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(12) Patent Application Publication (10) Pub. No.: US 2015/0267245 A1 Hogan (43) Pub US 20150267245A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2015/0267245 A1 Hogan (43) Pub. Date: Sep. 24, 2015 (54) PRESERVATION OF BIOLOGICAL (52) U.S. Cl. MATERIALS IN NON-AQUEOUS FLUID CPC .............. CI2O I/6806 (2013.01); C12O I/686 MEDIA (2013.01) (71) Applicant: GenTegra, LLC, Pleasanton, CA (US) (57) ABSTRACT (72) Inventor: Michael Hogan, Tucson, AZ (US) The invention provides compositions and methods for pre (73) Assignee: GenTegra, LLC, Pleasanton, CA (US) serving a biological material—Such as a protein, a nucleic acid or a biological sample, or any combination thereof in a (21) Appl. No.: 14/213,066 Substantially water-free, nonionic or ionic organic solvent. Improved preservation, including for example the stability (22) Filed: Mar 14, 2014 and/or the solubility of the biological material in the substan tially water-free fluid medium, is achieved with compositions Related U.S. Application Data comprising one or more Substances (e.g., an antioxidant) (60) Provisional application No. 61/786,171, filed on Mar. described in the disclosure, and/or a metal salt. The biological 14, 2013. material is soluble and stable, and retains its function and activity, when it is preserved in the substantially water-free Publication Classification fluid medium at ambient temperature or higher for extended periods of time. Therefore, the composition comprising the (51) Int. Cl. biological material does not need to be refrigerated or frozen CI2O I/68 (2006.01) during shipping or storage. Patent Application Publication Sep. 24, 2015 Sheet 1 of 7 US 2015/0267245 A1 ouuoo feN is g - X: Y. Oluo) 'SO &S eSO 3nS 6ug is eSoons 6U G. Sir 5 esoluons 6U g : esoions on Yi Jeppedd OGZ *" g Oluoo "So SN D esouons fug sur 8x o N eSoons fuga II e. - 8. Q eSoons bug' . eSOJOnS ON & as ouuo) '6en s OJuO3 "So . eSOJOnS 6Lug is: cE eSOJOnS fu ga 3 a ? esoluons fug' cy esoons ON Jeppedd OGC ~ Patent Application Publication US 2015/0267245 A1 Patent Application Publication Sep. 24, 2015 Sheet 3 of 7 US 2015/0267245 A1 s: 8XXAs. e 3 |||| Patent Application Publication Sep. 24, 2015 Sheet 4 of 7 US 2015/0267245 A1 as: §§§§§ | ?i ogos ©ocael Patent Application Publication Sep. 24, 2015 Sheet 5 of 7 US 2015/0267245 A1 s: ©Cae;L is sergeerases saazwsix-axiassroxsix&i:sage | ogo= ogo= Patent Application Publication Sep. 24, 2015 Sheet 6 of 7 US 2015/0267245 A1 s Patent Application Publication Sep. 24, 2015 Sheet 7 of 7 US 2015/0267245 A1 }} US 2015/0267245 A1 Sep. 24, 2015 PRESERVATION OF BIOLOGICAL 0010. It is another object, feature, and/or advantage of the MATERIALS IN NON-AQUEOUS FLUID present invention to provide compositions for preservation of MEDIA biological materials, wherein the biological materials are soluble and stable in the substantially water-free fluid media CROSS-REFERENCE TO RELATED at ambient temperature or higher for extended periods of APPLICATIONS time, and thus do not need to be refrigerated or frozen during shipping or storage. 0001. This application claims priority under 35 U.S.C. 0011. In one aspect, the present invention provides meth S119 of a provisional application Ser. No. 61/786,171 filed ods and compositions for preservation of biological materi Mar. 14, 2013, which is hereby incorporated by reference in als. Such as proteins, nucleic acids and biological samples, in its entirety. substantially water-free fluid media. The biological materials are soluble and stable in the substantially water-free fluid GRANT REFERENCE media at ambient temperature or higher for extended periods 0002 This invention was made with government support of time, and thus do not need to be refrigerated or frozen under Contract No. HR0011-12-C-0005 awarded by the during shipping or storage. The biological materials retain Defense Advanced Research Projects Agency (DARPA). The their structural integrity, function and activity after preserva government has certain rights in the invention. tion in the substantially water-free fluid media at ambient temperature or higher for extended periods of time. Because FIELD OF THE INVENTION the biological materials are preserved in a fluid medium, they may not need to be re-dissolved for use in fluid-phase reac 0003. The present invention relates to preservation of bio tions or assays, including nucleic acid amplification reactions logical materials, such as proteins, nucleic acids and biologi based on polymerase chain reaction (PCR) and analytical and cal samples, in Substantially water-free fluid media. diagnostic assays, such as immunoassays. 