Compositions for stabilizing DNA, RNA, and proteins in blood and other biological samples during shipping and storage at ambient temperatures

• US 9,999,217 B2
• Filed: 05/25/2016
• Issued: 06/19/2018
• Est. Priority Date: 07/26/2010
• Status: Active Grant

• Current Assignee: Biomatrica, Inc. (Exact Sciences Corporation)
• Original Assignee: Biomatrica, Inc. (Exact Sciences Corporation)
• Inventors: Whitney, Scott E., Wilkinson, Steven, Muller, Rolf
• Primary Examiner(s): HANLEY, SUSAN MARIE
• Application Number:
  US15/164,531
• Publication Number:
  US 20160338342A1
• Time in Patent Office: 755 Days
• CPC Class Codes: A01N 1/021 Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer

Abstract

Compositions and methods are disclosed for substantially liquid, gel, suspension, slurry, semisolid and/or colloid storage of biological samples following admixture with the herein disclosed storage composition, permitting substantial recovery of biological activity following storage without refrigeration. In certain embodiments, unfractionated blood or tissue samples may be stored without refrigeration for weeks, months or years in a form that permits recovery of intact DNA, RNA or protein components following the storage period.

Claims

1. A composition for stable storage of nucleic acid and polypeptide molecules in a biological sample at room temperature, comprising:

(a) an imidazolium salt comprising;

i) a compound selected from the group consisting of;

1-benzyl-3-hexylimidazolium,

1-methyl-3-carboxyethylimidazolium,

1-hexyl-3-methylimidazolium,

1-decyl-3-methylimidazolium, and

1-(2-hydroxyethyl)-3-methylimidazolium; and

ii) a pharmaceutically acceptable anion;

(b) two or all three of;

(i) at least one precipitating agent,

(ii) at least one lower alcohol, wherein the lower alcohol is a straight or branched carbon chain alcohol of no more than 8 carbons, and

(iii) at least one chaotrope;

(c) a chelating agent, a reducing agent, or a combination thereof;

(d) one or more pH buffers; and

(e) water,

wherein the composition is an aqueous solution and the imidazolium salt of (a) is present in the composition at a concentration of about 0.1% to about 10% (w/v).

2. The composition of claim 1 wherein the imidazolium salt of (a) is selected from the group consisting of:

1-benzyl-3-hexylimidazolium bromide,

1-methyl-3-carboxyethylimidazolium bromide,

1-hexyl-3-methylimidazolium bromide,

1-decyl-3-methylimidazolium bromide, and

1-(2-hydroxyethyl)-3-methylimidazolium bromide.

3. The composition of claim 1 wherein the precipitating agent is selected from the group consisting of:

5-(4-dimethyl)amino benzylidene rhodanine,

sulfosalicylic acid,

lithium chloride, and

lithium hydroxide.

4. The composition of claim 1 wherein

the lower alcohol is selected from the group consisting of methanol, ethanol, n-propanol, isopropanol, n-butanol, and isobutanol (2-methylpropan-1-ol);

the chaotrope is selected from the group consisting of;

guanidine hydrochloride, guanidine thiocyanate, potassium thiocyanate, sodium thiocyanate and urea;

the chelating agent is selected from the group consisting of diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), trans-1,2-diaminocyclohexane-N,N,N′

,N′

-tetraacetic acid (CDTA), 1,2-bis(2-aminophenoxy)ethane-N,N,N′

N′

-tetraacetic acid (BAPTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), N-(2-hydroxyethyl)ethylenediamine-N,N′

,N′

-triacetic acid, and nitrilotriacetic acid (NTA);

the reducing agent is selected from the group consisting of 2-mercaptoethanol, thiosulfate, TCEP (tris-(2-carboxyethyl) phosphine), dithiothreitol and dithioerythritol; and

the pH buffer is selected from the group consisting of citric acid, tartaric acid, malic acid, sulfosalicylic acid, sulfoisophtalic acid, oxalic acid, borate, CAPS (3-(cyclohexylamino)-1-propanesulfonic acid), CAPSO (3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid), EPPS (4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid), HEPES (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid), MES (2-(N-morpholino)ethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), MOPSO (3-morpholino-2-hydroxypropanesulfonic acid), PIPES (1,4-piperazinediethanesulfonic acid), TAPS (N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid), TAPSO (2-hydroxy-3-[tris(hydroxymethyl)methylamino]-1-propanesulfonic acid), TES (N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid), bicine (N,N-Bis(2-hydroxyethyl)glycine), tricine (N-[Tris(hydroxymethyl)methyl]glycine), tris (tris(hydroxymethyl)aminomethane) and bis-tris (2-[Bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-1,3-propanediol).

