WO2019217637A1 - Stabilization of proteins in biological samples - Google Patents

Abstract

Embodiments of the present disclosure relate to stabilization of one or more proteins in a biological sample at non-freezing temperatures. In particular, embodiments of the present disclosure include formulations, compositions, articles of manufacture, kits and methods for substantially stable storage and/or shipping of a biological sample containing one or more proteins at non-freezing temperatures.

Description

STABIUIZATION OF PROTEINS IN BIOUOGICAU SAMPUES

REUATED APPUICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Patent

Application No. 62/668,955 filed May 9, 2018, which is incorporated herein by reference in its entirety for all purposes.

BACKGROUND

[0002] There exists a need for improved formulations for and methods of stabilizing one or more proteins at non-freezing temperatures for a time sufficient for storage and shipping of the one or more proteins for research, diagnostic and therapeutic purposes.

SUMMARY

[0003] Embodiments of the present disclosure include formulations for the substantially stable storage and/or shipping of biological samples comprising one or more proteins at non- freezing temperatures. In accordance with these embodiments, the one or more proteins remain stabilized after storage in the formulations at non-freezing temperatures for a period of at least 1 day. In some embodiments, at least 80% of the substantially, stably stored one or more proteins remain stabilized after storage at non-freezing temperatures for a period of at least 1 day. In some embodiments, the one or more proteins remain stabilized after storage at ambient temperatures. In some embodiments, the biological sample is selected from blood, blood buffy coat, urine, feces, stool, serum, serosal fluid, plasma, lymph, cerebrospinal fluid, saliva, sputum, mucosal secretion, vaginal fluid, ascites fluid, pleural fluid, pericardial fluid, peritoneal fluid, abdominal fluid, cell culture medium, organ culture medium, buccal cells, bacteria, viruses, yeast cells, 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. In some embodiments, the biological sample comprises a blood sample. In some embodiments, the blood sample comprises whole blood, plasma, or serum. In some embodiments, the formulation comprises (i) at least one of an amino acid or amino acid derivative, a polyol or reducing sugar, or combinations thereof; and (ii) at least one buffer. In some embodiments, the amino acid or amino acid derivative is selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, betaine, sarcosine, dimethylglycine, taurine, and combinations thereof. In some embodiments, the amino acid or amino acid derivative is betaine. In some embodiments, the amino acid or amino acid derivative is sarcosine. In some embodiments, the amino acid or amino acid derivative is dimethylglycine. In some embodiments, the polyol or reducing sugar is selected from the group consisting of glycol, glycerol, erythritol, threitol, arabitol, xylitol, ribitol, adonitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol, glucose, dextrose, galactose, fructose, ribose, xylose, glyceraldehyde, and combinations thereof. In some embodiments, the polyol or reducing sugar is dextrose. In some embodiments, the at least one buffer is selected from the group consisting of citric acid, tartaric acid, malic acid, sulfosalicylic acid, sulfoisophthalic acid, oxalic acid, borate, CAPS (3-(cyclohexylamino)-l- propanesulfonic acid), CAPSO (3-(cyclohexylamino)-2-hydroxy-l-propanesulfonic acid), EPPS (4-(2-hydroxyethyl)-l-piperazinepropanesulfonic acid), HEPES (4-(2- hydroxyethyl)piperazine-l-ethanesulfonic acid), MES (2-(N-morpholino)ethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), MOPSO (3-morpholino-2- hydroxypropanesulfonic acid), PIPES (l,4-piperazinediethanesulfonic acid), TAPS (N- [tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid), TAPSO (2-hydroxy-3- [tris(hydroxymethyl)methylamino]-l-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), bis-tris (2-[bis(2-hydroxyethyl)amino]-2- (hydroxymethyl)-l, 3 -propanediol), monosodium citrate, disodium citrate, trisodium citrate, sodium dihydrogen phosphate, and combinations thereof. In some embodiments, the at least one buffer is trisodium citrate. In some embodiments, the at least one buffer is sodium dihydrogen phosphate. In some embodiments, the at least one buffer is citric acid. In some embodiments, the at least one buffer is trisodium citrate, sodium dihydrogen phosphate, and citric acid. In some embodiments, the formulation further comprises a purine. In some embodiments, the purine is adenine or guanine. In some embodiments, the purine is adenine. In some embodiments, the formulation comprises an anticoagulant (e.g., EDTA, sodium polyanethol sulfonate). In some embodiments, the formulation comprises betaine, dextrose, trisodium citrate, sodium dihydrogen phosphate, citric acid, and adenine. In some embodiments, the formulation comprises sarcosine, dextrose, trisodium citrate, sodium dihydrogen phosphate, citric acid, and adenine. In some embodiments, the formulation comprises dimethylglycine, dextrose, trisodium citrate, sodium dihydrogen phosphate, citric acid, and adenine. [0004] Embodiments of the present disclosure also include compositions comprising a substantially, stably stored one or more proteins admixed with a formulation for substantially stable storage of one or more proteins in a biological sample.

[0005] Embodiments of the present disclosure also include articles of manufacture, comprising a formulation for substantially stable storage of one or more proteins in a biological sample provided herein. In some embodiments, the biological sample is selected from blood, blood buffy coat, urine, feces, stool, serum, serosal fluid, plasma, lymph, cerebrospinal fluid, saliva, sputum, mucosal secretion, vaginal fluid, ascites fluid, pleural fluid, pericardial fluid, peritoneal fluid, abdominal fluid, cell culture medium, organ culture medium, buccal cells, bacteria, viruses, yeast cells, 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. In some embodiments, the biological sample is a blood sample, whole blood, blood plasma, or serum. In some embodiments, the formulation is contained within a blood collection tube. In some embodiments, the blood collection tube is an evacuated blood collection tube.

[0006] Embodiments of the present disclosure also include kits comprising an article of manufacture provided herein and a package insert.

[0007] Embodiments of the present disclosure also include methods for substantially stable storage of one or more proteins in a biological sample at non-freezing temperatures, comprising: admixing a sample from a subject with a formulation for substantially stable storage of one or more proteins in a biological sample provided herein, wherein the one or more proteins remain stabilized after storage at non-freezing temperatures for a period of at least 1 day. In some embodiments, at least 80% of the one or more proteins remain stabilized at non-freezing temperatures for a period of at least 1 day. In some embodiments, the one or more proteins remain stabilized after storage at ambient temperatures. In some embodiments, the biological sample is selected from blood, blood buffy coat, urine, feces, stool, serum, serosal fluid, plasma, lymph, cerebrospinal fluid, saliva, sputum, mucosal secretion, vaginal fluid, ascites fluid, pleural fluid, pericardial fluid, peritoneal fluid, abdominal fluid, cell culture medium, organ culture medium, buccal cells, bacteria, viruses, yeast cells, 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. In some embodiments, the biological sample comprises a blood sample. In some embodiments, the blood sample comprises whole blood, plasma, or serum. In some embodiments, the one or more proteins include, but are not limited to, complement proteins such as C3a, C3b, iC3b, C4d, C5a, sC5b-9, Clq, C4a, Ba or a combination thereof. In some embodiments, the subject is an animal. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0008] Embodiments of the present disclosure also include methods for substantially stable storage/shipping of one or more proteins in a biological sample at non-freezing temperatures, comprising: admixing a biological sample collected from a subject with any formulation provided herein (e.g., Table 1 and/or Table 2).

