Polymerized albumin and methods of making and using thereof

Abstract

Described herein compositions comprising polymerized albumin (e.g., polymerized human serum albumin) and / or polymer-modified polymerized albumin (e.g., PEGylated polymerized human serum albumin). Also provided are methods of making and using polymerized albumin and / or polymer-modified polymerized albumin.

Description

[0001] POLYMERIZED ALBUMIN AND METHODS OF MAKING AND USING THEREOF

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims benefit of priority of U.S. Provisional Application No. 63 / 593,718, filed October 27, 2023, which is hereby incorporated by reference in its entirety.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] This invention was made with government support under Grant Nos. R01HL138116, R01HL156526, R01HL158076, R01HL159862, and R01HL162120 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] BACKGROUND

[0007] Plasma expanders (PEs) are typically administered to patients suffering from hypovolemia, in order to replace lost blood volume and to increase blood colloid oncotic pressure (COP), which pulls additional fluid volume from the extravascular space into the vascular space and thus further expands the blood volume and keeps blood vessels in an open state which facilitates tissue perfusion. PEs can be subdivided into two classes: crystalloids and colloids. Crystalloids consist of fluids containing electrolytes and other small molecules, and are transient species in the circulation due to capillary leakage. Alternatively, non-protein, synthetic colloids such as hydroxyethyl starch (HES), which are larger in size compared to the molecules in crystalloid solutions reduce capillary leakage. However, hyperviscous urine was observed upon transfusion of high molecular weight (MW) HES which led to renal failure, while low MW HES can elicit an allergic reaction similar to gelatin. Dextran has similar negative side-effects, and also inhibits coagulation.

[0008] Since human serum albumin (HSA) accounts for -80% of the blood COP, it is a naturally occurring PE. HSA also possesses antioxidant properties that reduces inflammation when used as a resuscitation fluid. One study demonstrated that intensive care unit administration of either 4% HSA or saline resulted in the same 28 day -mortality rate; however, the type of resuscitation fluid becomes more important when treating hypovolemic patients. Such patients typically suffer from ischemia-reperfusion injury, septic shock, or burns that dysregulate endothelial permeability. HSA administered to patients with compromised endothelial function increased vascular permeability, and promoted HSA extravasation into the tissue space. In this scenario, tissues are directly exposed to toxic HSA-bound metabolites, blood COP decreases, and tissue edema becomes prevalent.

[0009] Accordingly improved PEs based on HSA are needed.

[0010] SUMMARY

[0011] Provided herein are compositions including polymerized albumin, wherein the composition comprises less than 5% by weight low molecular weight albumin species having a molecular weight less than 100 kDa; and less than 5% by weight high molecular weight albumin species.

[0012] In some aspects, the polymerized albumin can comprise polymerized human serum albumin (PolyHSA). In some aspects, the albumin can comprise a recombinant albumin.

[0013] In some aspects, the composition includes less than 5% by weight low molecular weight albumin species having a molecular weight less than 500 kDa and less than 5% by weight high molecular weight albumin species, such as less than 1% by weight low molecular weight albumin species having a molecular weight less than 500 kDa and less than 1% by weight high molecular weight albumin species. In some aspects, the low molecular weight albumin species include unreacted albumin, albumin oligomers, or combinations thereof.

[0014] In some aspects, the high molecular weight albumin species comprise albumin species that are retained on a filtration membrane having a pore size of 0.2 pm.

[0015] In some aspects, the high molecular weight albumin species comprise albumin species having a molecular weight greater than 10,000 kDa.

[0016] In some aspects, the high molecular weight albumin species comprise albumin species having a molecular weight greater than 2000 kDa.

[0017] In some aspects, the composition comprises less than 5% by weight albumin species having a molecular weight less than 100 kDa; and less than 5% by weight albumin species that are retained on a filtration membrane having a pore size of 0.2 pm.

[0018] In some aspects, the composition comprises less than 5% by weight albumin species having a molecular weight less than 100 kDa; and less than 5% by weight albumin species having a molecular weight greater than 10,000 kDa.