5. The composition of claim 1 which further comprises a surfactant or a detergent.

6. The composition of claim 1, wherein the imidazolium salt of (a) is 1-hexyl-3-methylimidazolium bromide, the chaotrope is guanidine hydrochloride, the pH buffer is tartaric acid and oxalic acid, the precipitating agent is lithium hydroxide, and the chelating agent is diethylenetriaminepentaacetic acid (DTPA).

7. An admixture of a biological sample and a composition of claim 1, comprising a stably-stored nucleic acid molecule from said biological sample.

8. An admixture of a vertebrate blood sample and a composition of claim 1, comprising a stably-stored nucleic acid molecule from said vertebrate blood sample.

9. An admixture of a human blood sample and a composition of claim 1, comprising a stably-stored protein molecule or polypeptide molecule from said human blood sample.

10. The admixture of claim 7, wherein the sample is selected from the group consisting of DNA, RNA, blood, buffy coat from blood, urine, feces, serum, serosal fluid, plasma, lymph, cerebrospinal fluid, saliva, mucosal secretion, vaginal fluid, ascites fluid, pleural fluid, pericardial fluid, peritoneal fluid, abdominal fluid, buccal cells, bacteria, organ culture, cell culture, viruses, yeast cells, plasmid DNA, mRNA, tRNA, rRNA, siRNA, micro RNA, hnRNA, cDNA, a vaccine, a cell, a sorted or selected cell, a tissue, a cell lysate, homogenate or extract, a tissue lysate, homogenate or extract, a blood sample, biopsy specimen, tissue explant, organ culture and a biological fluid.

11. A method for substantially stabilizing one or a plurality of nucleic acid or polypeptide molecules that are present in a biological sample, comprising:

(a) admixing the biological sample with the composition of claim 1 to obtain a mixture; and

(b) maintaining the mixture without refrigeration for a time period of at least 7 days, and thereby substantially stabilizing said one or a plurality of nucleic acid or polypeptide molecules that are present in the biological sample, wherein either one, two or all three of;

(i) degradation is substantially prevented of at least 70% of recoverable DNA in the mixture, relative to an amount of DNA or polypeptide that is recoverable from the sample when stored for the time period at −

20°

C. without the composition of claim 1,

(ii) degradation is substantially prevented of at least 70% of recoverable RNA in the mixture, relative to an amount of RNA that is recoverable from the sample when stored for the time period at −

80°

C. without the composition of claim 1, and

(iii) degradation is substantially prevented of at least 70% of recoverable polypeptide molecules in the mixture, relative to an amount of polypeptide molecules that is recoverable from the sample when stored for the time period at −

20°

C. without the composition of claim 1.

12. The method of claim 11 wherein the biological sample is selected from:

(a) a biological sample that comprises vertebrate blood,

(b) the biological sample of (a) wherein the vertebrate blood is unfractionated,

(c) a biological sample that comprises human blood,

(d) the biological sample of (c) wherein the human blood is unfractionated,

(e) a biological sample that is selected from the group consisting of DNA, RNA, blood, blood buffy coat, urine, feces, serum, serosal fluid, plasma, lymph, cerebrospinal fluid, saliva, mucosal secretion, vaginal fluid, ascites fluid, pleural fluid, pericardial fluid, peritoneal fluid, abdominal fluid, buccal cells, bacteria, organ culture, cell culture, viruses, yeast cells, plasmid DNA, mRNA, tRNA, rRNA, siRNA, micro RNA, hnRNA, cDNA, a protein, a polypeptide, a glycoprotein, a vaccine, a cell, a sorted or selected cell, a tissue, a cell lysate, homogenate or extract, a tissue lysate, homogenate or extract, a blood sample, biopsy specimen, tissue explant, organ culture and a biological fluid.

13. The method of claim 11 wherein the step of maintaining comprises maintaining for a time period of at least 10, 20, 30, 40, 50, 60 or 70 days.

14. The composition of claim 1 wherein the pharmaceutically acceptable anion is selected from the group consisting of bromide, chloride, iodide, C₁-C₁₂alkylsulfonate, hexafluorophosphate, methylsulfate, ethylsulfate, tetrafluoroborate, trifluoromethanesulfonate, and bis(trifluoromethylsulfonyl)imide.