[0009] Embodiments of the present disclosure also include methods for substantially stable storage of one or more proteins in a biological sample at non-freezing temperatures, comprising: admixing a biological sample collected from a subject with a formulation selected from the group consisting of: (i) dextrose and EDTA; (ii) melibiose and EDTA; (iii) glycine; (iv) betaine; (v) sorbitol; (vi) betaine, dextrose, trisodium citrate, sodium dihydrogen phosphate, citric acid, and adenine; (vii) sarcosine, dextrose, trisodium citrate, sodium dihydrogen phosphate, citric acid, and adenine; (viii) dimethylglycine, dextrose, trisodium citrate, sodium dihydrogen phosphate, citric acid, and adenine; and (ix) dimethylglycine, EDTA, trisodium citrate, sodium dihydrogen phosphate, citric acid, and adenine, wherein the one or more proteins remain stabilized after storage at non-freezing temperatures for a period of at least 1 day. In some embodiments, the one or more proteins remain stabilized after storage at ambient temperatures. In some embodiments, the biological sample is selected from blood, blood buffy coat, urine, feces, stool, serum, serosal fluid, plasma, lymph, cerebrospinal fluid, saliva, sputum, mucosal secretion, vaginal fluid, ascites fluid, pleural fluid, pericardial fluid, peritoneal fluid, abdominal fluid, cell culture medium, organ culture medium, buccal cells, bacteria, viruses, yeast cells, 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. In some embodiments, the biological sample comprises a blood sample. In some embodiments, the blood sample comprises whole blood, plasma, or serum.

INCORPORATION BY REFERENCE

[0010] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The novel features of the various embodiments of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the various embodiments of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of these embodiments are utilized, and the accompanying drawings of which:

[0012] FIGURE 1 illustrates stabilization of iC3b levels with formulations 012 and 013.

[0013] FIGURE 2 illustrates stabilization of sC5b-9 levels with formulations 012 and 013.

[0014] FIGURE 3 illustrates stabilization of C3a levels with formulation 014.

PET All, ED DESCRIPTION

[0015] Embodiments of the present disclosure relate to formulations, compositions, articles of manufacture, kits, and methods for substantially stable storage and/or shipping of a biological sample comprising one or more proteins, including but not limited to, a blood sample, a plasma sample, or a serum sample at non-freezing temperatures. In some embodiments, the one or more proteins remain stabilized after storage at ambient

temperatures. In some embodiments, the biological sample is selected from blood, blood huffy coat, urine, feces, stool, serum, serosal fluid, plasma, lymph, cerebrospinal fluid, saliva, sputum, mucosal secretion, vaginal fluid, ascites fluid, pleural fluid, pericardial fluid, peritoneal fluid, abdominal fluid, cell culture medium, organ culture medium, buccal cells, bacteria, viruses, yeast cells, 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. In some embodiments, the biological sample comprises a blood sample. In some embodiments, the blood sample comprises whole blood, plasma, or serum.

[0016] In some embodiments, the formulations provide for the substantially stable storage of one or more proteins, wherein at least 60%, 70%, 80%, 90%, 95%, or even 99% of the one of more proteins remain in a native, non-denatured state after storage at non-freezing temperatures.

[0017] In some embodiments, the levels of one or more proteins remain substantially unchanged after storage at non-freezing temperatures using the formulations and methods provided herein. In some embodiments, the formulations provide for substantially stable storage of one or more proteins that retain their native conformation. In some embodiments, the one or more proteins are functionally active after storage at non-freezing temperatures.

DEFINITIONS

[0018] 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, references to“the method” includes one or more methods, and/or steps of the type described herein which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

[0019]“About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, or ±10%, or ±5%, or even ±1% from the specified value, as such variations are appropriate for the disclosed compositions or to perform the disclosed methods.

[0020] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the various embodiments of the present disclosure belong.

[0021] As used herein, the term“non-freezing temperature” refers to temperatures at which the formulation or admixture of a formulation and sample is in a non-frozen state. In some embodiments the non-freezing temperature is greater than 0°C. In some embodiments, the “non-freezing temperature” is a temperature of at least 0°C. In some embodiments,“non- freezing temperature” is equal to or greater than 0°C. In some embodiments, the term“non- freezing temperature” refers to temperatures above the freezing point of the formulation or admixture of the formulation and sample. In some embodiments, the non-freezing temperature is less than 0°C.

[0022] The term“ambient temperature” as used herein refers to common indoor room temperatures. In some embodiments, ambient temperature is 15 to 32°C. In some embodiments, ambient temperature is 20 to 27°C. The term“ambient temperature” may be used interchangeably with the term“room temperature.”

[0023] Definitions of standard chemistry terms may be found in reference works, including Carey and Sundberg“ADVANCED ORGANIC CHEMISTRY 4TH ED.” Vols. A (2000) and B (2001), Plenum Press, New York. Unless otherwise indicated, conventional methods of mass spectroscopy, NMR, HPLC, protein chemistry, biochemistry, molecular biology, recombinant DNA techniques and pharmacology, within the skill of the art are employed. Unless specific definitions are provided, the nomenclature employed in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those known in the art. Standard techniques can be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients. Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, tissue culture and collection, and transformation (e.g., electroporation, lipofection). Reactions and purification techniques can be performed e.g., using kits of manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures can be generally performed using conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification.

[0024] As used herein, the terms“stabilization,”“stabilizing,” and“stabilized,” when used in reference to one or more proteins refer to the ability of a material to limit changes in the levels or biological activity of one or more proteins, often as measured over time (i.e. in the presence of a stabilizer, one or more proteins retain biological activity and/or protein levels of one or more proteins remain unchanged for a longer time period than in the absence of the stabilizer).“Stabilization of one or more proteins” also refers to the ability of a material to maintain biological activity or protein levels of one or more proteins under suboptimal conditions of temperature or pH. For example,“stabilizing one or more proteins” can refer to the ability of a material to maintain biological activity or protein levels of one or more proteins after being frozen (e.g., frozen at temperatures less than 0°C) and subsequently thawed (e.g., thawed at temperatures greater than 0°C). As another example,“stabilizing one or more proteins” refers to the ability of a material to enhance biological activity and/or protein levels under suboptimal conditions, as compared to activity or protein levels in the absence of a“stabilizing” compound or material. As a further example,“stabilizing one or more proteins” refers to the ability of a material to inhibit both the increase or decrease of protein levels or biological activity of one or more proteins for a longer period of time than in the absence of the stabilizer.

