Stabilization of Undenatured Polypeptides, Nucleic Acids, and Exosomes in Blood Samples at Ambient Temperature

Abstract

To provide formulations for stabilization of one or more polypeptides or nucleic acid molecules in a native, non-denatured state at ambient temperature.SOLUTION: The invention provides a formulation for substantially stable storage of one or more polypeptides in a native, non-denatured conformation in a blood sample at ambient temperature, where the one or more polypeptides are stabilized in a native, non-denatured conformation substantially free of contaminating intracellular polypeptides after storage at ambient temperature for at least three days. The formulation comprises (i) a pH buffer, (ii) a non-reducing sugar, (iii) a trisaccharide, and (iv) one or more of a water-soluble polymer, a cationic compound, a zwitterionic compound, a phosphatase inhibitor, and a combination thereof.SELECTED DRAWING: None

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 62 / 010,164, filed Jun. 10, 2014, which is incorporated herein by reference in its entirety. 1.Technical Field The present invention generally relates to the stabilization of one or more native nucleic acid molecules and / or polypeptides, or exosomes, in a blood sample at ambient temperature.The present invention particularly relates to formulations, compositions, products, kits, and methods for the substantially stable preservation of one or more native nucleic acid molecules and / or polypeptides, or exosomes, in a blood sample at ambient temperature. [Background technology]

[0002] 2.Background Whole blood is a complex mixture of cells, nucleic acids, proteins, and various other analytes. Blood components include, but are not limited to, cells such as white blood cells (monocytes, lymphocytes, and granulocytes), red blood cells, platelets, and circulating tumor cells; nucleic acid molecules such as circulating free DNA (cfDNA); polypeptides such as lipoproteins, albumin, and serum proteins, and various other analytes.

[0003] Due in part to the relatively short half-life and storage requirements for whole blood, there is a continuing need for donors for whole blood. For example, whole blood collected for transfusion must be transported under refrigerated conditions and stored under frozen conditions with constant rocking to maintain intact viable cells and protect cellular or acellular polypeptides, nucleic acid molecules, and other analytes from chemical or enzymatic degradation. This refrigeration requirement can limit the availability of whole blood supplies in areas that lack the necessary refrigeration facilities, for example, due to lack of sufficient equipment or the necessary continuous power to maintain the appropriate refrigeration temperatures.

[0004] Compositions and methods for stabilizing, transporting and storing whole blood and blood components at ambient temperature have been investigated.While currently used compositions can prevent degradation of polypeptides and nucleic acid molecules, their main limitation is that stabilized polypeptides and nucleic acid molecules are maintained in denatured conformation, such as single-stranded DNA lacking proper secondary and / or tertiary structure, and unfolded proteins, due to the reaction components used in the storage formulations and compositions.Another limitation is that these formulations can result in the lysis of blood cells, thereby releasing the contents of cells into whole blood samples, which can complicate the subsequent quantification and diagnostic analysis of freely circulating polypeptides and nucleic acid molecules due to contaminating genomic DNA and intracellular proteins.

[0005] Non-invasive diagnostic tests have been developed in recent years to determine fetal abnormalities using cell-free DNA circulating in maternal blood (e.g., MATERNIT21® PLUS Non-Invasive Fetal Anomaly Test, Sequenom, Inc). The impact of this tool has discouraged further research into the use of cell-free DNA and cell-free amino acid profiles to predict disease states and the relative progression of certain diseases. The use of blood samples taken within a pre-specified short time is paramount, as changes to the profile occur relatively quickly at ambient temperature, and exposure to 4° C. can cause spurious changes in diagnostic markers on the cell surface and increase the amount released into the plasma or serum fraction. Collection tubes exist to stabilize cell-free nucleic acids for up to 7 days at room temperature (Streck Labs), but the main component of the formulation provided in the tube is a formaldehyde-releasing agent (such as imidazolinyl urea or diazolidinyl urea) that reacts to fix the cells and make them less permeable to agents entering or leaving the cells.

[0006] Thus, there is a need to develop novel formulations, compositions, and methods for stabilization of nucleic acid molecules and polypeptides, either individually or together, where the substantially stable nucleic acid molecules and polypeptides are maintained in their native, undenatured conformation in blood samples at ambient temperature.The formulations, compositions, and methods of the present invention advantageously overcome the aforementioned limitations by preserving the integrity of blood cells to prevent contamination of circulating nucleic acid molecules and polypeptides with components of lysed cells, while protecting functionally active polypeptides and nucleic acid molecules in their native conformation at ambient temperature.These stable nucleic acid molecules and polypeptides may be transported and stored without the need for refrigeration or freezing, and are stable at ambient temperature for extended periods of time, e.g., days, weeks, months, or years, facilitating the quantification, analysis, and / or use of various nucleic acid molecules and polypeptides for diagnostic and potentially therapeutic applications. Summary of the Invention

[0007] In one aspect of the present invention, a formulation is provided for the substantially stable storage of one or more polypeptides in a natural, native conformation in a blood sample at ambient temperature, wherein the one or more polypeptides are stabilized in a natural, native conformation substantially free of contaminating intracellular polypeptides after storage at ambient temperature for at least 3 days. In some embodiments, at least 80% of the polypeptides are stabilized in a natural, native conformation after storage at ambient temperature for at least 3 days. In some embodiments, the one or more polypeptides remain in a natural, native conformation after storage at ambient temperature for at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days. In some embodiments, the formulation comprises one or more of the following: a pH buffer; a non-reducing sugar; a trisaccharide; and a water soluble polymer; a cationic compound; a zwitterionic compound; a phosphatase inhibitor, or a combination thereof. In some embodiments, the non-reducing sugar is sucrose. In some embodiments, the trisaccharide is selected from the group consisting of maltotriose, isomaltotriose, raffinose, melezitose, nigerotriose, and combinations thereof. In some embodiments, the trisaccharide is melezitose. In some embodiments, the water soluble polymer is polyvinyl alcohol. In some embodiments, the phosphatase inhibitor is 2-glycerol phosphate. In some embodiments, the zwitterionic compound is a compound of formula (I):

[0008] [ka]

[0009] wherein R1, R2, and R3 are independently selected from unsubstituted or substituted alkyl, unsubstituted or substituted aryl, unsubstituted or substituted arylalkyl, or R1 and R2 optionally form a ring, Y is CH2, CH(A), CH(A)-CH(A), CH(A)-CH(A)-CH(A), where A is unsubstituted or substituted alkyl, aryl, arylalkyl, or any side chain typically found in one of the 20 naturally occurring amino acids; and Z is CO2-, SO3-, or OPO3-. In some embodiments, the cationic compound is selected from the group consisting of compounds of the following formula: (a) A compound of formula (II)

[0010] [ka]

[0011] wherein R1, R2, and R3 are independently selected from unsubstituted or substituted alkyl, unsubstituted or substituted aryl, unsubstituted or substituted arylalkyl, or R1 and R2 optionally form a ring; Y is CH2, CH(A), CH(A)-CH(A), CH(A)-CH(A)-CH(A), where A is unsubstituted or substituted alkyl, aryl, arylalkyl, or any side chain typically found in one of the 20 naturally occurring amino acids; Z is CO2A; and X is a pharma- ceutically acceptable anion: (b) A compound of formula (III)

[0012] [ka]

[0013] wherein R1, R2, R3, and R4 are independently selected from unsubstituted or substituted alkyl, unsubstituted or substituted aryl, unsubstituted or substituted arylalkyl, or R1 and R2 optionally form a ring, Y is CH2, CH(A), CH(A)-CH(A), CH(A)-CH(A)-CH(A), where A is unsubstituted or substituted alkyl, aryl, arylalkyl, or any side chain typically found in one of the 20 naturally occurring amino acids; and X is a pharma- ceutically acceptable anion; and (c) a compound of formula (IV):

[0014] [ka]

