Apr. 15, 2013. Squalene avoids the need for repeated injections to ensure 10 good protection. The present invention relates to a method for refining These uses of squalene reinforce the determination of a squalene produced by fermentation, starting from microor person skilled in the art to have secure methods for producing ganisms, more particularly microalgae, even more particu squalene of high purity. larly those from the family Thraustochytriales sp. Moreover, this quality may open up other routes of appli In the sense of the invention, “microalgae of the family 15 cation in the medical field. Thraustochytriales sp. means microalgae belonging to the Chemical coupling of squalene with nucleoside analogs species Schizochytrium sp., Aurantiochytrium sp. and might thus constitute, in the future, a considerable advance in Thraustochytrium sp. the treatment of certain cancers or inviral diseases of the HIV Squalene is a lipid present in all higher organisms, and is type. the common precursor of Steroid hormones, both animal and The Various Sources of Squalene Vegetable, and of some vitamins, such as the D vitamins. Squalene is extracted conventionally from deep-sea shark It is present in many cell membranes, thus providing them livers. with fluidity. However, the liver accumulates numerous toxic com This unsaturated linear hydrocarbon is an isoprenoid with pounds, such as heavy metals (including mercury) and other thirty carbon atoms and fifty hydrogen atoms, of formula: 25 fat-soluble toxins. 2,6,10,15,19,23-hexamethyl-2,6,10,14, 18.22-tetracosahex Toxicological studies have shown that at the concentra ene, CoHo, i.e. it consists of 6 isoprene units, all in trans tions used in cosmetics, squalene and its hydrogenated form conformation. squalane are not toxic and are not irritant or sensitizing for Like all the terpenes, it is formed from isopentyl pyrophos human skin. phate, which couples with dimethylallyl pyrophosphate to 30 However, the level of purity of squalene is essential when Supply successively the geranyl pyrophosphates, then the far used in the medical field, notably as an adjuvant for vaccines. nesyl pyrophosphates, two molecules of which condense It is therefore absolutely essential to have squalene of high after reduction by NADPH to form squalene under the action quality, free from impurities (traces of metals, notably mer of squalene synthase. cury, and other toxins). 35 Several routes for production of squalene, other than its In plants and numerous microorganisms, this pathway extraction from shark livers, are proposed in the literature. coexists with other metabolic pathways leading to phytoene, As a first alternative, it may be isolated from olive oil, palm the precursor of chlorophyll, of carotenoid pigments and of oil, and other cereal oils or oils derived from amaranth, seeds, the terpenes in lattices. rice bran, and wheat germ. Squalene, and its derivative epoxidized on the terminal 40 However, the major drawback here is that squalene is double bond, possess the property of being transformed, extracted from these in very small amounts, on the order of owing to specialized enzymes (the cyclases), remarkably 0.1 to 0.7 wt %, and requires a great many arduous and regio- and stereoselectively, into polycyclic triterpenes of expensive purification steps. considerable structural variety: hopene and diplopterol in the As a second alternative, first methods of production of eukaryotes and tetrahymanol in the protozoa (pentacyclic 45 squalene have been proposed from microorganisms, more triperpenes); lanosterol in the yeasts, fungi and mammals and particularly from natural yeasts or recombinant yeasts, nota cycloartenol in plants (tetracyclic triperpenes). bly of the Saccharomyces type. The Applications of Squalene Thus, Saccharomyces cerevisiae is known to be able to Squalene has long been used, notably in Japan, as a food produce squalene, but in very Small amounts: on the order of Supplement. 50 0.04.1 mg/g of biomass (BHATTACHARJEE, P. et al., 2001, Moreover, it was a Japanese chemist, Mitsumaru World J. Microb. Biotechnol., 17, pp 811-816). Tsujimoto, who discovered it in 1906 and determined its Optimization of these capacities for production has there structure in 1916. fore been attempted by means of genetic recombination. It is considered to be an effective antioxidant, with many However, as presented in patent application WO 2010/ beneficial properties in natural medicines. 55 023551 for the medical field (production of squalene of purity Its conventional uses include cosmetics, although there it is above 97% as an adjuvant for vaccines), this first alternative commoner to use its hydrogenated derivative, squalane, can only be applied industrially if recombinant yeasts that which does not oxidize, and therefore does not become ran overproduce squalene (at more than 15 wt % of dry cells) are cid. available. When it is of high purity, combined with adjuvants that 60 Now, obtaining these recombinant cells requires the appli stimulate the immune system, squalene has been, and still is, cation of numerous arduous, long and complex steps of meta used in certain vaccines: in the form of “oil-in-water emul bolic engineering, by employing tools of molecular biology, Sion, it acts as a Surfactant, thus increasing the response of the leading to stimulation of the squalene biosynthesis pathways vaccine. and to inhibition of the squalene catabolism pathways. It is used in experimental vaccines targeting emerging 65 In fact, as is moreover recalled in said patent application viruses, such as H5N1 and H1N1, but especially in combina WO 2010/023551, multiple genes are involved in the biosyn tion with the antigens of seasonal influenza, in the composi thesis of squalene: including mevalonate kinase, phosphom US 9,346,722 B2 3 4 evalonate kinase, pyrophosphomevalonate decarboxylase, Thus, although the microalgae of the family Thraustochy isopentenyl pyrophosphate isomerase, HMGR (3-hydroxy-3- triales sp. now allow production of squalene in appreciable methylglutaryl-CoA reductase), and squalene synthetase. amounts, it is still necessary to refine it to meet food, cosmetic For the catabolism pathways, the genes coding for numer and especially medical requirements.