[0025] As described 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” encompasses native or artificial proteins, protein fragments and polypeptide analogs of a protein sequence. A polypeptide may be monomeric or polymeric. The term“polypeptide” encompasses fragments and variants (including fragments of variants) thereof, unless otherwise contradicted by context.

[0026] As used herein, the term“biological sample” includes, but is not limited to, blood, blood huffy coat, urine, feces, stool, serum, serosal fluid, plasma, lymph, cerebrospinal fluid, saliva, sputum, mucosal secretion, vaginal fluid, ascites fluid, pleural fluid, pericardial fluid, peritoneal fluid, abdominal fluid, cell culture medium, organ culture medium, buccal cells, bacteria, viruses, yeast cells, 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.

[0027] As used herein, the term“blood sample” refers to whole blood, plasma, serum, or any other blood-based sample or any other sample derived from blood.

FORMULATION REAGENTS

pH Buffers

[0028] According to certain embodiments, the herein described formulations and

compositions for substantially stable storage/shipping of one or more proteins in biological sample, a blood sample, a plasma sample, or a serum sample include one or more pH buffers. In some embodiments, the pH buffer is any of a large number of compounds known in the art for their ability to resist changes in the pH of a solution, such as an aqueous solution, in which the pH buffer is present. Selection of one or more particular pH buffers for inclusion in a stable storage composition may be done based on the present disclosure and according to routine practices in the art, and may be influenced by a variety of factors including the pH that is desirably to be maintained, the nature of the sample to be stabilized, the solvent conditions to be employed, the other components of the formulation to be used, and other criteria. For example, typically a pH buffer is employed at a pH that is within about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0 pH unit of a proton dissociation constant (pKa) that is a characteristic of the buffer.

[0029] Non-limiting examples of pH buffers include citric acid, tartaric acid, malic acid, sulfosalicylic acid, sulfoisophthalic acid, oxalic acid, borate, CAPS (3-(cyclohexylamino)-l- propanesulfonic acid), CAPSO (3-(cyclohexylamino)-2-hydroxy-l-propanesulfonic acid), EPPS (4-(2-hydroxyethyl)-l-piperazinepropanesulfonic acid), HEPES (4-(2- hydroxyethyl)piperazine-l-ethanesulfonic acid), MES (2-(N-morpholino)ethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), MOPSO (3-morpholino-2- hydroxypropanesulfonic acid), PIPES (l,4-piperazinediethanesulfonic acid), TAPS (N- [tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid), TAPSO (2-hydroxy-3- [tris(hydroxymethyl)methylamino]-l-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), bis-tris (2-[bis(2-hydroxyethyl)amino]-2- (hydroxymethyl)-l, 3 -propanediol), monosodium citrate, disodium citrate, trisodium citrate, and sodium dihydrogen phosphate. Certain embodiments contemplated herein feature a formulation 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.

Amino Acids and Amino Acid Derivatives

[0030] Amino acids include, for example, the natural amino acids alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and their derivatives. Examples of amino acid derivatives include betaine

(trimethylglycine), sarcosine, dimethylglycine, and taurine, for example. In some embodiments, the amino acid is glycine. In some embodiments, the amino acid derivative is betaine. In some embodiments, the amino acid derivative is sarcosine. In some embodiments, the amino acid derivative is dimethylglycine. In some embodiments, the amino acid or amino acid derivative is present at a concentration between about 10 mM - 100 mM, or between about 0.1 M - 1.0 M, or between about 1.0 M - 10.0 M.

Chelating Agents

[0031] Chelating agents or chelators are, according to certain embodiments, included in the presently described formulations and compositions for substantially stable storage/shipping of one or more proteins in a biological sample, a blood sample, a plasma sample, or a serum sample. Such chelating agents are known to those familiar with the art for their ability to complex with and hinder the reactivity of metal cations. Exemplary chelating agents include diethylenetriaminepentaacetic acid (DTP A), ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), trans-l,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA), l,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), 1,4,7,10- tetraazacyclododecane-l,4,7,l0-tetraacetic acid (DOTA), N-(2- hydroxyethyl)ethylenediamine-N,N',N'-triacetic acid, sodium gluconate, polystyrene sulfonate, and nitrilotriacetic acid (NT A). In some embodiments, the chelating agent is present at a concentration of about 0.01 - 10 mg/mL. In some embodiments, the chelating agent is present at a concentration of about 0.05 - 1 mg/mL. In some embodiments, the chelating agent is present at a concentration of about 0.1 - 0.5 mg/mL. In some embodiments, the chelating agent is EDTA. In some embodiments, the chelating agent is present at a concentration from about 1.0 mM - 50 mM, from about 10 mM - 40 mM, from about 5.0 mM - about 25 mM, from about 10 mM - 20 mM, or from about 10 - 100 mM.

Purines and Purine Derivatives

[0032] In some embodiments, a purine or purine derivative is included in the presently described formulations and compositions for substantially stable storage/shipping of one or more proteins in a biological, blood, plasma, or serum sample. In some embodiments, the purine is adenine, guanine, or both. In some embodiments, the purine is adenine. In some embodiments, the purine is guanine. In some embodiments, the purine is a purine derivative. In some embodiments, the purine derivative is 2-mercaptopurine. In some embodiments, the purine or purine derivative is present at a concentration of about 0.1 - 10 mM, or about 1 - 20 mM, or about 2 mM.

Polyols

[0033] Also, as described herein, certain embodiments include at least one polyol in the formulations and compositions for substantially stable storage/shipping of one or more proteins in a biological, blood, plasma, or serum sample at non-freezing temperatures.

Polyols are polyhydric alcohols containing two or more hydroxyl groups and have the general formula H(CHOH)_nH, wherein n is an integer selected from 2 to 7 inclusive. Polyols differ in chain length with most polyols having five- or six-carbon chains being derived from pentoses (five-carbon sugars) and hexoses (six-carbon sugars); however 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 substantially stable storage composition may be done based on the present disclosure and according to routine 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 is a hexose polyol. In some embodiments, the polyol is sorbitol. In some embodiments, the polyol is present at a concentration between about 10 mM - 100 mM, or between about 0.1 M - 1.0 M, or between about 1.0 M - 10.0 M. In some embodiments, the polyol is a hexose polyol and is present at a concentration between about 10 mM - 100 mM, or between about 0.1 M - 1.0 M, or between about 1.0 M - 10.0 M. In some embodiments, the polyol is sorbitol and is present at a concentration between about 10 mM - 100 mM, or between about 0.1 M - 1.0 M, or between about 1.0 M - 10.0 M.