[0019] In some aspects, the composition comprises less than 5% by weight albumin species having a molecular weight less than 100 kDa; and less than 5% by weight albumin species having a molecular weight greater than 500 kDa. In some aspects, the composition comprises less than 5% by weight albumin species having a molecular weight less than 500 kDa; and less than 5% by weight albumin species that are retained on filtration membrane having a pore size of 0.2 pm.

[0020] In some aspects, the composition comprises less than 5% by weight albumin species having a molecular weight less than 500 kDa; and less than 5% by weight albumin species having a molecular weight greater than 10,000 kDa.

[0021] In some aspects, the polymerized albumin is prepared by a process that includes: polymerizing albumin; filtering the polymerized albumin by ultrafiltration against a first filtration membrane having a pore size that separates the low molecular weight albumin species from the polymerized albumin; and filtering the polymerized albumin by ultrafiltration against a second filtration membrane having a pore size that separates the high molecular weight albumin species from the polymerized albumin. In certain aspects, the ultrafiltration includes tangential- flow filtration.

[0022] In some aspects, the first filtration membrane is rated for retaining solutes having a molecular weight greater than 100 kDa, thereby forming a retentate fraction comprising the polymerized albumin and a permeate fraction comprising low molecular weight albumin species having a molecular weight less than 100 kDa. In some aspects, the second filtration membrane is rated for retaining solutes having a molecular weight greater than 500 kDa, thereby forming a retentate fraction comprising the high molecular weight albumin species and a permeate fraction comprising the polymerized albumin. In other aspects, the second filtration membrane has a pore size of 0.2 pm, thereby forming a retentate fraction comprising the high molecular weight albumin species and a permeate fraction comprising the polymerized albumin.

[0023] In some aspects, the first filtration membrane is rated for retaining solutes having a molecular weight greater than 500 kDa, thereby forming a retentate fraction comprising the polymerized albumin and a permeate fraction comprising low molecular weight albumin species having a molecular weight less than 500 kDa. In some aspects, the second filtration membrane has a pore size of 0.2 pm, thereby forming a retentate fraction comprising the high molecular weight albumin species and a permeate fraction comprising the polymerized albumin.

[0024] In some aspects, the polymerized albumin has a cross-linker to albumin molar ratio of at least about 10: 1, such as a cross-linker to albumin molar ratio of from 20: 1 to 80: 1. In some aspects, the cross-linker includes glutaraldehyde, succindialdehyde, activated forms of polyoxyethylene and dextran, -hydroxy aldehydes, such as glycolaldehyde, N-maleimido-6- aminocaproyl-(2'-nitro,4'-sulfonic acid)-phenyl ester, m-maleimidobenzoic acid-N- hydroxy succinimide ester, succinimidyl 4-(N-maleimidomethyl)cyclohexane-l -carboxylate, sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-l -carboxylate, m-maleimidobenzoyl-N- hydroxysuccinimide ester, m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester, N- succinimidyl(4-iodoacetyl)aminobenzoate, sulfosuccinimidyl(4-iodoacetyl)aminobenzoate, succinimidyl 4-(p-maleimidophenyl)butyrate, sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate, l-ethyl-3-(3-dimethylarninopropyl)carbodiimide hydrochloride, N,N'-phenylene dimal eimide, or a combination thereof. In certain aspects, the cross-linker is from at least one of a dialdehyde class, bisimidate class, the acyl diazide class, or the aryl dihalide class. In certain aspects, the cross-linker includes glutaraldehyde.

[0025] In some aspects, the polymerized albumin is covalently modified with a polymer. In some aspects, the polymer comprises a polyalkylene oxide, such as polyethylene glycol (PEG). In other aspects, the polymer comprises a zwitterionic polymer. In some aspects, the polymer has a molecular weight of from 200 Da to 1,000,000 Da, such as from 1,000 Da to 10,000 Da, or from 3,000 Da to 6,000 Da, or about 5,000 Da.