0012. In one aspect, the present invention provides com BACKGROUND OF THE INVENTION positions comprising a biological material in a Substantially 0004 Biological materials, such as proteins and nucleic water-free fluid medium, wherein the fluid medium com acids, may in certain instances be freeze dried (lyophilized) to prises a non-ionic organic solvent (e.g., an alcohol solvent) or enhance their stability in the absence of refrigeration. Freeze an ionic organic solvent comprising an organic salt and an drying comprises freezing of an aqueous mixture containing organic hydrogen bond donor. To enhance, e.g., the Stability a biological material and removal of water via Sublimation. and/or the solubility of the biological material in the substan Biological materials can Suffer denaturation including for tially water-free fluid medium, the compositions can further example, partial denaturation—as a result of freeze-drying, comprise a metal salt, and/or one or more Substances selected for example, as a result of the freezing step and/or the Subli from the group consisting of reducing agents, antioxidants, mation step. Thus, there is a need in the art for methods, free radical scavengers, oxygen radical scavengers, hydroxyl compositions, and systems for stable preservation of biologi radical scavengers, singlet oxygen quenchers, hydroperoX ide-removing agents, protease inhibitors, nuclease inhibitors, cal materials that prevents denaturation. ribonuclease (RNase) inhibitors, deoxyribonuclease (DNase) inhibitors, metal chelators, preservatives, anti-microbials, SUMMARY OF THE INVENTION buffers (or buffering agents), detergents, and chaotropes. 0005. It is therefore a primary object, feature, and/or 0013. In another aspect, the present invention provides advantage of the present invention to improve on or overcome methods of preserving a biological material, comprising mix the deficiencies in the art. ing an aqueous mixture comprising a biological material with 0006. It is another object, feature, and/or advantage of the a non-ionic organic solvent (e.g., an alcohol Solvent) or an present invention to provide methods for preservation of bio ionic organic solvent comprising an organic salt and an logical materials, such as proteins, nucleic acids and biologi organic hydrogen bond donor to produce an aqueous organic cal samples, in Substantially water-free fluid media. mixture, and removing water from the aqueous organic mix 0007. It is another object, feature, and/or advantage of the ture to produce a substantially water-free fluid medium com present invention to provide methods for preservation of bio prising the biological material and the non-ionic or ionic logical materials, wherein the biological materials are soluble organic solvent. The fluid medium can further comprise a and stable in the substantially water-free fluid media at ambi metal salt and/or one or more Substances as described herein. ent temperature or higher for extended periods of time, and 0014. In another aspect, the present invention provides thus do not need to be refrigerated or frozen during shipping containers and kits containing compositions that comprise or Storage. biological materials in substantially water-free fluid media. 0008. It is another object, feature, and/or advantage of the 0015 While multiple embodiments are disclosed, still present invention to provide methods of preserving biological other embodiments of the present invention will become material in a non-aqueous fluid media, including, for apparent to those skilled in the art from the following detailed example, a non-ionic organic solvent such as an alcohol Sol description, which shows and describes illustrative embodi vent or an ionic organic solvent comprising an organic salt ments of the invention. Accordingly, the drawings and and an organic hydrogen bond donor to produce an aqueous detailed description are to be regarded as illustrative in nature organic mixture. and not restrictive. 0009. It is another object, feature, and/or advantage of the present invention to provide compositions for preservation of BRIEF DESCRIPTION OF THE DRAWINGS biological materials, such as proteins, nucleic acids and bio 0016. The following figures are included to illustrate cer logical samples, in Substantially water-free fluid media. tain aspects of the present invention, and should not be viewed US 2015/0267245 A1 Sep. 24, 2015 as exclusive embodiments. The subject matter disclosed is applies to each one of the numerical values in that series of capable of considerable modifications, alterations, combina numerical values or in that series of ranges of numerical tions, and equivalents in form and function, as will occur to values. In certain embodiments, the term “about' or “approxi those skilled in the art and having the benefit of this disclo mately” means within 10% or 5% of the specified value. SUC. (0027. Whenever the term “at least” or “greater than' pre 0017 FIG. 1 shows electrophoresis results of reverse tran cedes the first numerical value in a series of two or more scription PCR (RT-PCR) for analysis of human 18S riboso numerical values, the term “at least or “greater than applies mal RNA (rRNA) after preservation of RT-PCR reagents in to each one of the numerical values in that series of numerical glycerol, with addition of no Sucrose or varying amounts of values. Sucrose, at ambient temperature for varying periods of time. 0028. Whenever the term “no more than or “less than 0018 FIGS.
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