Disaccharide Derivatives

[0034] In certain embodiments, the formulations or compositions for substantially stable storage/shipping of one or more proteins in a biological, blood, plasma, or serum sample include at least one halogenated disaccharide derivative. In some embodiments, the halogenated disaccharide derivative is a di- or tri-chlorinated disaccharide. In some embodiments, such di- or tri-chlorinated disaccharides unexpectedly are capable of substantially stable storage of one or more proteins either alone or in the presence of only a buffer or in the presence of only one other formulation component. Halogenated disaccharide derivatives are known, e.g., see US Patent Publication No. 2014/0065062, and include sucralose (l,6-dichloro-l,6-dideoxy- -D-fructofuranosyl-4-chloro-4-deoxy-a-D- galactopyranoside), trichloronated maltose, l,6-dichloro-l,6-dideoxy- -D-fructofuranosyl-4- chloro-4-deoxy-6-0-monododecanoate-a-D-galactopyranoside, and l,6-dichloro-l,6- dideoxy- -D-fructofuranosyl-4-chloro-4-deoxy-6-0-monotetradecanoate-a-D- galactopyranoside. Selection of one or more particular halogenated disaccharide derivative for inclusion in a substantially stable storage composition may be done based on the present disclosure and according to routine practices in the art, and may be influenced by a variety of factors including other formulation components. In some embodiments, the functionalized carbohydrate is sucralose and is present at about 1.0 - 50.0 mM. In some embodiments, the functionalized carbohydrate is sucralose and is present at about 5.0 - 100 mM. In some embodiments, the functionalized carbohydrate is sucralose and is present at about 10.0 mM.

Non-reducing Sugars

[0035] In some embodiments, the formulations and compositions for substantially stable storage/shipping of one or more proteins in a biological, blood, plasma, or serum sample at non-freezing temperatures include at least one non-reducing sugar. As used herein,“non reducing sugars” refers to carbohydrate molecules that lack a functional aldehyde group. Exemplary non-reducing sugars include sucrose and trehalose. In some embodiments, the non-reducing sugar is sucrose. In some embodiments, the non-reducing sugar is trehalose. In some embodiments, the non-reducing sugar is present at a concentration of about 1.0 - 50 mM. In some embodiments, the non-reducing sugar is present at a concentration of about 50.0 100 mM. In some embodiments, the non-reducing sugar is present at a concentration of 0.1 M - 1.0 M. In some embodiments, the non-reducing sugar is present at a concentration of 1.0 M - 10.0 M. In some embodiments, the non-reducing sugar is present at a

concentration of about 1 to about 50

Reducing Sugars

[0036] In some embodiments, the formulations and compositions for substantially stable storage/shipping of one or more proteins in a biological, blood, plasma, or serum sample at non-freezing temperatures include at least one reducing sugar. As used herein,“reducing sugars” include monosaccharides, disaccharides, oligosaccharides, and polysaccharides. Exemplary monosaccharides that are reducing sugars include, but are not limited to, glucose, dextrose, galactose, fructose, ribose, xylose, and glyceraldehyde. Exemplary disaccharides that are reducing sugars include, but are not limited to, lactose, maltose, cellobiose, and melibiose. In some embodiments, the reducing sugar is dextrose. In some embodiments, the reducing sugar is melibiose. In some embodiments, the reducing sugar is present at a concentration between about 1.0 mM - 50 mM, or between about 20 100 mM, or between about 0.1 M - 1.0 M, or between about 1.0 M - 10.0 M.

Anticoagulants

[0037] In some embodiments, an anticoagulant is included in the presently described formulations or compositions. In other embodiments, an anticoagulant is included as part of a collection tube. Such anticoagulants are known in the art. Exemplary anticoagulants include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), hirudin, heparin, sodium citrate, and sodium polyanethol sulfonate (SPS). In some embodiments, the anticoagulant is present at a concentration from about 1.0 1000 pg/mL, from about 10 500 pg/mL, from about 25 400 pg/mL, from about 50 300 pg/mL, from about 100 300 pg/mL, or from about 200 300 pg/mL. In some embodiments, the anticoagulant is present at a concentration of about 1.0 25 pg/mL. In some embodiments, the anticoagulant is present at a concentration of about 10 - 20 pg/mL. In some embodiments, the anticoagulant is present at a concentration from about 1.0 mM - 100 mM, from about 1.0 mM - 50 mM, from about 5.0 mM - about 25 mM, from about 10 mM - 20 mM, from about 0.05 M - 0.15 M, or from about 0.1 M - 1.0 M. EXEMPLARY FORMULATIONS FOR STABILIZATION OF ONE OR MORE

PROTEINS IN A BLOOD PLASMA OR SERUM SAMPLE AT AMBIENT TEMPERATURES

[0038] Described herein, in some embodiments, are formulations for substantially stable storage and/or shipping of a biological sample comprising one or more proteins at non- freezing temperatures, wherein the one or more proteins remain stabilized after

storage/shipping at non-freezing temperatures for a period of at least 1 day. In some embodiments, at least 80% of the substantially, stably stored one or more proteins remain stabilized after storage/shipping at non-freezing temperatures for a period of at least 1 day. In some embodiments, the one or more proteins remain stabilized after storage/shipping at ambient temperatures. In some embodiments, the biological sample is selected from blood, blood buffy coat, urine, feces, stool, serum, serosal fluid, plasma, lymph, cerebrospinal fluid, saliva, sputum, mucosal secretion, vaginal fluid, ascites fluid, pleural fluid, pericardial fluid, peritoneal fluid, abdominal fluid, cell culture medium, organ culture medium, buccal cells, bacteria, viruses, yeast cells, 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. In some embodiments, the biological sample comprises a blood sample. In some embodiments, the blood sample comprises whole blood, plasma, or serum.