[0015] wherein R1 and R2 are independently selected from unsubstituted or substituted alkyl, unsubstituted or substituted aryl, unsubstituted or substituted arylalkyl; and X is a pharma- ceutically acceptable anion. In some embodiments, R1 and R2 of the compound of Formula (I), Formula (II), or Formula (III) form a morpholino ring, a pyrrolidinium ring, a piperidinium ring, or an oxazinium ring. In some embodiments, the zwitterionic compound is selected from the zwitterionic compounds set forth in Table 1. In some embodiments, the cationic compound is selected from the cationic compounds set forth in Table 1. In some embodiments, the zwitterionic compound is N,N-dimethyl-N-(2-hydroxyethyl)-3-ammonium-propionate or N-ethyl-piperidinium-4-butylsulfonate. In some embodiments, the one or more polypeptides are selected from the group consisting of antibodies, enzymes, plasma proteins, serum proteins, and combinations thereof. In some embodiments, the composition comprises: a pH buffer; a non-ionic starch; and one or more of a polyol, a phosphatase inhibitor, an amino acid, or a combination thereof. In some embodiments, the polyol is selected from the group consisting of glycol, glycerol, erythritol, threitol, arabitol, xylitol, ribitol, adonitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol, and combinations thereof. In some embodiments, the polyol is glycerol. In some embodiments, the phosphatase inhibitor is 2-glycerol phosphate. In some embodiments, the amino acid is glycine. In some embodiments, the amino acid is sarcosine. In some embodiments, the non-reducing sugar is sucrose and the trisaccharide is selected from the group consisting of maltotriose, isomaltotriose, raffinose, melezitose, and nigerotriose, more preferably melezitose. In some embodiments, the water soluble polymer is polyvinyl alcohol, the phosphatase inhibitor is 2-glycerol phosphate, and the zwitterionic compound is a quaternary inner salt.In some embodiments, the water-soluble polymer is polyvinyl alcohol, the phosphatase inhibitor is 2-glycerol phosphate, and the zwitterionic compound is N,N-dimethyl-N-(2-hydroxyethyl)-3-ammonium-propionate or N-ethyl-piperidinium-4-butylsulfonate. In certain embodiments, the polyol is selected from the group consisting of glycol, glycerol, erythritol, threitol, arabitol, xylitol, ribitol, adonitol, mannitol, sorbitol, galactitol, fucitol, iditol, and inositol, the phosphatase inhibitor is 2-glycerol phosphate, and the amino acid is glycine or sarcosine. In certain embodiments, the polyol is glycerol, the phosphatase inhibitor is 2-glycerol phosphate, and the amino acid is glycine or sarcosine. In some embodiments, the formulation is selected from those specified in Table 2. In some embodiments, a substantially stably stored composition of one or more purified, native polypeptides is provided, comprising one or more purified, native polypeptides mixed with any of the aforementioned formulations.

[0016] In some embodiments, a method for substantially stable preservation of one or more polypeptides in a blood sample in a natural, native conformation at ambient temperature is described herein, the method comprising: mixing a sample of blood taken from a subject with a formulation for substantially stable preservation of one or more polypeptides in a blood sample in a natural, native conformation described herein, wherein the one or more polypeptides remain in a natural, native state after storage at room temperature for at least 3 days. In some embodiments, the polypeptide is selected from the group consisting of an antibody, an enzyme, a plasma protein, a serum protein, and combinations thereof. In some embodiments, at least 80% of the polypeptides remain in a natural, native conformation after storage at ambient temperature for at least 3 days. In some embodiments, the subject is an animal. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0017] In another aspect of the present invention, a formulation is provided for the substantially stable storage of one or more nucleic acid molecules in a natural, native state in a blood sample at ambient temperature, wherein the one or more nucleic acid molecules are stabilized in a natural, native state substantially free of contaminating intracellular nucleic acid molecules after storage at ambient temperature for at least 3 days. In some embodiments, at least 80% of the nucleic acid molecules are stabilized in a natural, native state substantially free of contaminating intracellular nucleic acid molecules after storage at ambient temperature for at least 3 days. In some embodiments, the one or more nucleic acid molecules remain in a natural, native conformation after storage at room temperature for at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days. In some embodiments, the formulation comprises: a pH buffer; a phosphatase inhibitor; a purine; a zwitterionic compound; a cationic compound; or a combination thereof; and an apoptosis inhibitor or a caspase inhibitor. In some embodiments, the phosphatase inhibitor is 2-glycerol phosphate. In some embodiments, the zwitterionic compound is a quaternary inner salt. In some embodiments, the quaternary inner salt is 2-(benzyl(2-hydroxyethyl)(methyl)ammonio)acetate. In some embodiments, the zwitterionic compound is a compound of formula (I):

[0018] [ka]

[0019] wherein R1, R2, and R3 are independently selected from unsubstituted or substituted alkyl, unsubstituted or substituted aryl, unsubstituted or substituted arylalkyl, or R1 and R2 optionally form a ring, Y is CH2, CH(A), CH(A)-CH(A), CH(A)-CH(A)-CH(A), where A is unsubstituted or substituted alkyl, aryl, arylalkyl, or any side chain typically found in one of the 20 naturally occurring amino acids; and Z is CO2-, SO3-, or OPO3-. In some embodiments, the cationic compound is selected from the group consisting of compounds of the following formula: (a) A compound of formula (II):

[0020] [ka]

[0021] wherein R1, R2, and R3 are independently selected from unsubstituted or substituted alkyl, unsubstituted or substituted aryl, unsubstituted or substituted arylalkyl, or R1 and R2 optionally form a ring, Y is CH2, CH(A), CH(A)-CH(A), CH(A)-CH(A)-CH(A), where A is unsubstituted or substituted alkyl, aryl, arylalkyl, or any side chain typically found in one of the 20 naturally occurring amino acids; Z is CO2A; and X is a pharma- ceutically acceptable anion; (b) A compound of formula (III):

[0022] [ka]

[0023] wherein R1, R2, R3, and R4 are independently selected from unsubstituted or substituted alkyl, unsubstituted or substituted aryl, unsubstituted or substituted arylalkyl, or R1 and R2 optionally form a ring, Y is CH2, CH(A), CH(A)-CH(A), CH(A)-CH(A)-CH(A), where A is unsubstituted or substituted alkyl, aryl, arylalkyl, or any side chain typically found in one of the 20 naturally occurring amino acids; and X is a pharma- ceutically acceptable anion; and (c) a compound of formula (IV):

[0024] [ka]

[0025] wherein R1 and R2 are independently selected from unsubstituted or substituted alkyl, unsubstituted or substituted aryl, unsubstituted or substituted arylalkyl; and X is a pharma- ceutically acceptable anion. In some embodiments, R1 and R2 of the compound of Formula (I), Formula (II), or Formula (III) form a morpholino ring, a pyrrolidinium ring, a piperidinium ring, or an oxazinium ring. In some embodiments, the zwitterionic compound is selected from the zwitterionic compounds specified in Table 1. In some embodiments, the cationic compound is selected from the cationic compounds specified in Table 1. In some embodiments, the formulation further comprises a nuclease inhibitor. In some embodiments, the purine is adenine. In some embodiments, the nuclease inhibitor is aurintricarboxylic acid. In some embodiments, the composition further comprises an agent selected from the group consisting of an antibiotic, a purine derivative, and combinations thereof. In some embodiments, the antibiotic is selected from the group consisting of rifampicin, actinomycin D, 5-hydroxy-1,4-naphthoquinone, and combinations thereof. In some embodiments, the purine derivative is 5-mercaptopurine. In some embodiments, the caspase inhibitor is Q-VD-OPH. In some embodiments, the one or more nucleic acid molecules are circulating-free DNA molecules. In certain embodiments, the phosphatase inhibitor is 2-glycerol phosphate, the zwitterionic compound is a quaternary inner salt, more preferably 2-(benzyl(2-hydroxyethyl)(methyl)ammonio)acetate, the apoptosis inhibitor is Apotosis Inhibitor (Calbiochem Cat #178488), the caspase inhibitor is Q-VD-OPH, and the pH buffer is MOPS. In certain embodiments, the phosphatase inhibitor is 2-glycerol phosphate, the zwitterionic compound is 2-(benzyl(2-hydroxyethyl)(methyl)ammonio)acetate, the apoptosis inhibitor is Apoptosis Inhibitor (Calbiochem Cat#178488), the caspase inhibitor is Q-VD-OPH, and the pH buffer is MOPS.In some embodiments, the formulation comprises: a pH buffer; a phosphatase inhibitor; a purine; a cationic or zwitterionic compound; and a nuclease inhibitor. In certain embodiments, the phosphatase inhibitor is 2-glycerol phosphate, the purine is adenine, the zwitterionic compound is a quaternary inner salt, the nuclease inhibitor is aurintricarboxylic acid, and the pH buffer is MOPS. In certain embodiments, the phosphatase inhibitor is 2-glycerol phosphate, the purine is adenine, the zwitterionic compound is 2-(benzyl(2-hydroxyethyl)(methyl)ammonio)acetate, the nuclease inhibitor is aurintricarboxylic acid, and the pH buffer is MOPS. In some embodiments, the formulation further comprises a polar solvent and a chelating agent. In some embodiments, the formulation further comprises dimethylacetamide and tripotassium EDTA. In some embodiments, the formulation is selected from the formulations specified in Table 3. In some embodiments, a substantially stably stored composition of purified, native one or more nucleic acid molecules is provided, comprising the purified, native one or more nucleic acid molecules admixed with any of the aforementioned formulations.