[0026] In some aspects, the polymerized albumin is covalently modified with the polymer in a process that comprises: conjugating one or more polymers to the polymerized albumin to form a polymer-modified polymerized albumin; filtering the polymer-modified polymerized albumin by ultrafiltration against a first filtration membrane having a pore size that separates low molecular weight contaminants from the polymer-modified polymerized albumin; and filtering the polymer-modified polymerized albumin by ultrafiltration against a second filtration membrane having a pore size that separates the high molecular weight contaminants from the polymer- modified polymerized albumin. In some aspects, the ultrafiltration comprises tangential -flow filtration.

[0027] In some aspects, the first filtration membrane is rated for retaining solutes having a molecular weight greater than 100 kDa, thereby forming a retentate fraction comprising the polymer-modified polymerized albumin and a permeate fraction comprising low molecular weight contaminants having a molecular weight less than 100 kDa. In some aspects, the second filtration membrane is rated for retaining solutes having a molecular weight greater than 500 kDa, thereby forming a retentate fraction comprising the high molecular weight contaminants and a permeate fraction comprising the polymer-modified polymerized albumin. In other aspects, the second filtration membrane has a pore size of 0.2 pm, thereby forming a retentate fraction comprising the high molecular weight contaminants and a permeate fraction comprising the polymer-modified polymerized albumin.

[0028] In some aspects, the first filtration membrane is rated for retaining solutes having a molecular weight greater than 500 kDa, thereby forming a retentate fraction comprising the polymer-modified polymerized albumin and a permeate fraction comprising low molecular weight contaminants having a molecular weight less than 500 kDa. In some aspects, the second filtration membrane has a pore size of 0.2 pm, thereby forming a retentate fraction comprising the high molecular weight albumin species and a permeate fraction comprising the polymerized albumin.

[0029] Also provided herein are methods of treating subjects in need thereof using the compositions described herein.

[0030] For example, provided herein are methods of treating hypercytokinemia in a subject in need thereof. In some aspects, these methods can include administering an effective amount of a composition described herein to reduce circulating cytokine levels by at least 5%, such as from 5% to 70%.

[0031] In some aspects, the hypercytokinemia is induced by an infectious agent such as influenza (e.g., H1N1 influenza or H5N1 influenza), coronavirus infection (e.g., avian coronavirus (IBV), porcine coronavirus HKU15 (PorCoV HKU15), Porcine epidemic diarrhea virus (PEDV), HCoV-229E, HCoV-OC43, HCoV-HKUl, HCoV-NL63, SARS-CoV, SARS- CoV-2, or MERS-CoV), Influenza B, Parainfluenza virus, Ebola, Epstein-Barr virus, cytomegalovirus, or group A streptococcus. In some aspects, the hypercytokinemia is associated with graft-versus-host disease.

[0032] Also described herein are methods of preventing hypercytokinemia in a subject include administering to the subject a therapeutically effective amount of a composition described herein to reduce or prevent an increase circulating cytokine levels. In some embodiments, the subject is infected with or has been exposed to an infectious agent such as influenza (e.g., H1N1 influenza or H5N1 influenza), coronavirus infection (e.g., avian coronavirus (IBV), porcine coronavirus HKU15 (PorCoV HKU15), Porcine epidemic diarrhea virus (PEDV), HCoV-229E, HCoV- OC43, HCoV-HKUl, HCoV-NL63, SARS-CoV, SARS-CoV-2, or MERS-CoV), Influenza B, Parainfluenza virus, Ebola, Epstein-Barr virus, cytomegalovirus, or group A streptococcus. In some embodiments, the subject has received or will receive transplanted cells, transplanted tissue, a transplanted organ, or any combination thereof. In some embodiments, the transplanted cells, transplanted tissue, a transplanted organ, or any combination thereof comprise an allograft or a xenograft.

[0033] Also described herein are methods of treating endothelial dysfunction in a subject comprising administering to the subject a therapeutically effective amount of a composition described herein to reduce circulating levels of a biomarker for endothelial dysfunction in the subject. Also described herein are methods of preventing endothelial dysfunction in a subject comprising administering to the subject a therapeutically effective amount of a composition described herein to reduce or prevent an increase in circulating levels of a biomarker for endothelial dysfunction in the subject. In some embodiments, the composition described herein can be administered in an effective amount to prevent circulating levels of the biomarker for endothelial dysfunction rising above normal levels for subjects without endothelial dysfunction. In some embodiments, the biomarker for endothelial dysfunction comprises syndecan-1.