[0039] In some embodiments, the formulation comprises (i) at least one of an amino acid or amino acid derivative, a polyol or reducing sugar, or combinations thereof; and (ii) at least one buffer. In some embodiments, the amino acid or amino acid derivative is selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, betaine, sarcosine, dimethylglycine, taurine, and combinations thereof. In some embodiments, the amino acid or amino acid derivative is betaine. In some embodiments, the amino acid or amino acid derivative is sarcosine. In some embodiments, the amino acid or amino acid derivative is dimethylglycine. In some embodiments, the polyol or reducing sugar is selected from the group consisting of glycol, glycerol, erythritol, threitol, arabitol, xylitol, ribitol, adonitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol, glucose, dextrose, galactose, fructose, ribose, xylose, glyceraldehyde, and combinations thereof. In some embodiments, the polyol or reducing sugar is dextrose. In some embodiments, the at least one buffer is selected from the group consisting of citric acid, tartaric acid, malic acid, sulfosalicylic acid, sulfoisophthalic acid, oxalic acid, borate, CAPS (3-(cyclohexylamino)-l-propanesulfonic acid), CAPSO (3- (cyclohexylamino)-2-hydroxy-l-propanesulfonic acid), EPPS (4-(2-hydroxyethyl)-l- piperazinepropanesulfonic acid), HEPES (4-(2-hydroxyethyl)piperazine-l-ethanesulfonic acid), MES (2-(N-morpholino)ethanesulfonic acid), MOPS (3-(N- morpholino)propanesulfonic acid), MOPSO (3-morpholino-2-hydroxypropanesulfonic acid), PIPES (l,4-piperazinediethanesulfonic acid), TAPS (N-[tris(hydroxymethyl)methyl]-3- aminopropanesulfonic acid), TAPSO (2-hydroxy-3-[tris(hydroxymethyl)methylamino]-l- 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), bis-tris (2-[bis(2-hydroxyethyl)amino]-2- (hydroxymethyl)-l, 3 -propanediol), monosodium citrate, disodium citrate, trisodium citrate, sodium dihydrogen phosphate, and combinations thereof. In some embodiments, the at least one buffer is trisodium citrate. In some embodiments, the at least one buffer is sodium dihydrogen phosphate. In some embodiments, the at least one buffer is citric acid. In some embodiments, the at least one buffer is trisodium citrate, sodium dihydrogen phosphate, and citric acid. In some embodiments, the formulation further comprises a purine. In some embodiments, the purine is adenine or guanine. In some embodiments, the purine is adenine. In some embodiments, the formulation comprises betaine, dextrose, trisodium citrate, sodium dihydrogen phosphate, citric acid, and adenine. In some embodiments, the formulation comprises sarcosine, dextrose, trisodium citrate, sodium dihydrogen phosphate, citric acid, and adenine. In some embodiments, the formulation comprises dimethylglycine, dextrose, trisodium citrate, sodium dihydrogen phosphate, citric acid, and adenine. In some embodiments, the formulation comprises dimethylglycine, EDTA, trisodium citrate, sodium dihydrogen phosphate, citric acid, and adenine.

[0040] Exemplary compositions of formulations for substantially stable storage/shipping of one or more proteins in a biological, blood, plasma, or serum sample at non-freezing temperatures are shown in Table 1.

Table 1: Exemplary Compositions of Formulations for Stabilizing One or More

Proteins in a Biological Sample

[0041] Exemplary formulations for substantially stable storage/shipping of one or more proteins in a biological, blood, plasma, or serum sample at non-freezing temperatures are shown in Table 2.

Table 2: Exemplary Formulations for Stabilizing One or More Proteins in a

Biological Sample

[0042] Exemplary formulations for substantially stable storage/shipping of one or more proteins in a biological, blood, plasma, or serum sample at non-freezing temperatures also include formulations comprising one or more of sodium citrate, sodium phosphate, citric acid, adenine, dimethylglycine, EDTA, SPS, and combinations thereof. In some

embodiments, the exemplary formulation includes any of formulations 001-014 described herein, and any combinations or modifications thereof. In some embodiments, the formulation includes sodium citrate at concentration ranging from about 50-150 mM, from 75-125 mM, from about 85-115 mM, or from about 90-105 mM. In some embodiments, the formulation includes sodium phosphate at a concentration ranging from about 10-20 mM, from about 12-20 mM, from about 14-20 mM, from about 16-20 mM, from about 18-20 mM, from about 16-18 mM, from about 15-19 mM, from about 15-17 mM, or from about 17-19 mM. In some embodiments, the formulation includes citric acid at a concentration ranging from about 10-20 mM, from about 12-20 mM, from about 14-20 mM, from about 16-20 mM, from about 18-20 mM, from about 16-18 mM, from about 15-19 mM, from about 15-17 mM, or from about 17-19 mM. In some embodiments, the formulation includes adenine at a concentration ranging from about 0.5-5.0 mM, from about 0.5-4.0 mM, from about 0.5-3.0 mM, from about 1.0-5.0 mM, from about 1.0-2.5 mM, from about 1.0-2.0 mM, from about 1.5-5.0 mM, from about 1.5-3.0 mM, from about 1.5-2.5 mM, or from about 2.0-3.0 mM. In some embodiments, the formulation includes dimethylglycine at a concentration ranging from about 0.5-5.0 M, from about 0.5-4.0 M, from about 0.5-3.5 M, from about 0.5-3.0 M, from about 0.5-2.5 M, from about 0.5-2.0 M, from about 0.5-1.5 M, from about 1.0-3.0 M, from about 1.0-2.5 M, or from about 1.0-2.0 M. In some embodiments, the formulation includes EDTA at a concentration ranging from about 1-20 mM, from about 2-18 mM, from about 4-16 mM, from about 8-12 mM, from about 10-12 mM, from about 5-15 mM, from about 10-15 mM, from about 9-11 mM, from about 9-10 mM, or from about 10-11 mM. In some embodiments, the formulation includes SPS at a concentration ranging from about 0.010-0.050%, from about 0.015-0.045%, from about 0.020-0.040%, from about 0.025- 0.035%, from about 0.020-0.035%, from about 0.020-0.030%, from about 0.030-0.040%, from about 0.015-0.030%, or from about 0.015-0.025%.

METHODS FOR PREPARING FORMULATIONS FOR STABILIZING ONE OR

MORE PROTEINS IN A BIOLOGICAL SAMPLE AT NON-FREEZING

TEMPERATURES

[0043] Methods for preparing the formulations described herein for substantially stable storage/shipping of one or more proteins in a biological sample, a blood sample, a plasma sample, or a serum sample at non-freezing temperatures employ techniques that are well- known to those skilled in the art and generally use commercially available reagents. In some embodiments, the formulations are prepared as concentrated stock solutions of the formulation reagents, e.g., 2X, 5X, 10X, 20X or the like, so as to be admixed with the biological sample, the blood sample, the plasma sample, or the serum sample at the appropriate ratios to form the desired concentrations. In some embodiments, the formulations are prepared in concentrations sufficient to produce a IX concentration when mixed with a fixed amount of sample.

PURIFIED STABILIZED ONE OR MORE PROTEINS IN A BIOLOGICAL SAMPLE

[0044] In some embodiments, the substantially stable one or more proteins in a biological sample at non-freezing temperatures are further purified using well-known conventional methods routinely employed by those skilled in the art. Apparatus, kits, and methods for purifying one or more proteins from biological samples including, but not limited to, blood, plasma, or serum are well-known. In some embodiments, the purified one or more proteins are subsequently stored in the formulations described herein for extended periods before analysis.