[0026] In some embodiments, a method for substantially stable storage of one or more nucleic acid molecules in a natural, native state in a blood sample at ambient temperature is described herein, the method comprising: mixing a sample of blood taken from a subject with a formulation for substantially stable storage of one or more nucleic acid molecules in a natural, native state in a blood sample at ambient temperature as described herein, wherein the one or more nucleic acid molecules remain in a natural, native state substantially free of contaminating intracellular nucleic acid molecules after storage at room temperature for at least 3 days. In some embodiments, the nucleic acid molecule encodes a polypeptide selected from the group consisting of an antibody, an enzyme, a plasma protein, and a serum protein. In some embodiments, at least 80% of the nucleic acid molecule remains in a natural, native conformation substantially free of contaminating intracellular nucleic acid molecules after storage at ambient temperature for at least 3 days. In some embodiments, the nucleic acid molecule is a circulating free DNA molecule. In some embodiments, the subject is an animal. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0027] In some embodiments, a formulation for the substantially stable storage of one or more exosomes in a natural state in a blood sample at ambient temperature is described herein, the formulation comprises: a pH buffer; a phosphatase inhibitor; a purine; a zwitterionic compound; a cationic compound; or a combination thereof; and an apoptosis inhibitor or a caspase inhibitor, and one or more exosomes are stabilized after storage at room temperature for at least 3 days. In some embodiments, at least 80% of the exosomes are stabilized after storage at room temperature for at least 3 days. In some embodiments, one or more exosomes are stabilized after storage at room temperature for at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, or at least 18 days. In some embodiments, the phosphatase inhibitor is 2-glycerol phosphate. In some embodiments, the zwitterionic compound is a quaternary inner salt. In some embodiments, the quaternary inner salt is 2-(benzyl(2-hydroxyethyl)(methyl)ammonio)acetate. In some embodiments, the zwitterionic compound is a compound of formula (I):

[0028] [ka]

[0029] wherein R1, R2, and R3 are independently selected from unsubstituted or substituted alkyl, unsubstituted or substituted aryl, unsubstituted or substituted arylalkyl, or R1 and R2 optionally form a ring, Y is CH2, CH(A), CH(A)-CH(A), CH(A)-CH(A)-CH(A), where A is unsubstituted or substituted alkyl, aryl, arylalkyl, or any side chain typically found in one of the 20 naturally occurring amino acids; and Z is CO2-, SO3-, or OPO3-. In some embodiments, the cationic compound is selected from the group consisting of compounds of the following formula: (a) A compound of formula (II):

[0030] [ka]

[0031] wherein R1, R2, and R3 are independently selected from unsubstituted or substituted alkyl, unsubstituted or substituted aryl, unsubstituted or substituted arylalkyl, or R1 and R2 optionally form a ring, Y is CH2, CH(A), CH(A)-CH(A), CH(A)-CH(A)-CH(A), where A is unsubstituted or substituted alkyl, aryl, arylalkyl, or any side chain typically found in one of the 20 naturally occurring amino acids; Z is CO2A; and X is a pharma- ceutically acceptable anion; (b) A compound of formula (III):

[0032] [ka]

[0033] wherein R1, R2, R3, and R4 are independently selected from unsubstituted or substituted alkyl, unsubstituted or substituted aryl, unsubstituted or substituted arylalkyl, or R1 and R2 optionally form a ring, Y is CH2, CH(A), CH(A)-CH(A), CH(A)-CH(A)-CH(A), where A is unsubstituted or substituted alkyl, aryl, arylalkyl, or any side chain typically found in one of the 20 naturally occurring amino acids; and X is a pharma- ceutically acceptable anion; and (c) a compound of formula (IV):

[0034] [ka]

[0035] wherein R1 and R2 are independently selected from unsubstituted or substituted alkyl, unsubstituted or substituted aryl, unsubstituted or substituted arylalkyl; and X is a pharma- ceutically acceptable anion. In some embodiments, R1 and R2 of the compound of Formula (I), Formula (II), or Formula (III) form a morpholino ring, a pyrrolidinium ring, a piperidinium ring, or an oxazinium ring. In some embodiments, the zwitterionic compound is selected from the zwitterionic compounds specified in Table 1. In some embodiments, the cationic compound is selected from the cationic compounds specified in Table 1. In some embodiments, the formulation further comprises a nuclease inhibitor. In some embodiments, the purine is adenine. In some embodiments, the nuclease inhibitor is aurintricarboxylic acid. In some embodiments, the composition further comprises an agent selected from the group consisting of an antibiotic, a purine derivative, and combinations thereof. In some embodiments, the antibiotic is selected from the group consisting of rifampicin, actinomycin D, 5-hydroxy-1,4-naphthoquinone, and combinations thereof. In some embodiments, the purine derivative is 5-mercaptopurine. In certain embodiments, the phosphatase inhibitor is 2-glycerol phosphate, the zwitterionic compound is a quaternary inner salt, more preferably 2-(benzyl(2-hydroxyethyl)(methyl)ammonio)acetate, the apoptosis inhibitor is Apotosis Inhibitor (Calbiochem Cat #178488), the caspase inhibitor is Q-VD-OPH, and the pH buffer is MOPS. In certain embodiments, the phosphatase inhibitor is 2-glycerol phosphate, the zwitterionic compound is 2-(benzyl(2-hydroxyethyl)(methyl)ammonio)acetate, the apoptosis inhibitor is Apotosis Inhibitor (Calbiochem Cat #178488), the caspase inhibitor is Q-VD-OPH, and the pH buffer is MOPS. In certain embodiments, the formulation comprises a pH buffer, a phosphatase inhibitor, a purine, a cationic compound, or a zwitterionic compound; and a nuclease inhibitor.In certain embodiments, the phosphatase inhibitor is 2-glycerol phosphate, the purine is adenine, the zwitterionic compound is a quaternary inner salt, the nuclease inhibitor is aurintricarboxylic acid, and the pH buffer is MOPS. In certain embodiments, the phosphatase inhibitor is 2-glycerol phosphate, the purine is adenine, the zwitterionic compound is 2-(benzyl(2-hydroxyethyl)(methyl)ammonio)acetate, the nuclease inhibitor is aurintricarboxylic acid, and the pH buffer is MOPS. In some embodiments, the formulation further comprises a polar solvent and a chelating agent. In some embodiments, the formulation further comprises dimethylacetamide and tripotassium EDTA. In some embodiments, the formulation is selected from the formulations set forth in Table 3. In some embodiments, a substantially stably preserved composition of one or more purified exosomes is provided, comprising one or more purified exosomes mixed with any of the aforementioned formulations.

[0036] In some embodiments, disclosed herein is a method for substantially stably preserving one or more exosomes in their native state in a blood sample at ambient temperature, the method comprising: mixing a sample of blood taken from a subject with a formulation for substantially stable preservation of one or more exosomes in their native state in a blood sample described herein, wherein the one or more exosomes remain in their native state after storage at room temperature for at least 3 days. In some embodiments, the subject is an animal. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0037] In some embodiments, described herein is an article of manufacture comprising any one of the formulations described herein, including those set forth in Tables 2 and 3, contained within a blood collection tube. In some embodiments, the blood collection tube is an evacuated blood collection tube.

[0038] In some embodiments, a kit comprising any one of the articles of manufacture and a package insert is described herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] The present invention relates to formulations, compositions, products, kits, and methods for the substantially stable storage of one or more natural, undenatured polypeptides or nucleic acid molecules in a blood sample at ambient temperature.The present invention also relates to formulations, compositions, products, kits, and methods for the substantially stable storage of one or more exosomes in a blood sample at ambient temperature.In some embodiments, the formulations provide substantially stable storage of the polypeptides and / or nucleic acid molecules, where at least 60%, 70%, 80%, 90%, 95%, or even 99% of the polypeptides and / or nucleic acid molecules remain in a natural, undenatured state, substantially free of contaminating intracellular components, after storage at room temperature.In some embodiments, the formulations provide substantially stable storage of exosomes, where at least 50%, 60%, 70%, 80%, 90%, 95%, or even 99% of the exosomes are stable after storage at room temperature.

[0040] In some embodiments, the formulations provide substantially stable storage of polypeptides that retain native conformation and are functionally active. In some embodiments, the compositions described herein also provide substantially stable storage conditions for native full-length nucleic acid molecules, such as circulating free DNA, free of contaminating intracellular DNA, for improved quantification or diagnostic analysis. definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents, patent applications, and publications cited herein are incorporated by reference in their entirety.

[0041] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes one or more methods, and / or steps of the type described herein that would be apparent to a person of ordinary skill in the art upon reading this disclosure, etc.

[0042] As used herein, "about" when referring to measurable values ​​such as amounts, temporal durations, and the like, is meant to encompass variations of ±20%, or ±10%, or ±5%, or ±1% from the specified value, such variations being appropriate for the disclosed compositions or for practicing the disclosed methods.