[0034] Also described herein are methods of treating endothelial dysfunction in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a composition described herein to reduce endothelial barrier permeability.

[0035] Also described herein are methods of protecting endothelial tissue in a subject comprising administering to the subject a composition described herein in a therapeutically effective amount to protect endothelial tissue from damage.

[0036] Also provided herein are methods of using the compositions described herein as plasma expanders or plasma substitutes.

[0037] Also provided herein are methods of replacing lost blood volume in a subject in need thereof that comprise administering to the subject a therapeutically effective amount of a composition described herein.

[0038] In some embodiments, the subject has a normal blood pressure. In some embodiments, the composition described herein is administered via infusion or exchange transfusion. In some embodiments, the composition described herein is administered via infusion. In some embodiments, the infusion comprises infusion of a volume of a composition described herein, and wherein the volume comprises from 10% to 30% of the subject’s total blood volume. In some embodiments, the composition described herein is administered via exchange transfusion. In some embodiments, the exchange transfusion comprises exchange transfusion of from 5% to 90% of the subject’s total blood volume with a composition described herein. In some embodiments, the composition described herein is administered in an amount effective to reduce circulating cytokine levels by at least 5%, such as from 5% to 70%. In some embodiments, the composition described herein can be administered in a therapeutically effective amount to reduce an immune response. In some embodiments, the composition described herein can be administered in a therapeutically effective amount to reduce the number of leukocytes adhered to endothelial tissue in the subject. In some embodiments, the compositions described herein can be administered in a therapeutically effective amount to improve vascular integrity. Also described herein are methods of using the compositions described herein for the ex vivo perfusion of an organ or tissue.

[0039] Also described herein are methods of using the compositions described herein for ex vivo cell or tissue culture.

[0040] Also described herein are methods of using the compositions described herein in extracorporeal devices, such as extracorporeal membrane oxygenation (ECMO) devices.

[0041] Also described herein are methods of binding or sequestering a metabolite (e.g., free heme) in vivo or ex vivo, the method comprising contacting the metabolite with the compositions described herein.

[0042] Additional advantages will be set forth in part in the description that follows, and in part will be obvious from the description, or may be learned by practice of the aspects described below. The advantages described below will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive.

[0043] BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate certain examples of the present disclosure and together with the description, serve to explain, without limitation, the principles of the disclosure. The application includes reference to the accompanying figures, in which:

[0045] Figures 1 A-1B illustrate methods for the synthesis and fractionation of PolyHSA.

[0046] Figures 2A-2C show the SEC-HPLC analysis of HSA and PolyHSA. (2A) PolyHSA 43 : 1. (2B) PolyHSA 60: 1. (2C) HSA with and without tryptophan. The gray shaded areas represent the standard deviation from triplicate HSA / PolyHSA preparations. N=3.

[0047] Figure 3 shows an SDS-PAGE analysis of representative HSA and PolyHSA fractions.

[0048] Figure 4 shows the hydrodynamic diameter measured via DLS for HSA and PolyHSA fractions. N=3, * p < 0.05.

[0049] Figure 5 shows the zeta potential of HSA and PolyHSA fractions. N=3, * p < 0.05.

[0050] Figure 6 shows the viscosity of HSA and PolyHSA brackets. The viscosity was measured at a total protein concentration of 100 mg / mL. N=3, * p < 0.05.

[0051] Figure 7 shows the osmolality of HSA and PolyHSA brackets. The osmolality was measured at a total protein concentration of 100 mg / mL. N=3. Figure 8 shows the COP for HSA and PolyHSA brackets. The COP was measured at a total protein concentration of 100 mg / mL. N=3, * p < 0.05.

[0052] Figure 9 shows circular dichroism (CD) spectroscopy of HSA and PolyHSA brackets measured at 3 pM. N=3.

[0053] Figure 10 illustrates the synthesis and fractionation of PPHSA.

