(12) Patent Application Publication (10) Pub. No.: US 2014/0227686 A1 Saghbini Et Al

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(12) Patent Application Publication (10) Pub. No.: US 2014/0227686 A1 Saghbini Et Al US 20140227686A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0227686 A1 Saghbini et al. (43) Pub. Date: Aug. 14, 2014 (54) STABILIZED CHEMICAL DEHYDRATION OF Publication Classification BOLOGICAL MATERAL (51) Int. Cl. (71) Applicant: GENTEGRALLC, Pleasanton, CA CI2O I/68 (2006.01) (US) (52) U.S. Cl. CPC .................................... CI2O I/6806 (2013.01) (72) Inventors: Michael Saghbini, Poway, CA (US); USPC ........................................................... 435/6.1 Michael Hogan, Tucson, AZ (US); Chunnian Shi, San Diego, CA (US); (57) ABSTRACT Brian Dalby, Carlsbad, CA (US); David Wong, San Marcos, CA (US) The present invention provides compositions and methods that enable the stabilization and storage of samples by con (73) Assignee: GENTEGRALLC, Pleasanton, CA tacting a sample with an assembly of particles, and reducing (US) the water activity level of the contacted sample. By reducing the water activity level of the sample, the assembly of par (21) Appl. No.: 14/254,478 ticles minimizes the degradation of the sample. Stabilizers may or may not be added to the assembly of particles to (22) Filed: Apr. 16, 2014 further minimize the degradation of the sample. Subsequently to storage in the assembly of particles, the samples are recov Related U.S. Application Data erable by eluting the assembly of particles with a fluid solu tion. In one embodiment, the entire assembly of particles will (63) y apps No. 13/081,436, filed on dissolve into the solution. In another embodiment, only part pr. o. s of the assembly of particles will dissolve into the solution. (60) Provisional application No. 61/321,269, filed on Apr. The assembly of particles provides the advantage that while it 6, 2010. is porous, it comprises non-porous particulate material. Patent Application Publication Aug. 14, 2014 Sheet 1 of 4 US 2014/022768.6 A1 FIGURE 1: Fig. 1.1 Biolodicalinioniral fluid &- Close & id Fig. 1.2 water soluble crystalline compoundstabilizers it Solid tissue s Š Closeid is Swabbed l Close biological material id FIGURE 2: Biaiogist Fisic: S & assa:&ssiss&s Rs 3&s &&ssssssss & s&sassissississirees 88sixxx 888 & 388xxiiise & six & &SS &SSSS Patent Application Publication Aug. 14, 2014 Sheet 2 of 4 US 2014/022768.6 A1 FIGURE3 &sess &: Rsriko. Sigtskarsge::cess. Six. FIGURE 4: Patent Application Publication Aug. 14, 2014 Sheet 3 of 4 US 2014/022768.6 A1 FIGURES: X S. S SS S Š SS S Sucrose Saliva Cotton tipped swabs Polyester tipped swabs Collection FIGURE 6: US $3. Šs 338. E3:Sf 8: S&S Risis: &S ESE issisp. Šssis Patent Application Publication Aug. 14, 2014 Sheet 4 of 4 US 2014/022768.6 A1 FG US 2014/022768.6 A1 Aug. 14, 2014 STABILIZED CHEMICAL DEHYDRATION OF tions from biological samples, including DNA, RNA, BOLOGICAL MATERAL polypeptides, viral samples, cell extracts, antibodies, and cell cultures. The invention further provides methods for stabiliz CROSS-REFERENCE TO RELATED ing biological samples in fluid Suspension. APPLICATIONS 0007. In one aspect, the invention may comprise anassem 0001. This application in a continuation application of bly of particles for stabilizing one or more biomolecules U.S. Ser. No. 13/081,436 filed Apr. 6, 2011, which claims the comprising: particulate material comprising particles and benefit of U.S. Provisional Application No. 61/321,269 filed said one or more biomolecules, wherein said biomolecules Apr. 6, 2010, which is incorporated herein by reference. are retained on an outer Surface layer of said particles and wherein said biomolecules have a water activity level sub FIELD OF THE INVENTION stantially less than 1. The above invention may further com prise an outer Surface layer comprising one or more stabiliz 0002 This invention relates generally to a method for ers. In some instances, one or more of the biomolecules may stabilizing biological samples. In particular the invention pro comprise a nucleic acid, a polypeptide, blood, serum, plasma, vides a method for stabilizing blood and blood components cell, tissue, sputum, mucus, cerebrospinal fluid, hair, urine, and other bodily fluids, bacterial, fungal, viral, animal and stool, semen, a metabolite, an antibody, a lipid, or a combi plant cell cultures in fluid Suspension. The invention also nation thereof. In other instances, one or more biomolecules provides a method for stabilizing tissue and organ samples. are selected form the group consisting of a bodily fluid, a tissue homogenate, a cell culture, a crude biological extract, a BACKGROUND OF THE INVENTION purified biologic, and any combination thereof. In yet other 0003 Water is a major component contributing to insta instances, one or more biomolecules are selected from