[0043] As described herein, a nucleic acid molecule refers to a polymer of two or more modified and / or unmodified deoxyribonucleotides or ribonucleotides, either in the form of separate fragments or as a component of a larger structure. Nucleic acid molecules, oligonucleotides, and polynucleotides include RNA or DNA (either single-stranded or double-stranded, coding, complementary, or antisense), or RNA / DNA hybrid sequences of more than one nucleotide in single-stranded or double-stranded form, DNA analogs such as PNA (peptide nucleic acid), and any chemical modifications thereof. DNA can be single-stranded or double-stranded DNA, cDNA, or DNA amplified by any amplification technique or any DNA polymer. In some embodiments, the nucleic acid molecule is circulating free DNA (cfDNA). As used herein, cfDNA refers to DNA that circulates in the blood of a subject and is not contained within a cell. RNA can be mRNA, rRNA, tRNA, siRNA, total RNA, small nuclear RNA (snRNA), RNAi, microRNA, genomic RNA, RNA isolated from a cell or tissue, ribozyme, or any RNA polymer. In some embodiments, naturally occurring nucleic acid molecules are included, such as those that can be isolated from natural sources. In some embodiments, the nucleic acid molecules are forms, fragments, and derivatives derived from natural sources, as well as recombinant forms and artificial molecules, so long as at least one characteristic of the naturally occurring molecules is present. In some embodiments, the nucleic acid molecules are in biological samples and can be applied for analytical, diagnostic, and / or pharmaceutical purposes, such as, but not limited to, nucleic acids and their derivatives (e.g., oligonucleotides, DNA, cDNA, PCR products, genomic DNA, plasmids, chromosomes, artificial chromosomes, gene transfer vectors, RNA, mRNA, tRNA, siRNA, miRNA, hnRNA, ribozymes, genomic RNA, peptide nucleic acid (PNA), and bacterial artificial chromosomes (BAC). In some embodiments, the biological sample is blood.

[0044] As used in "purified polypeptide", or "purified nucleic acid molecule", or "purified exosome", the term "purified" refers to recovery of a polypeptide, nucleic acid molecule, or exosome, respectively, that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 98%, or at least 99% purified with respect to removal of impurities, e.g., cellular components such as proteins, lipids, or salts. The term "substantially purified" generally refers to the separation of most of the cellular proteins or reactive impurities from a blood sample, resulting in the removal of compounds that may interfere with the subsequent use of the separated biological molecule (such as a nucleic acid molecule).

[0045] As used herein, the term "polypeptide" refers to any polymeric chain of amino acids. The terms "peptide" and "protein" are used interchangeably with the term polypeptide and also refer to a polymeric chain of amino acids. The term "polypeptide" includes natural or artificial protein protein fragments and polypeptide analogs of protein sequences. Polypeptides can be monomeric or polymeric. Unless otherwise contradicted by the context, the term "polypeptide" includes fragments and variants thereof (including fragments of variants).

[0046] The term "isolated protein" or "isolated polypeptide" refers to a protein or polypeptide that is not associated with naturally associated components with which it is naturally associated by virtue of its origin (e.g., a cell or biological sample) or source of derivation. Thus, a chemically synthesized polypeptide will be "isolated" from its naturally associated components. Proteins can also be purified or substantially purified substantially free of naturally associated components by isolation purified to at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 98%, or at least 99% with respect to the removal of impurities, e.g., cellular components such as nucleic acids, lipids, or salts, using protein purification techniques well known in the art. Formulations and Compositions for Stabilization of Polypeptides, Nucleic Acids, or Exosomes in Their Native, Undenatured State in Blood Samples at Ambient Temperature In one aspect of the present invention, formulations and compositions are provided for the substantially stable storage of one or more polypeptides in native conformation in a blood sample at ambient temperature, where one or more polypeptides are stabilized in native, native conformation after storage at ambient temperature for at least 3 days. In some embodiments, formulations and compositions are provided for the substantial stability of polypeptides in native, native conformation in a blood sample at ambient temperature, where at least 80% of the polypeptides are stabilized in native, native conformation substantially free of contaminating intracellular polypeptides after storage at room temperature for at least 3 days. In some embodiments, one or more polypeptides remain in native, native conformation after storage at room temperature for at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, or at least 18 days.

[0047] The term "ambient temperature" as used herein refers to typical room temperature. In some embodiments, the ambient temperature is between 15° C. and 32° C. In some embodiments, the ambient temperature is between 20° C. and 27° C.

[0048] In certain embodiments, a formulation is described herein for the substantially stable storage of one or more nucleic acid molecules in a natural, native state in a blood sample at ambient temperature, the formulation comprising: a pH buffer; a non-reducing sugar; a trisaccharide; and at least one of a water-soluble polymer, a cationic compound, a zwitterionic compound, and a phosphatase inhibitor. In certain embodiments, a formulation is provided for the substantially stable storage of one or more polypeptides and / or nucleic acid molecules in a natural, native state in a blood sample at ambient temperature, the formulation comprising a pH buffer; a non-ionic starch; and one or more of a polyol, a phosphatase inhibitor, and an amino acid, wherein the one or more polypeptides and / or nucleic acid molecules are stabilized in a natural, native state after storage at room temperature for at least 3 days. In certain embodiments, the polyol is selected from the group consisting of glycol, glycerol, erythritol, threitol, arabitol, xylitol, ribitol, adonitol, mannitol, sorbitol, galactitol, fucitol, iditol and inositol, the phosphatase inhibitor is 2-glycerol phosphate and the amino acid is glycine or sarcosine.In certain embodiments, the polyol is glycerol, the phosphatase inhibitor is 2-glycerol phosphate and the amino acid is glycine or sarcosine.

[0049] In some embodiments, a formulation is provided for substantially stable storage of one or more nucleic acid molecules in a natural, native state in a blood sample at ambient temperature, where the one or more nucleic acid molecules remain in a natural, native state substantially free of contaminating intracellular nucleic acid molecules after storage at room temperature for at least 3 days. In some embodiments, a formulation is provided for substantially stable storage of one or more nucleic acid molecules in a natural, native state in a blood sample at ambient temperature, where at least 80% of the nucleic acid molecules are stabilized in a natural, native state substantially free of contaminating intracellular nucleic acid molecules after storage at room temperature for at least 3 days. In other embodiments, the one or more nucleic acid molecules remain in a natural, native conformation after storage at room temperature for at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, or at least 18 days.

[0050] In certain embodiments, the formulation comprises: a pH buffer; a phosphatase inhibitor; a purine; a zwitterionic compound, a cationic compound or a combination thereof; and an apoptosis inhibitor or a caspase inhibitor. In certain preferred embodiments, the phosphatase inhibitor is 2-glycerol phosphate, the zwitterionic compound is a quaternary inner salt, more preferably 2-(benzyl(2-hydroxyethyl)(methyl)ammonio)acetate, the apoptosis inhibitor is Apotosis Inhibitor (Calbiochem Cat #178488), the caspase inhibitor is Q-VD-OPH, and the pH buffer is MOPS.

[0051] In certain other embodiments, a formulation for the substantially stable preservation of one or more nucleic acids in a natural, undenatured state in a blood sample at ambient temperature comprises: a pH buffer; a phosphatase inhibitor; a purine; a zwitterionic compound, a cationic compound, or a combination thereof; and a nuclease inhibitor. In certain embodiments, the phosphatase inhibitor is 2-glycerol phosphate, the purine is adenine, the zwitterionic compound is a quaternary inner salt, more preferably 2-(benzyl(2-hydroxyethyl)(methyl)ammonio)acetate, the nuclease inhibitor is aurintricarboxylic acid, and the pH buffer is MOPS. In some embodiments, the formulation further comprises one of the following: an antibiotic, a purine derivative, an apoptosis inhibitor, a caspase inhibitor, or a combination thereof. In some embodiments, the formulation comprises a polar solvent and a chelating agent. In some embodiments, the formulation further comprises dimethylacetamide and tripotassium EDTA.

[0052] In some embodiments, compositions are provided herein in which a blood sample is mixed with a nucleic acid, a polypeptide, or an exosome, and a stabilizing formulation to generate one or more substantially stable native nucleic acids, polypeptides, or exosomes, respectively, in the preparation of whole blood. In some embodiments, compositions are provided that include one or more purified or substantially purified nucleic acid molecules, polypeptides, or exosomes mixed with the corresponding stabilizing formulation. Formulation Reagents A.pH buffer According to certain embodiments, the formulations and compositions described herein for the substantially stable preservation of nucleic acid molecules, polypeptides, or exosomes in blood samples at ambient temperature contain one or more pH buffers. In some embodiments, the pH buffer is any of a number of compounds known in the art for their ability to resist changes in pH of a solution, such as an aqueous solution, in which the pH buffer is present. The selection of one or more specific pH buffers for inclusion in the composition for stable preservation can be made based on the present disclosure and by practice in the art, and can be influenced by a variety of factors, including the pH desired to be maintained, the nature of the sample to be stabilized, the state of the solvent utilized, other components of the formulation used, and other criteria. For example, typically, a pH buffer is selected based on the proton dissociation constant (pK a ) at a pH within a range of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 pH units.

[0053] Non-limiting examples of pH buffers include citric acid, tartaric acid, malic acid, sulfosalicylic acid, sulfoisophthalic 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). Certain embodiments contemplated herein, including many of the formulations set forth in Table 2 or Table 3, may be characterized as formulations having a pH of about 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, or 9.0. B. Cationic and Zwitterionic Compounds In certain embodiments described herein, the zwitterionic compound is a quaternary inner salt. In some embodiments, the quaternary inner salt for the substantially stable storage of nucleic acids is one disclosed in WO 2012 / 018638.