[0054] Figures 11 A-l IB show the SEC-HPLC analysis of HSA, PHSA, and PPHSA. (11 A) 43 : 1 glutaraldehyde to HSA molar ratio. (1 IB) 60: 1 glutaraldehyde to HSA molar ratio. At the same glutaraldehyde to HSA molar ratio and bracket, PPHSA exhibited an increase in size compared to PHSA. At the same glutaraldehyde to HSA molar ratio, fraction A eluted earlier than fraction B for the same material PHSA / PPHSA. At the same glutaraldehyde to HSA molar ratio, the elution order from largest to smallest size: PPHSA A, PHSA A, PPHSA B, PHSA B, HSA. N=3

[0055] Figure 12 shows the SDS-PAGE analysis of HSA, PHSA, and PPHSA brackets.

[0056] Figure 13 shows the hydrodynamic diameter measured via DLS for HSA, PHSA, and PPHSA brackets. * p < 0.05, N=3.

[0057] Figure 14 shows the zeta potential of HSA, PHSA, and PPHSA brackets. * p < 0.05, N=3.

[0058] Figure 15 shows the viscosity of HSA, PHSA, and PPHSA brackets measured at 50 mg / mL of total protein. * p < 0.05, N=2.

[0059] Figure 16 shows the osmolality of HSA, PHSA, and PPHSA brackets measured at 50 mg / mL of total protein. * p < 0.05, N=2.

[0060] Figure 17 shows the COP of HSA, PHSA, and PPHSA brackets measured at 50 mg / mL of total protein. * p < 0.05, N=2.

[0061] Figure 18 shows circular dichroism (CD) spectroscopy of HSA, PHSA, and PPHSA brackets measured at 3 pM. N=2.

[0062] Reference will now be made in detail to specific aspects of the disclosed materials, compounds, compositions, formulations, articles, and methods, examples of which are illustrated in the accompanying Examples and Figures.

[0063] DETAILED DESCRIPTION

[0064] The following description of the disclosure is provided as an enabling teaching of the disclosure in its best, currently known embodiments. Many modifications and other embodiments disclosed herein will come to mind to one skilled in the art to which the disclosed compositions and methods pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the disclosures are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. The skilled artisan will recognize many variants and adaptations of the aspects described herein. These variants and adaptations are intended to be included in the teachings of this disclosure and to be encompassed by the claims herein.

[0065] Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0066] As can be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure.

[0067] Any recited method can be carried out in the order of events recited or in any other order that is logically possible. That is, unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.

[0068] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided herein can be different from the actual publication dates, which can require independent confirmation.

[0069] It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed compositions and methods belong. It can be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly defined herein.

[0070] Prior to describing the various aspects of the present disclosure, the following definitions are provided and should be used unless otherwise indicated. Additional terms may be defined elsewhere in the present disclosure.

[0071] General Definitions

[0072] As used herein, “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps, or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. Moreover, each of the terms “by”, “comprising,” “comprises”, “comprised of,” “including,” “includes,” “included,” “involving,” “involves,” “involved,” and “such as” are used in their open, nonlimiting sense and may be used interchangeably. Further, the term “comprising” is intended to include examples and aspects encompassed by the terms “consisting essentially of’ and “consisting of.” Similarly, the term “consisting essentially of’ is intended to include examples encompassed by the term “consisting of.”

[0073] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a compound”, “a composition”, or “a disorder”, includes, but is not limited to, two or more such compounds, compositions, or disorders, and the like.

[0074] It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It can be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it can be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0075] When a range is expressed, a further aspect includes from the one particular value and / or to the other particular value. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g., the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g., ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘less than x’, less than y’, and ‘less than z’. Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’. In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.

[0076] It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the sub-ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.

[0077] As used herein, the terms “about,” “approximate,” and “at or about” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact but may be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In such cases, it is generally understood, as used herein, that “about” and “at or about” mean the nominal value indicated ±10% variation unless otherwise indicated or inferred. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0078] As used herein, “molecular weight” refers to the sum of the atomic masses of all atoms in a molecule, based on a scale in which the atomic masses of hydrogen, carbon, nitrogen, and oxygen are 1, 12, 14, and 16, respectively.