ARTICLES OF MANUFACTURE

[0045] In certain embodiments, articles of manufacture are provided, which comprise a formulation provided herein, contained within a suitable blood collection tube, container or vessel for collection of a biological sample. Exemplary compositions of formulations for substantially stable storage/shipping of one or more proteins in a biological sample at non- freezing temperatures are shown in Table 1 and Table 2. In some embodiments, these articles of manufacture are used for substantially stable storage/shipping of one or more proteins at the time of biological sample collection. In certain embodiments, the blood collection tube is an evacuated blood tube having less than atmospheric pressure to withdraw a predetermined volume of whole blood. In some embodiments, these articles of manufacture are used in the kits and methods described herein.

KITS

[0046] In certain aspects, embodiments of the present disclosure include kits comprising any one of the articles of manufacture comprising the formulations of the present disclosure and a package insert. In some embodiments, the components of the kit are supplied in a container, such as a compartmentalized plastic enclosure. In some embodiments, the container has a hermetically sealable cover so that the contents of the kit can be sterilized and sealed for storage prior to use.

METHODS FOR SUBSTANTIALLY STABLE STORAGE/SHIPPING OF ONE OR

MORE PROTEINS IN A BIOLOGICAL SAMPLE AT NON-FREEZING

TEMPERATURES

[0047] In another aspect, embodiments of the present disclosure include methods for substantially stable storage and/or shipping of a biological sample comprising one or more proteins at non-freezing temperatures. In some embodiments, the one or more proteins remain stabilized after storage/shipping at ambient temperatures. In some embodiments, the biological sample is selected from blood, blood huffy coat, urine, feces, stool, serum, serosal fluid, plasma, lymph, cerebrospinal fluid, saliva, sputum, mucosal secretion, vaginal fluid, ascites fluid, pleural fluid, pericardial fluid, peritoneal fluid, abdominal fluid, cell culture medium, organ culture medium, buccal cells, bacteria, viruses, yeast cells, 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. In some embodiments, the biological sample comprises a blood sample. In some

embodiments, the blood sample comprises whole blood, plasma, or serum.

[0048] In certain embodiments, the methods comprise admixing a biological sample, a blood sample, a plasma sample, or serum sample with formulations described herein for substantially stable storage of one or more proteins in a biological sample, a blood sample, a plasma sample, or a serum sample at non-freezing temperatures, wherein the one or more proteins and/or their protein levels remain stabilized for a period of at least 1 day. In some embodiments, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, at least 50% of the one or more proteins remain stabilized at non-freezing temperatures for a period of at least 1 day. In some embodiments, at least 80% of the one or more proteins remain stabilized at non-freezing temperatures for a period of at least 1 day. In some embodiments, the one or more proteins and/or their protein levels remain stabilized at ambient temperatures.

[0049] In some embodiments, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, at least 50% of the one or more proteins and/or their protein levels remain stabilized at non-freezing or ambient temperatures for a period of at least 1 day, at least 2 days, at least 3 days, 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, at least 19 days, at least 20 days, at least 21 days, at least 22 days, at least 23 days, at least 24 days, at least 25 days, at least 26 days, at least 27 days, at least 28 days, at least 29 days, at least 30 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 15 months, at least 18 months, at least 21 months, at least 24 months.

[0050] In some embodiments, the one or more proteins include, but are not limited to, one or more complement proteins. For example, the one or more complement proteins can include one or more of C3a, C3b, iC3b, C4d, C5a, sC5b-9, Clq, C4a, Ba or any combinations thereof.

[0051] Exemplary formulations for use in the methods described herein are provided in Table 1. In certain embodiments, the formulation is one of the formulations set forth in Table 2. In certain embodiments, the formulation selected from the group consisting of: (i) dextrose and EDTA; (ii) melibiose and EDTA; (iii) glycine; (iv) betaine; (v) sorbitol; (vi) betaine, dextrose, trisodium citrate, sodium dihydrogen phosphate, citric acid, and adenine; (vii) sarcosine, dextrose, trisodium citrate, sodium dihydrogen phosphate, citric acid, and adenine; (viii) dimethylglycine, dextrose, trisodium citrate, sodium dihydrogen phosphate, citric acid, and adenine and (ix) dimethylglycine, EDTA, trisodium citrate, sodium dihydrogen phosphate, citric acid, and adenine. [0052] Any suitable method of biological sample collection may be used. Methods of biological sample collection, blood collection tubes, bags, containers and vessels are well- known in the art and have been employed by medical practitioners for decades. Blood collected for substantially stable storage of one or more protein may be obtained using any method or apparatus commonly employed by those skilled in the art such as venipuncture or finger prick. In some embodiments, when the blood is collected by venipuncture, the formulation is located inside the blood collection tube, e.g., an evacuated tube

(VACUTAINER blood collection tube, Becton Dickenson or VACUETTE blood collection tube, Greiner Bio-One) at the time that the blood sample is obtained from the subject. In some embodiments, when the blood is collected by venipuncture, the formulations are added to an already obtained whole blood sample, either immediately or shortly after it is withdrawn.

[0053] Any suitable method of assaying or measuring protein concentration and/or detecting a protein or fragment thereof (e.g., peptide/polypeptide) can be used in conjunction with the compositions, formulations, and methods described herein. For example, one or more proteins or fragments thereof (e.g., one or more peptides/polypeptides) can be stabilized using the formulations provided herein and according to the methods provided herein, such that the measurement or detection of the one or more proteins is improved. In some embodiments, protein detection/quantification methods include, but are not limited to, an enzyme-linked immunosorbent assay (ELISA), a lateral flow assay, chromatography, mass spectrometry, high-performance liquid chromatography (HPLC), liquid chromatography- mass spectrometry (LC/MS), immunoprecipitation, western blotting, immunoelectrophoretic methods, and the like, as well as other methods that would be recognized by one of ordinary skill in the art based on the present disclosure.

[0054] The methods as described herein may use the articles of manufacture and kits disclosed herein.

[0055] The following Examples are presented by way of illustration and not limitation.

EXAMPLE 1

STABILIZATION OF COMPLEMENT PROTEIN iC3b LEVELS WITH

FORMULATIONS 012 AND 013

[0056] This Example describes stabilization of complement protein iC3b levels with formulations 012 and 013.

[0057] Samples [0058] Fresh blood was collected from a normal healthy volunteer into an EDTA evacuated blood collection tube. Approximately 500 pL of whole blood was aliquoted into centrifuge tubes and diluted equal parts with formulation 012 or formulation 013. Whole blood samples were either processed for plasma immediately or incubated for 24-48 hours at 2-8°C, 25°C and 37°C. To determine stabilization of iC3b, the levels of the complement proteins were determined at the indicated time points and compared to the protein concentrations in plasma at time zero.

[0059] Assay

[0060] Commercially available solid-phase antibody capture assays (Quidel, San Diego, CA, USA) were used for iC3b-specific enzyme-linked immunosorbent assays (ELISAs).