[0054] In some embodiments, the zwitterionic compound is a compound of formula (I):

[0055] [ka]

[0056] wherein R1, R2, and R3 are independently selected from unsubstituted or substituted alkyl, unsubstituted or substituted aryl, unsubstituted or substituted arylalkyl, or R1 and R2 optionally form a ring, Y is CH2, CH(A), CH(A)-CH(A), CH(A)-CH(A)-CH(A), where A is unsubstituted or substituted alkyl, aryl, arylalkyl, or any side chain typically found in one of the 20 naturally occurring amino acids; and Z is CO2-, SO3-, or OPO3-. In some embodiments, R1 and R2 form a morpholino ring, a pyrrolidinium ring, a piperidinium ring, or an oxazinium ring.

[0057] In some embodiments, the cationic compound is selected from the group consisting of compounds of the following formula: (a) A compound of formula (II):

[0058] [ka]

[0059] wherein R1, R2, and R3 are independently selected from unsubstituted or substituted alkyl, unsubstituted or substituted aryl, unsubstituted or substituted arylalkyl, or R1 and R2 optionally form a ring, Y is CH2, CH(A), CH(A)-CH(A), CH(A)-CH(A)-CH(A), where A is unsubstituted or substituted alkyl, aryl, arylalkyl, or any side chain typically found in one of the 20 naturally occurring amino acids; Z is CO2A; and X is a pharma- ceutically acceptable anion; (b) A compound of formula (III):

[0060] [ka]

[0061] wherein R1, R2, R3, and R4 are independently selected from unsubstituted or substituted alkyl, unsubstituted or substituted aryl, unsubstituted or substituted arylalkyl, or R1 and R2 optionally form a ring, Y is CH2, CH(A), CH(A)-CH(A), CH(A)-CH(A)-CH(A), where A is unsubstituted or substituted alkyl, aryl, arylalkyl, or any side chain typically found in one of the 20 naturally occurring amino acids; and X is a pharma- ceutically acceptable anion; and (c) a compound of formula (IV):

[0062] [ka]

[0063] wherein R1 and R2 are independently selected from unsubstituted or substituted alkyl, unsubstituted or substituted aryl, unsubstituted or substituted arylalkyl; and X is a pharma- ceutically acceptable anion. In some embodiments, R1 and R2 of the compound of Formula (II) or Formula (III) form a morpholino ring, a pyrrolidinium ring, a piperidinium ring, or an oxazinium ring.

[0064] An "alkyl" group refers to an aliphatic hydrocarbon group. The alkyl moiety includes a "saturated alkyl" group, meaning that it does not contain an alkene or alkyne moiety. The alkyl moiety also includes an "unsaturated alkyl" moiety, meaning that it contains at least one alkene or alkyne moiety. An "alkene" moiety refers to a group having at least one carbon-carbon double bond, and an "alkyne" moiety refers to a group having at least one carbon-carbon triple bond. The alkyl moiety includes branched, straight, or cyclic moieties, whether saturated or unsaturated. Depending on the structure, an alkyl group includes a monoradical or a diradical (i.e., an alkylene group), and is a "lower alkyl" having from 1 to 6 carbon atoms. As used herein, C1-C x is C1-C2, C1-C3...C1-C x The "alkyl" moiety optionally has 1 to 10 carbon atoms (whenever it appears herein, a number range such as "1 to 10" refers to each integer within the given range; for example, "1 to 10 carbon atoms" means that the alkyl group is selected from moieties having 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to 10 carbon atoms, but this definition also encompasses occurrences of the term "alkyl" where no number range is specified). The alkyl group of the compounds described herein may be designated as "C1-C4 alkyl" or a similar designation. By way of example only, "C1-C4 alkyl" indicates that there are 1 to 4 carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, and t-butyl. Thus, C1-C4 alkyl includes C1-C2 alkyl and C1-C3 alkyl. The alkyl group is optionally substituted or unsubstituted. Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl, ethenyl, propenyl, butenyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.

[0065] As used herein, the term "ring" refers to a covalently closed structure. Rings include, for example, carbocycles (e.g., aryl and cycloalkyl), heterocycles (e.g., heteroaryl and non-aromatic heterocycles), aromatics (e.g., aryl and heteroaryl), and non-aromatics (e.g., cycloalkyl and non-aromatic heterocycles). Rings can be optionally substituted. Rings can be monocyclic or polycyclic.

[0066] The term "aryl," used alone or as part of a larger moiety, as in "arylalkyl," "arylalkoxy," or "aryloxyalkyl," refers to a monocyclic, bicyclic, or tricyclic carbocyclic ring system containing fused rings in which at least one ring is aromatic. The term "aryl" may be used interchangeably with the term "aryl ring." In one embodiment, aryl includes groups having 6-12 carbon atoms. In another embodiment, aryl includes groups having 6-10 carbon atoms. Examples of aryl groups include phenyl, naphthyl, anthracyl, phenanthrenyl, naphthacenyl, 1,2,3,4-tetrahydronaphthalenyl, 1H-indenyl, 2,3-dihydro-1H-indenyl, and the like. A particular aryl is phenyl. In another embodiment, aryl includes indanyl, naphthyl, and tetrahydronaphthyl, where the radical or point of attachment is on the aromatic ring.

[0067] The term "optionally substituted" or "substituted" means that the referenced group can be substituted with one or more additional groups individually and independently selected from alkyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, alkylthio, arylthio, alkylsulfoxide, arylsulfoxide, alkylsulfone, arylsulfone, cyano, halo, acyl, nitro, haloalkyl, fluoroalkyl, amino, and protected derivatives thereof, including mono- and di-substituted amino groups. As an example, the optional substituents may be LsRs, where each Ls is independently selected from a single bond, -O-, -C(=O)-, -S-, -S(=O)-, -S(=O)2-, -NH-, -NHC(O)-, -C(O)NH-, S(=O)2NH-, -NHS(=O)2, -OC(O)NH, -NHC(O)O-, -(substituted or unsubstituted C1-C6 alkyl), or -(substituted or unsubstituted C2-C6 alkenyl); and each Rs is independently selected from H, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, heteroaryl, or heteroalkyl.

[0068] In certain other embodiments of the formulations described herein, including those preparations set forth in Table 2 or Table 3, the cationic or zwitterionic compound is selected from one of the exemplary compounds in Table 1.

[0069] [Table 1-1]

[0070] [Table 1-2]