the bility of collected biological material. Such biological mate group consisting of a plant extract, a microbial extract, an rial tends to be complex in nature and often contains damag animal extract, and any combination thereof. In the above ing entities such as nucleases, proteases, and other degrading invention, the biomolecules do not comprise d-Lysergic Acid and modifying enzymes and other chemicals that require an Diethylamide or polio virus. In the above invention, one or aqueous environment for activity. The damaging entities must more biomolecules may have a higher resistance from deg be immediately inactivated following sample collection to radation than a biomolecule not retained by said assembly. maintain biological material integrity. Additionally, certain The above invention may further comprise one or more bio biological materials, such as RNA, can spontaneously hydro molecules in contact with a solid support, wherein said solid lyze in the absence of exogenous enzyme activity, due to Support is selected from the group consisting of a Swab, a direct or metal-catalyzed addition of free water. sponge or a paper. In some instances, least a portion of said 0004 Some level of nuclease inactivation can beachieved biomolecules are recoverable from said assembly of the in the liquid state (patent 652864-RNA later). However, above invention. excess free water content can still cause hydrolysis. Dehydra 0008. In one aspect, the invention may comprise anassem tion has been used historically to achieve dry state stability. bly of particles comprising: particulate material comprising However, even active dehydration systems, using vacuum or one or more stabilizers on at least an outer Surface of said forced air, take hours to achieve Such stable state and require particulate material. The above invention may further com expensive equipment and thus are hard to implement at the prise one or more stabilizers located only on said outer Sur site of sample collection. Additionally, the time needed to face. In some instances, the above invention comprises an achieve dryness increases proportionately with an increase in assembly of particles that absorbs liquid upon contact of said sample size, thus contributing to further instability for large liquid with said assembly. The invention may further com samples. Thus, there is a need for a scalable method for prise an assembly of particles comprising a biomolecule that biological sample stabilization without refrigeration, via coexists as a thin chemically dehydrated surface film on the sample dehydration and the addition of stabilizers and inhibi particulate material. In different embodiments of this inven tors of degradation that can be performed in the seconds-to tion, a stabilizer may be selected from the group consisting of minutes time frame, without the use of mechanical drying anti-microbial agent, anti-oxidant, apoptosis inhibitors, equipment. buffer, chaotrope chelating agent, denaturing agent, deter 0005. The present invention provide instantaneous stabi gent, hydroxyl radical scavengers, hydroperoxide-removing lization of a biological sample, by rapid complexion of free entities, metal chelator, nuclease inhibitor, plasticizers, pro water in the sample and by diffusional addition of stabilizers tease inhibitors, protein modification inhibitor, protein pre to the sample, for transport and or archiving for Subsequent cipitants, protein stabilizers, reactive oxygen scavengers, and analysis of their constituents components, and propagation of reducing agent and any combination thereof. In some living entities, if stabilizing cultured cells. instances, the stabilizer is an oxidation inhibitor, a pyruvate inhibitor, an enzymatic activity inhibitor or a combination SUMMARY OF THE INVENTION thereof. In some embodiments of the invention, the particu 0006. The present invention provides methods, products, late material is a crystalline compound. In other embodiments and kits (having components described herein) for stabilizing of the invention, the particulate material is selected from the biological samples, including Solid tissues derived from group consisting of a monosaccharide, a disaccharide, a humans, animals and plants, as well as biological fluids Such polysaccharide, an organic salt, an inorganic salt, and any as blood urine, saliva, sputum, nasal discharges, lavages, tis combination thereof. In the above invention, a random pack Sue homogenates, by completely covering the sample in a ing of said particulate material may leave at least 20%, 25%, crystalline water-soluble compound and reducing the water 30%, 35%, 40% or more as interstitial space. The above activity level of the biological sample. The present invention invention may further comprise individual particles of said also provides methods for stabilizing extracts
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