Approximately 100 pL of standards, controls and diluted test specimens (n=2) were incubated at room temperature in polystyrene (PS) microtiter wells pre-coated with a capture antibody specific for the target fragment (iC3b). During the initial incubation step, iC3b was bound to the coated wells. Unbound material was removed via successive wash cycles. During the second step, a volume of analyte-specific enzyme-conjugated-antibody was added to each well and incubated at room temperature. Analyte-specific enzyme-conjugated antibody reacted with the captured analytes, and unbound conjugate was removed via successive wash cycles. During a third step, a volume of enzyme substrate was added to each well and incubated at room temperature. The bound enzyme reacted with substrate producing a signal proportional to the amount of specimen analytes bound. The enzymatic reaction was stopped upon addition of a volume of acid. The substrate color intensity present in each well was measured spectrophotometrically at analyte-specific wavelength using a Synergy -HTX- S1LFA automated microplate reader (Biotek Instruments Inc., Winooski, VT, USA), followed by determination of analyte concentration for each analyte from the corresponding standard curve. A final dilution of 1 :25 was used in the iC3b fragment test.

[0061] As shown in Figure 1, iC3b concentration in non-protected blood samples (NP) rose after storage of the blood samples for 48 hours at all temperatures tested. For blood samples mixed 1: 1 with stabilizing formulation 012 and formulation 013, iC3b concentrations stayed close to the original time zero concentrations after storage at 2-8°C, 25°C, and 37°C for 48 hours (Figure 1). EXAMPLE 2

STABILIZATION OF COMPLEMENT PROTEIN sC5b-9 LEVELS WITH

FORMULATIONS 012 AND 013

[0062] This Example describes stabilization of complement protein sC5b-9 levels with formulations 012 and 013.

[0063] Samples

[0064] Fresh blood was collected from a normal healthy volunteer into an EDTA evacuated blood collection tube. Approximately 500 pL of whole blood was aliquoted into centrifuge tubes and diluted equal parts with formulation 012 or formulation 013. Whole blood samples were either processed for plasma immediately or incubated for 24-48 hours at 2-8°C, 25°C and 37°C. To determine stabilization of sC5b-9, the levels of the complement proteins were determined at the indicated time points and compared to the protein concentrations in plasma at time zero.

[0065] Assay

[0066] Commercially available solid-phase antibody capture assays (Quidel, San Diego, CA, USA) were used for sC5b-9-specific enzyme-linked immunosorbent assays (ELISAs).

Approximately 100 pL of standards, controls and diluted test specimens (n=2) were incubated at room temperature in polystyrene (PS) microtiter wells pre-coated with a capture antibody specific for the target fragment (sC5b-9). During the initial incubation step, sC5b-9 was bound to the coated wells. Unbound material was removed via successive wash cycles. During the second step, a volume of analyte-specific enzyme-conjugated-antibody was added to each well and incubated at room temperature. Analyte-specific enzyme-conjugated antibody reacted with the captured analytes, and unbound conjugate was removed via successive wash cycles. During a third step, a volume of enzyme substrate was added to each well and incubated at room temperature. The bound enzyme reacted with substrate producing a signal proportional to the amount of specimen analytes bound. The enzymatic reaction was stopped upon addition of a volume of acid. The substrate color intensity present in each well was measured spectrophotometrically at analyte-specific wavelength using a Synergy -HTX- S1LFA automated microplate reader (Biotek Instruments Inc., Winooski, VT, USA), followed by determination of analyte concentration for each analyte from the corresponding standard curve. A final dilution of 1 : 10 was used in the sC5b-9 fragment test.

[0067] As shown in Figure 2, sC5b-9 concentration in non-protected blood samples (NP) rose after storage of the blood samples for 48 hours at all temperatures tested. For blood samples mixed 1 :1 with stabilizing formulation 012 and formulation 013, sC5b-9

concentrations stayed close to the original time zero concentrations after storage at 2-8°C, 25°C, and 37°C for 48 hours (Figure 2).

EXAMPLE 3

STABILIZATION OF COMPLEMENT PROTEIN C3a LEVELS WITH

FORMULATION 014

[0068] This Example describes stabilization of complement protein C3a levels with formulation 014.

[0069] Samples

[0070] Fresh blood was drawn from healthy donors into EDTA evacuated blood tubes (BD, Franklin Lakes, NJ), BD P100 tubes, or syringes pre-loaded with formulation 014. Whole blood samples were either processed immediately to plasma (2,200g / 15 min) and stored at - 80°C or incubated for 48 hours at ambient temperature before plasma isolation. For shipping studies, matched samples were shipped overnight at ambient temperature from San Diego to Florida and back before processing.

[0071] Assay

[0072] Commercially available solid-phase antibody capture assays (Quidel, San Diego, CA, USA) were used for C3a-specific enzyme-linked immunosorbent assays (ELIS As). 100 pL of standards, controls and diluted test specimens (n=2) were incubated at room temperature in polystyrene (PS) microtiter wells pre-coated with a capture antibody specific for the target complement fragment (C3a). During the initial incubation step, C3a was bound to the coated wells. Unbound material was removed via successive wash cycles. During the second step, a volume of analyte-specific enzyme-conjugated-antibody was added to each well and incubated at room temperature. Analyte-specific enzyme-conjugated antibody reacted with the captured analytes, and unbound conjugate was removed via successive wash cycles. During a third step, a volume of enzyme substrate was added to each well and incubated at room temperature. The bound enzyme reacted with substrate producing a signal proportional to the amount of specimen analytes bound. The enzymatic reaction was stopped upon addition of a volume of acid. The substrate color intensity present in each well was measured spectrophotometrically at analyte-specific wavelength using a Synergy-HTX-SlLFA automated microplate reader (Biotek Instruments Inc., Winooski, VT, USA), followed by determination of analyte concentration for each analyte from the corresponding standard curve. A final dilution of 1:200 was used in the C3a fragment test. Protein fold changes were calculated by dividing the concentration of the complement protein at time 48 hours or after shipping post-draw by the protein concentration at time 0.

[0073] As shown in Figure 3, C3a protein levels increased in non-protected blood samples (EDTA) either after storage of the blood samples for 48 hours or after shipment of blood samples as expressed with fold change >1.0. For blood samples mixed 1 : 1 with stabilizing formulation 014, C3a fold changes are close to 1.0, indicating stabilization of the concentration of the protein levels (Figure 3). Commercially-available protein-stabilization blood tube P100 did not prevent increase of C3a levels upon shipping of blood samples indicated by fold change >2.0.