[0071] In certain embodiments for the substantially stable storage of one or more polypeptides, including formulations set forth in Table 2, the zwitterionic compound is a quaternary inner salt. In certain embodiments, the quaternary inner salt is N,N-dimethyl-N-(2-hydroxyethyl)-3-ammonium-propionate or N-ethyl-piperidinium-4-butylsulfonate at a concentration of about 1.0-100 mg / mL, 1.0-50 mg / mL, or 10.0-50 mg / mL. In certain embodiments for the substantially stable storage of one or more nucleic acid molecules, including formulations set forth in Table 3, the quaternary inner salt is 2-(benzyl(2-hydroxyethyl)(methyl)ammonio)acetate. C. Non-reducing sugars Also, as described herein, in some embodiments, there is a formulation comprising at least one non-reducing sugar in a composition for the substantially stable preservation of one or more polypeptides in a native conformation in a blood sample at ambient temperature. A non-reducing sugar is a carbohydrate molecule that lacks a functional aldehyde group. Exemplary non-reducing sugars include sucrose and trehalose. In some embodiments, the non-reducing sugar is trehalose and is present at a concentration of about 1.0-50 mM, or about 10.0-30 mM, or about 25 mM. In some embodiments for the substantially stable preservation of one or more polypeptides in a native conformation, the non-reducing sugar is sucrose that is present at a concentration of about 1.0-50 mM, about 1.0-30 mM, or about 10 mM. D. trisaccharide As described herein, in certain embodiments, the formulation includes at least one trisaccharide in the formulation or composition for the substantially stable preservation of one or more polypeptides in their native conformation in a whole blood sample at ambient temperature. Trisaccharides are oligosaccharides consisting of three monosaccharide monomers with glycosidic bonds attached thereto. Glycosidic bonds can be formed between hydroxyl groups on the constituent monosaccharides, and different bond combinations (regiochemistry) and stereochemistry (alpha- or beta-) result in diastereoisomeric trisaccharides with different chemical and physical properties. Selection of one or more particular trisaccharides for inclusion in the composition for stable preservation may be made based on the present disclosure and in accordance with practices in the art, and may be influenced by a variety of factors, including other formulation components. Exemplary trisaccharides include, but are not limited to, maltotriose, isomaltotriose, raffinose, melezitose, and nigerotriose. In certain embodiments for stabilizing polypeptides, including the formulations set forth in Table 2, the trisaccharide is melezitose. In some embodiments, melezitose is present at a concentration of about 1%-20%, or about 5.0-15%. E. Polyol Also, as described herein, certain embodiments include at least one polyol in a composition for the substantially stable preservation of one or more polypeptides in their native conformation in a whole blood sample at ambient temperature. A polyol is a polyhydric alcohol containing two or more hydroxyl groups and has the general formula H(CHOH) n H, where n is an integer selected from 2 to 7. Polyols vary in chain length, with most polyols having 5 or 6 carbon chains being derived from pentoses (5 carbon sugars) and hexoses (6 carbon sugars), although shorter and longer carbon chain polyols also exist. Exemplary polyols include, but are not limited to, glycol, glycerol, erythritol, threitol, arabitol, xylitol, ribitol, adonitol, mannitol, sorbitol, galactitol, fucitol, iditol, and inositol. Selection of one or more particular polyols for inclusion in a composition for substantially stable storage may be made based on the present disclosure and in accordance with practices in the art, and may be influenced by a variety of factors, including other formulation components. In certain embodiments, the polyol present in the formulation, including the formulation in Table 2, is a pentose polyol and is present at a concentration between 20-100 mM, or about 25-75 mM. In certain embodiments, the polyol present in the formulation, including the formulations of Table 2, is adonitol and is present at a concentration of between about 20-100 mM, or about 25-75 mM. F. Water-soluble polymers As described herein, certain embodiments include at least one water-soluble polymer in formulations and compositions for substantially stable preservation of one or more polypeptides in their native conformation in a whole blood sample at ambient temperature. Such water-soluble polymers include polyvinyl alcohol. From this disclosure, it will be understood that one of skill in the art may select other water-soluble polymers for use in formulations and compositions for stable preservation, which may vary based on other components of the composition utilized, the particular biological sample being preserved, whether the nucleic acid molecules or polypeptide molecules, or both, are desired to be retrieved, and other factors. Certain embodiments, including but not limited to the formulations shown in Table 2, contemplate the inclusion of a water-soluble polymer at a concentration (volumetric basis, i.e., vol / vol) of about 0.1-10% (vol / vol), about 0.1-5% (vol / vol), or 1.0% (vol / vol). In certain embodiments, the water-soluble polymer is polyvinyl alcohol having a molecular weight range of about 30-70,000 daltons and about 87-90% hydrolyzed. G. Phosphatase Inhibitors The formulation described herein for the substantially stable preservation of one or more nucleic acid molecules or polypeptides in a native state in a blood sample at ambient temperature comprises, in certain embodiments, a phosphatase inhibitor.In certain embodiments, the phosphatase inhibitor is an inhibitor of the serine-threonine class of phosphatases.In certain embodiments, the phosphatase inhibitor is 2-glycerol phosphate.For stabilizing polypeptides, the concentration is about 1.0-100mM, about 25-75mM, or about 50mM; for nucleic acid molecules, the concentration is about 1.0-200mM, about 25-150mM, or about 125mM; and for exosomes, the concentration is about 1.0-200mM, about 25-150mM, or about 125mM. H. Nonionic starch In certain embodiments, the formulation for the substantially stable preservation of one or more polypeptides in a native conformation in a blood sample at ambient temperature comprises, in certain embodiments, a non-ionic starch. In certain embodiments, the non-ionic starch is hydroxyethyl starch (HES). Hydroxyethyl starch is one of the most frequently used bulking agents under the trade name HESPAN by B. Braun Medical Inc. In certain embodiments, HES is present at a concentration of about 1.0-10%, about 1.0-5.0%, or about 1.0-2.0%. I. Chelating Agents Chelating agents or chelating agents are included in the formulations and compositions described herein for the substantially stable preservation of one or more polypeptides or nucleic acid molecules in a native state in a blood sample, according to certain embodiments, and are known to those skilled in the art for their ability to complex and prevent the reactivity of metallic cations. Exemplary chelating agents include diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), ethyleneglycoltetraacetic 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, sodium gluconate, and nitrilotriacetic acid (NTA). In some embodiments, the chelating agent is disodium or tripotassium EDTA and is present at a concentration of about 1.0-100 mM, about 10-90 mM, or about 70 mM. J. Nuclease Inhibitors In certain embodiments, the formulation for the substantially stable preservation of one or more native nucleic acid molecules in blood samples at ambient temperature comprises a nuclease inhibitor.Nuclease inhibitors are well known to those skilled in the art.In some embodiments, such nuclease inhibitors are used in the formulations, compositions and methods of the present invention, including the formulations in Table 3.In some embodiments, the nuclease inhibitor is aurintricarboxylic acid. K. Purines and purine derivatives Purines or purine derivatives are included in the compositions described herein for the substantially stable preservation of nucleic acid molecules in blood samples according to certain embodiments. In some embodiments, the purines are adenine, guanine, or combinations thereof. In some embodiments, the purines are purine derivatives. In some embodiments, the purine derivatives are 2-mercaptopurine. L. Antibiotics In certain embodiments, the formulation for the substantially stable preservation of one or more native nucleic acid molecules in a blood sample at ambient temperature comprises an antibiotic. Antibiotics are well known to those skilled in the art. In some embodiments, such antibiotics are used in the formulations, compositions and methods of the present invention. In some embodiments, the antibiotic is rifampicin, actinomycin D, 5-hydroxy-1,4-naphthoquinone, or a combination thereof. M. Polar solvents In certain embodiments, the formulation for the substantially stable preservation of one or more native nucleic acid molecules in a blood sample at ambient temperature comprises a polar solvent. In some embodiments, the polar solvent is dimethylacetamide. In some embodiments, the polar solvent is present at a concentration of 25%. In some embodiments, the polar solvent is dimethylacetamide and is present at a concentration of 25%. A formulation that stabilizes native, naturally occurring polypeptides, nucleic acid molecules, or exosomes in a blood sample at ambient temperature. Described herein, in some embodiments, is a formulation for the substantially stable storage of one or more native polypeptides in a blood sample at ambient temperature, the formulation comprising a pH buffer; a non-reducing sugar; a trisaccharide; and at least one of a water soluble polymer, a zwitterionic compound, a cationic compound, or a phosphatase inhibitor, wherein the one or more polypeptides and / or nucleic acid molecules remain in a native, native state after storage at room temperature for a period of at least 3 days. In other embodiments, the one or more polypeptides or nucleic acid molecules remain in a native, native conformation substantially free of contaminating intracellular polypeptides after storage at room temperature for a period of at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, or at least 18 days.

[0072] In a particular embodiment, the disaccharide is sucrose and the trisaccharide is selected from the group consisting of maltotriose, isomaltotriose, raffinose, melezitose, and nigerotriose, more preferably melezitose. In yet another preferred embodiment, the water-soluble polymer is polyvinyl alcohol, the phosphatase inhibitor is 2-glycerol phosphate, and the zwitterionic compound is a quaternary amine, preferably N,N-dimethyl-N-(2-hydroxyethyl)-3-ammonium-propionate or N-ethyl-piperidinium-4-butylsulfonate.

[0073] In certain preferred embodiments, the formulation for substantially stable storage of a native polypeptide in a blood sample at ambient temperature is selected from Table 2.

[0074] [Table 2]

[0075] In another aspect, a formulation is provided for the substantially stable storage of one or more nucleic acid molecules in a natural, undenatured state in a blood sample at ambient temperature, wherein the one or more nucleic acid molecules are stabilized in a natural, undenatured state substantially free of intracellular nucleic acid after storage at room temperature for a period of at least 3 days. In certain embodiments, the formulation comprises a pH buffer; a phosphatase inhibitor; a purine; a zwitterionic compound, a cationic compound, or a combination thereof; and an apoptosis inhibitor or a caspase inhibitor. In certain embodiments, the phosphatase inhibitor is 2-glycerol phosphate, the zwitterionic compound is a quaternary inner salt, the apoptosis inhibitor is Apoptosis Inhibitor (Calbiochem Cat #178488), the caspase inhibitor is Q-VD-OPH, and the pH buffer is MOPS. In certain embodiments, the phosphatase inhibitor is 2-glycerol phosphate, the zwitterionic compound is 2-(benzyl(2-hydroxyethyl)(methyl)ammonioacetate, the apoptosis inhibitor is Apoptosis Inhibitor (Calbiochem Cat #178488), the caspase inhibitor is Q-VD-OPH, and the pH buffer is MOPS. In other embodiments, the one or more nucleic acid molecules remain in a native, undenatured conformation substantially free of intracellular nucleic acid after storage at room temperature for a period of at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, or at least 18 days.