[0074] Unless the context requires otherwise, throughout the present specification and claims, the word“comprise” and variations thereof, such as,“comprises” and“comprising,” which is used interchangeably with“including,”“containing,” or“characterized by,” is inclusive or open-ended language and does not exclude additional, unrecited elements or method steps. The phrase“consisting of’ excludes any element, step, or ingredient not specified in the claim. The phrase“consisting essentially of’ limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the claimed subject matter. The present disclosure contemplates embodiments of compositions and methods corresponding to the scope of each of these phrases. Thus, a composition or method comprising recited elements or steps contemplates particular embodiments in which the composition or method consists essentially of or consists of those elements or steps.

[0075] Reference throughout this specification to“one embodiment” or“an embodiment” or “an aspect” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. 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, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0076] 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-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.

[0077] While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the various embodiments of the present disclosure. It should be understood that various alternatives to the embodiments described herein may be employed in practicing the subject matter. It is intended that the following claims define the scope of the subject matter and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A formulation for substantially stable storage of one or more proteins in a biological sample at non-freezing temperatures, wherein the one or more proteins remain stabilized after storage at non-freezing temperatures for a period of at least 1 day.

2. The formulation of Claim 1, wherein at least 80% of the substantially, stably stored one or more proteins remain stabilized after storage at non-freezing temperatures for a period of at least 1 day.

3. The formulation of any of Claims 1-2, wherein the biological sample comprises a blood sample, whole blood, plasma, or serum.

4. The formulation of any of Claims 1-3, comprising:

(i) at least one of an amino acid or amino acid derivative, a polyol or reducing sugar, or combinations thereof; and

(ii) at least one buffer.

5. The formulation of Claim 4, wherein the amino acid or amino acid derivative is selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, betaine, sarcosine, dimethylglycine, taurine, and combinations thereof.

6. The formulation of Claim 5, wherein the amino acid or amino acid derivative is dimethylglycine, sarcosine, or betaine.

7. The formulation of Claim 4, wherein the polyol or reducing sugar is selected from the group consisting of glycol, glycerol, erythritol, threitol, arabitol, xylitol, ribitol, adonitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol, glucose, dextrose, galactose, fructose, ribose, xylose, glyceraldehyde, and combinations thereof.

8. The formulation of Claim 7, wherein the polyol or reducing sugar is dextrose.

9. The formulation of Claim 4, wherein the at least one buffer is selected from the group consisting of citric acid, tartaric acid, malic acid, sulfosalicylic acid,

sulfoisophthalic acid, oxalic acid, borate, CAPS (3-(cyclohexylamino)-l-propanesulfonic acid), CAPSO (3-(cyclohexylamino)-2-hydroxy-l-propanesulfonic acid), EPPS (4-(2- hydroxyethyl)-l-piperazinepropanesulfonic acid), HEPES (4-(2-hydroxyethyl)piperazine-l- ethanesulfonic acid), MES (2-(N-morpholino)ethanesulfonic acid), MOPS (3-(N- morpholino)propanesulfonic acid), MOPSO (3-morpholino-2-hydroxypropanesulfonic acid), PIPES (l,4-piperazinediethanesulfonic acid), TAPS (N-[tris(hydroxymethyl)methyl]-3- aminopropanesulfonic acid), TAPSO (2-hydroxy-3-[tris(hydroxymethyl)methylamino]-l- 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), bis-tris (2-[bis(2-hydroxyethyl)amino]-2- (hydroxymethyl)-l, 3 -propanediol), monosodium citrate, disodium citrate, trisodium citrate, sodium dihydrogen phosphate, and combinations thereof.

10. The formulation of Claim 9, wherein the at least one buffer is trisodium citrate.

11. The formulation of Claim 9, wherein the at least one buffer is sodium dihydrogen phosphate.

12. The formulation of Claim 9, wherein the at least one buffer is citric acid.

13. The formulation of Claim 9, wherein the at least one buffer is trisodium citrate, sodium dihydrogen phosphate, and citric acid.

14. The formulation of Claim 4, further comprising at least one anticoagulant.

15. The formulation of Claim 14, wherein the at least one anticoagulant is selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), hirudin, heparin, sodium citrate, and sodium polyanethol sulfonate (SPS).

16. The formulation of claim 4, further comprising a purine.

17. The formulation of claim 16, wherein the purine is adenine or guanine.

18. The formulation of claim 17, wherein the purine is adenine.

19. The formulation of claim 16, wherein the formulation comprises

dimethylglycine, EDTA, trisodium citrate, sodium dihydrogen phosphate, citric acid, and adenine.

20. A composition comprising a substantially, stably stored one or more protein admixed with the formulation of any one of Claims 1-19.

21. An article of manufacture, comprising the formulation of any of Claims 1-19 contained within a blood collection tube.

22. The article of manufacture of Claim 21, wherein the blood collection tube is an evacuated blood collection tube.

23. A kit, comprising any one of the articles of manufacture of any of Claims 21 or 22 and a package insert.

24. A method for substantially stable storage of one or more proteins in a biological sample at non-freezing temperatures, comprising: admixing a biological sample collected from a subject with the formulations of any one of Claims 1-19, wherein the one or more proteins remain stabilized after storage at non-freezing temperature for a period of at least 1 day.

25. The method of Claim 24, wherein at least 80% of the one or more proteins remain stabilized at non-freezing temperatures for a period of at least 1 day.

26. The method of claim 24, wherein the biological sample comprises a blood sample, whole blood, plasma, or serum.

27. The method of Claim 24, wherein the one or more proteins is a complement protein.

28. The method of any of Claims 24-27, wherein the subject is an animal.

29. The method of any of Claims 24-27, wherein the subject is a mammal.

30. The method of any of Claims 24-27, wherein the subject is a human.

31. A method for substantially stable storage of one or more proteins in a biological sample at non-freezing temperatures, comprising: admixing a biological sample collected from a subject with a formulation of Table 1.

32. A method for substantially stable storage of one or more proteins in a biological sample at non-freezing temperatures, comprising: admixing a biological sample collected from a subject with a formulation of Table 2.

33. A method for substantially stable storage of one or more proteins in a biological sample at non-freezing temperatures, comprising: admixing a biological sample collected from a subject with a formulation selected from the group consisting of:

(i) dextrose and EDTA;

(ii) melibiose and EDTA;

(hi) glycine;

(iv) betaine;

(v) sorbitol;

(vi) betaine, dextrose, trisodium citrate, sodium dihydrogen phosphate, citric acid, and adenine;

(vii) sarcosine, dextrose, trisodium citrate, sodium dihydrogen phosphate, citric acid, and adenine;

(viii) dimethylglycine, dextrose, trisodium citrate, sodium dihydrogen phosphate, citric acid, and adenine;

(ix) dimethylglycine, EDTA, trisodium citrate, sodium dihydrogen phosphate, citric acid, and adenine;

wherein the one or more proteins remain stabilized after storage at non- freezing temperatures for a period of at least 1 day.