[0076] In certain other embodiments, the formulation comprises a pH buffer; a phosphatase inhibitor; a purine; a zwitterionic compound, a cationic compound, or a combination thereof; and a nuclease inhibitor. In certain embodiments, the phosphatase inhibitor is 2-glycerol phosphate, the purine is adenine, the zwitterionic compound is a quaternary inner salt, more preferably 2-(benzyl(2-hydroxyethyl)(methyl)ammonio)acetate, the nuclease inhibitor is aurintricarboxylic acid, and the pH buffer is MOPS. In other embodiments, the formulation may further comprise one of the following: an antibiotic, a purine derivative, an apoptosis inhibitor, or a caspase inhibitor. In some embodiments, the formulation further comprises a polar solvent and a chelating agent. In some embodiments, the formulation comprises dimethylacetamide and tripotassium EDTA.

[0077] In certain embodiments, the formulation for substantially stable storage of native nucleic acid molecules in a blood sample at ambient temperature is selected from Table 3.

[0078] [Table 3]

[0079] In another aspect, a formulation is provided for the substantially stable storage of one or more exosomes in a native state in a blood sample at ambient temperature for a period of at least three days. In certain embodiments, the formulation comprises a pH buffer; a phosphatase inhibitor; a purine; a zwitterionic compound, a cationic compound, or a combination thereof; and an apoptosis inhibitor or a caspase inhibitor. In certain embodiments, the phosphatase inhibitor is 2-glycerol phosphate, the zwitterionic compound is a quaternary inner salt, the apoptosis inhibitor is Apoptosis Inhibitor (Calbiochem Cat #178488), the caspase inhibitor is Q-VD-OPH, and the pH buffer is MOPS. In certain embodiments, the phosphatase inhibitor is 2-glycerol phosphate, the zwitterionic compound is 2-(benzyl(2-hydroxyethyl)(methyl)ammonioacetate, and the apoptosis inhibitor is Apoptosis Inhibitor (Calbiochem Cat #178488). #178488), the caspase inhibitor is Q-VD-OPH, and the pH buffer is MOPS. In other embodiments, the exosomes remain in a native state after storage at room temperature for a period of at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, or at least 18 days. In certain embodiments, the formulation comprises a pH buffer; a phosphatase inhibitor; a purine; a zwitterionic ionomer; an anionic or cationic compound; and a nuclease inhibitor. In certain embodiments, the phosphatase inhibitor is 2-glycerol phosphate, the purine is adenine, the zwitterionic compound is a quaternary inner salt, more preferably 2-(benzyl(2-hydroxyethyl)(methyl)ammonio)acetate, the nuclease inhibitor is aurintricarboxylic acid, and the pH buffer is MOPS. In other embodiments, the formulation may further comprise one of the following: an antibiotic and a purine derivative. In some embodiments, the formulation further comprises a polar solvent and a chelating agent.In some embodiments, the formulation comprises dimethylacetamide and tripotassium EDTA.

[0080] In certain embodiments, the formulation for substantially stable storage of exosomes in a blood sample at ambient temperature is selected from Table 3. Methods for preparing formulations for stabilizing native nucleic acid molecules and polypeptides at ambient temperature The formulation of the present invention can be prepared using commercially available reagents and methods well known to those skilled in the art.In some embodiments, the formulation is prepared as a concentrated stock solution (e.g., 2X, 5X, 10X, 20X, etc.) of the formulation reagent and mixed with the blood sample at the appropriate concentration.In some embodiments, the blood sample is mixed with the formulation stock in equal amounts (1:1). Purified Nucleic Acid Molecules and Polypeptides In some embodiments, the substantially stable one or more polypeptides and / or nucleic acid molecules in the blood sample at ambient temperature are further purified using well-known conventional methods routinely used by those skilled in the art. Devices, kits and methods for purifying nucleic acid molecules and polypeptides from blood are well-known. For example, the substantially stable polypeptides and nucleic acids are purified in some embodiments by affinity chromatography, gel electrophoresis, etc. In some embodiments, the purified one or more polypeptides or nucleic acid molecules are then stored in the formulations described herein for an extended period of time before analysis. <Product> In certain embodiments, articles of manufacture are provided in which one of the formulations described herein, including those specified in Tables 2 or 3, is contained in a suitable blood collection tube, container or vessel. In some embodiments, these articles are used for substantially stable storage of one or more blood components by stabilizing one or more blood components upon collection. In certain embodiments, the blood collection tube is an evacuated blood tube having subatmospheric pressure to withdraw a predetermined amount of whole blood. In some embodiments, these articles are used in the kits and methods described herein. <Kit> In certain aspects of the present invention, a kit is provided that comprises any one of the products that comprise the formulation of the present invention and package insert.In some embodiments, the components of the kit are provided in a container, such as a compartmentalized plastic enclosure.In some embodiments, the container has a sealed cover, so that the contents of the kit can be sterilized and sealed for storage before use. <Method for substantially stable storage of blood components> In another aspect of the present invention, a method is provided for substantially stable preservation of one or more polypeptides, nucleic acid molecules, or exosomes in a blood sample in a native, native conformation or state.

[0081] In certain embodiments, the method comprises mixing a blood sample with a formulation for substantially stable storage of one or more polypeptides in a blood sample at ambient temperature, wherein the one or more polypeptides are stabilized in a native, undenatured state after storage at room temperature for a period of at least 3 days. In some embodiments, at least 80% of the polypeptides remain in a native, undenatured state for a period of at least 3 days. In certain embodiments, the formulation for substantially stable storage of one or more native polypeptides in a blood sample at ambient temperature comprises a pH buffer; a non-reducing sugar; a trisaccharide; and at least one or more of a water-soluble polymer, a zwitterionic compound, a cationic compound, or a phosphatase inhibitor. In certain embodiments, the formulation is one of the formulations specified in Table 2. In some embodiments of the method, the one or more polypeptides remain in a native, non-denatured conformation after storage at room temperature for a period of at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, or at least 18 days.

[0082] In some embodiments, the method includes mixing a blood sample with a formulation for substantially stable storage of one or more nucleic acid molecules in a blood sample at ambient temperature, wherein the one or more nucleic acid molecules are stabilized in a native, undenatured state, substantially free of contaminating intracellular nucleic acids, after storage at room temperature for a period of at least 3 days. In some embodiments, at least 80% of the nucleic acids remain in a native, undenatured state for a period of at least 3 days. In certain embodiments, the formulation for substantially stable storage of one or more native nucleic acid molecules in a blood sample at ambient temperature includes a pH buffer; a phosphatase inhibitor; a purine; a zwitterionic compound, a cationic compound, or a combination thereof; and an apoptosis inhibitor or a caspase inhibitor. In certain embodiments, the formulation includes a pH buffer; a phosphatase inhibitor; a purine; a zwitterionic compound or a cationic compound; and a nuclease inhibitor. In some embodiments, the formulation further includes a polar solvent and a chelating agent. In some embodiments, the formulation further includes dimethylacetamide and tripotassium EDTA. In certain embodiments, the formulation is one of the formulations specified in Table 3. In some embodiments of the methods, the one or more nucleic acid molecules remain in a native, non-denatured conformation after storage at room temperature for a period of at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, or at least 18 days.

[0083] In some embodiments, the method includes mixing a blood sample with a formulation for substantially stable storage of one or more exosomes in a blood sample at ambient temperature, wherein the one or more exosomes are stabilized for a period of at least 3 days at ambient temperature. In some embodiments, at least 80% of the exosomes are stabilized for a period of at least 3 days. In certain embodiments, the formulation for substantially stable storage of one or more exosomes in a blood sample at ambient temperature includes a pH buffer; a phosphatase inhibitor; a purine; a zwitterionic compound, a cationic compound, or a combination thereof; and an apoptosis inhibitor or a caspase inhibitor. In certain embodiments, the formulation includes a pH buffer; a phosphatase inhibitor; a purine; a zwitterionic compound or a cationic compound; and a nuclease inhibitor. In some embodiments, the formulation further includes a polar solvent and a chelating agent. In some embodiments, the formulation further includes dimethylacetamide and tripotassium EDTA. In certain embodiments, the formulation is one of the formulations specified in Table 3. In some embodiments of the method, the one or more exosomes are stabilized after storage at room temperature for a period of at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, or at least 18 days.

[0084] Blood collection tubes, bags, containers and vessels are well known in the art and have been used by physicians for decades. Blood can be collected using methods or devices commonly used by those skilled in the art, such as venipuncture or finger stick. When blood is collected by venipuncture, the formulation of the present invention can be located inside the blood collection tube, for example, vacuum tube (VACUTAINER blood collection tube, Becton Dickenson or VACUETTE, Greiner), when blood sample is obtained from subject, or can be added to the whole blood sample already obtained immediately or shortly after it is drawn.

[0085] The methods as described herein may use the articles of manufacture and kits disclosed herein.

[0086] The following examples are offered by way of illustration and not by way of limitation. EXAMPLES

[0087] Example 1. Stabilization of functionally active polypeptides in human blood samples for a period of at least 9 days at ambient temperature. This example demonstrates formulations of the invention, including formulations specified to prepare substantially stable storage of native, functionally active polypeptides in their native, native conformation in blood samples for a period of at least 9 days at ambient temperature.

[0088] The ability of the exemplary formulation AG specified in Table 2 to stabilize multiple polypeptides at ambient temperature (e.g., survey of 29 cytokines and chemokines) was examined using the Millipex MAP Human cytokine / chemokine panel HCYTOMAG-60K-PX29 kit from Luminex Corp, according to the manufacturer's instructions.

[0089] Briefly, human whole blood samples were collected from donors, the blood was fractionated, and the plasma layer was analyzed. Aliquots of 75 or 135 (9:1 samples in Table 2) microliters of plasma were added to 1.7 mL microtubes, to which solutions of 29 different cytokines were added, the samples were spiked with 400 pg / mL of internal standard, and the volume was adjusted to 150 microliters. Control samples containing internal standard in the absence (stored at -80°C) or presence of plasma (fresh on the day of assay) were prepared and processed at the same time as the test samples. In addition, standard curves were prepared using 16, 80, 400, 2,000, and 10,000 pg / mL to ensure linearity of the assay and aid in proper quantification of the concentrations of bound cytokines and chemokines.

[0090] Control and test samples were placed at room temperature and 25 microliter aliquots were removed on days 0, 3 and 6. On days 0 and 3, samples were stored at -80°C and processed on day 6. To each 25 mL aliquot, 25 microliters of the respective assay buffer was added, followed by a 25 microliter volume of human cytokine / chemokine antibody-immobilized magnetic beads coated with one of 29 antibodies directed against each member of a panel of cytokines / chemokines. The antibodies only recognize native cytokine and chemokine polypeptides; denatured polypeptides do not bind to the beads. The mixture was incubated overnight at 4°C with shaking. The 96-well plate was placed against a magnet to sequester the bound magnetic beads, the supernatant was removed, and the beads were washed twice with wash buffer.

[0091] A 25 microliter aliquot of Detection Antibody solution was added to each sample to the washed beads and incubated at room temperature for 1 hour. A 25 microliter aliquot of Streptavidin-Phycoerythrin solution was added and the samples were stored at room temperature for 30 minutes. To isolate the beads, a magnet was placed next to the 96-well plate, the supernatant was removed, and the samples were washed twice and resuspended in 150 microliters of wash buffer.

[0092] The amount of bound cytokine / chemokine from each plasma sample was determined using a Luminex Bio-Tek ELx405 instrument according to the manufacturer's instructions, and the amount of each of the 29 members of the native cytokine and chemokine family for each sample was calculated. The results for a subset of cytokines are shown in Table 4.

[0093] [Table 4]

[0094] Example 2. Stabilization of circulating free nucleic acids in human blood samples for a period of at least 8 days at ambient temperature. This example demonstrates the use of formulations of the present invention, including those set forth in Table 3, to prepare substantially stable storage of native, undenatured nucleic acid molecules in blood samples while preventing significant contamination from intracellular nucleic acids from lysed blood cells for a period of at least 8 days at ambient temperature.

[0095] The ability of exemplary formulations 1-10 set forth in Table 3 to stabilize circulating free nucleic acids substantially free of contaminating intracellular nucleic acids in blood samples was examined in two separate studies. Briefly, 1.0 mL of formulation was placed into three 5 mL Eppendorf tubes for each formulation. Unprotected tubes at room temperature and 4° C. did not receive formulation. Fresh human whole blood was drawn from healthy donors. The blood was pooled, mixed, and then aliquoted into 4.0 mL portions into each tube. After filling 10 tubes with blood, the tubes were closed and mixed by inversion 10 times before adding blood to more tubes. The blood was mixed again before adding to the next set of tubes. Once all tubes were filled and mixed, the time zero tube was spun at 3000 rpm for 10 minutes. Two 1.25 mL aliquots of plasma were removed without disturbing white and red blood cells, transferred to individual 1.5 mL Eppendorf microtubes, and spun at 16,000 g for 10 minutes at 4° C. Room temperature time zero blood for NP was prepared by adding 1 mL of 1× PBS pH 7.4. The tubes were mixed by inversion 10 times, after which plasma was prepared as described above. Two 1 mL aliquots of cell-free plasma for each formulation tested were transferred to the MagNA Pure Compact Nucleic Acid Isolation Kit on the Roche MagNA Pure Compact Instrument from Roche Molecular Systems. 1, Large Volume (Roche Cat# 03 730 972 001). Nucleic acids were eluted in 100 μL of elution buffer. Time zero samples were kept at 4°C until day 4 samples were prepared as described above. DNA extracted from each sample was analyzed by qPCR on a Bio-Rad CFX96 Real Time System (C1000 Touch Thermal Cycler) using a set of 18s rRNA primers from Qiagen (Cat# PPH05666E-200) and Bio-Rad iQ SYBR Green Supermix (Cat# 170-8882). 5 μL of each sample was used to generate the following plots. Additionally, a 1:10 dilution of isolated DNA was prepared and 5 μL of the dilution was also amplified by qPCR.

[0096] [Table 5]

[0097] [Table 6]

[0098] As shown in Tables 5 and 6, the addition of the exemplary formulations of Table 3 to human blood resulted in the recovery of circulating free nucleic acid that remained substantially free of contaminating intracellular DNA. On day 0, the amount of circulating free nucleic acid present in the test samples was essentially the same as the control and other test samples, but on day 8, blood stored at 4°C or room temperature showed a significant increase in the levels of circulating free nucleic acid, as indicated by the increased yield in these samples from contaminating intracellular nucleic acid from lysed blood cells. In contrast, formulations 1-10 maintained circulating free nucleic acid substantially free of intracellular nucleic acid with only a slight increase on day 8, and all of these formulations performed as well as or better than the unprotected samples stored at 4°C for 8 days.

[0099] Unless the context otherwise requires, throughout the present specification and claims, the term "comprise" is used interchangeably with "including," "containing," or "characterized by," and variations such as "comprises" and "comprising" are inclusive or open-ended language and do not exclude additional, unrecited elements or method steps. The phrase "consisting of" excludes any element, step, or ingredient not explicitly stated in the claim. The phrase "consisting essentially of" limits the scope of the claim to those materials or steps explicitly stated and that do not materially affect the underlying and novel characteristics of the claimed invention. The present disclosure contemplates embodiments of the compositions and methods of the present invention that fall within the scope of each of these phrases. Thus, a composition or method that includes recited elements or steps contemplates specific embodiments in which the composition or method consists essentially of those elements or steps.

[0100] References throughout this specification to "one embodiment," "a embodiment," or "one aspect" mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment of the invention. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0101] The various embodiments described above can be combined to provide further embodiments. These and other changes can be made to the embodiments in light of the above description. In general, the terms used in the following claims should not be construed to limit the claims to the specific embodiments disclosed in the specification and claims, but should be construed to include all possible embodiments to which such claims are entitled along with the full scope of equivalents to which they are entitled. Thus, the claims are not limited by this disclosure.

[0102] From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the claims.

Claims

1. 1. A formulation for stabilization of one or more polypeptides in their native, non-denatured conformation in a blood sample at ambient temperature for at least three days, the formulation comprising: (i) pH buffer; (ii) sucrose; (iii) melezitose; (iv) a polyol selected from the group consisting of glycol, glycerol, erythritol, threitol, arabitol, xylitol, ribitol, adonitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol, and combinations thereof; and (v) A formulation comprising polyvinyl alcohol or a phosphatase inhibitor.

2. 10. The formulation of claim 1, wherein at least 80% of the one or more polypeptides are stabilized in a native, non-denatured conformation after storage at ambient temperature for at least three days.

3. The formulation of claim 1 or 2, wherein the phosphatase inhibitor is 2-glycerol phosphate.

4. 4. The formulation of claim 1, wherein the one or more polypeptides are selected from the group consisting of an antibody, an enzyme, a plasma protein, a serum protein, and any combination thereof.

5. 5. The formulation of claim 1, further comprising one or more of non-ionic starch, amino acids, or any combination thereof.

6. The formulation of claim 5 , wherein the amino acid is glycine or sarcosine.

7. A composition comprising one or more polypeptides in their native, non-denatured conformation in admixture with the formulation of any one of claims 1 to 6.

8. 1. A method for stabilizing one or more polypeptides in a native, non-denatured conformation in a blood sample at ambient temperature, the method comprising: A method comprising the step of mixing a sample of blood taken from a subject with a formulation of any one of claims 1 to 6, wherein one or more polypeptides remain in their native, undenatured state after storage at room temperature for at least three days.

9. 9. The method of claim 8, wherein the polypeptide is selected from the group consisting of an antibody, an enzyme, a plasma protein, a serum protein, and any combination thereof.

10. 10. The method of claim 8 or 9, wherein at least 80% of the polypeptide remains in its native, undenatured state after storage at ambient temperature for at least 3 days.

11. The method of any one of claims 8 to 10, wherein the subject is an animal.

12. 12. The method of any one of claims 8 to 11, wherein the subject is a mammal.

13. 13. The method of any one of claims 8 to 12, wherein the subject is a human.

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