[0079] As used herein, “osmolarity” refers to the concentration of a solution expressed as the total number of solute particles per liter. As used herein, “viscosity'’ refers to a quantity expressing the magnitude of internal friction, as measured by the force per unit area resisting a flow in which parallel layers unit distance apart have unit speed relative to one another.

[0080] As used herein, “colloid osmotic pressure” refers to the physiochemical phenomenon that occurs when two solutions with different colloid concentrations are separated by a semipermeable membrane. It is a type of osmotic pressure induced by colloids, which can include protein and more specifically albumin, in a blood vessel's plasma that cause a pull on fluid back into the capillary.

[0081] As used herein, “glutaraldehyde” refers to CsHsO?. or OCH( Cl bflCHO, is a transparent oily, liquid with a pungent odor. It is a dialdehyde comprised of pentane with aldehyde functions at C-l and C-5. Alternatives to glutaraldehyde can include carboiimide, diisocyanates and polyepoxy compounds, as well as Genipin (Challenge Bioproducts Co., Ltd., Taiwan), epigallocatechin gallate (Sigma, St. Louis, MO), and grape seed proanthocyanidin (PureBulk, Inc., Roseburg, OR).

[0082] As used herein, the term "substantially" means that the subsequently described event or circumstance completely occurs or that the subsequently described event or circumstance generally, typically, largely, or approximately occurs. For example, the term “substantially” in the context “substantially free from” or “substantially free of’ refers to a circumstance in which very little or none of a particular component (e.g., less than 5% by weight of the component, less than 4% by weight of the component, less than 3% by weight of the component, less than 2.5% by weight of the component, less than 2% by weight of the component, less than 1% by weight of the component, less than 0.5% by weight of the component, or less than 0.1 % by weight of the component) is present in a composition.

[0083] It is understood that in aspects disclosed herein, the terms “diacycle” and “diafiltration cycles” are used interchangeably.

[0084] As used herein, the term "ultrafiltration" is used for processes and systems employing membranes rated for removing solutes having a molecular weight between about 1 kDa and 1000 kDa.

[0085] As used herein, the term "tangential-flow filtration" refers to a process in which the fluid mixture containing the components to be separated by filtration is recirculated at velocities tangential to the plane of the filtration membrane to reduce fouling of the filter. In such filtrations a pressure differential is applied along the length of the filtration membrane to cause the fluid and filterable solutes to flow through the membrane (i.e., filter). This filtration is suitably conducted as a batch process as well as a continuous-flow process. For example, the solution may be passed repeatedly over the membrane while that fluid which passes through the filter is continually drawn off into a separate unit or the solution is passed once over the membrane and the fluid passing through the filter is continually processed downstream.

[0086] As used herein, the term “PEGylation” generally refers to linking to polyethylene glycol (PEG). Thus, a “PEGylated” material refers to a material, such as a protein, that has PEG conjugated (e.g., covalently bonded) to it.

[0087] While aspects of the present invention can be described and claimed in a particular statutory class, such as the system statutory class, this is for convenience only, and one of ordinary skill in the art will understand that each aspect of the present invention can be described and claimed in any statutory class. Unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is in no way intended that an order be inferred in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.

[0088] The present invention may be understood more readily by reference to the following detailed description of various aspects of the invention and the examples included therein and to the Figures and their previous and following description.

[0089] Compositions

[0090] Described herein are compositions including polymerized albumin (e.g., polymerized human serum albumin) and / or polymer-modified polymerized albumin (e.g., PEGylated polymerized human serum albumin). In some embodiments, the composition can further include one or more pharmaceutically acceptable carriers. In some embodiments, the composition described herein can also be mixed with blood compositions, which includes whole blood, red blood cells, plasma, blood fractions, crystalloid solutions, or plasma expanders (PEs), or any combination thereof.

[0091] In some embodiments, a suitable plasma expander can include but is not limited to human serum albumin.

[0092] In some embodiments, the composition does not need a plasma expander. In some embodiments, the composition without a plasma expander can reduce clot formation compared to plasma expanders such as human serum albumin. The albumin can comprise any albumin known to those of skill in the art. In some embodiments, the albumin may be serum albumin isolated from a host species and purified for use in the formation of a conjugate. The serum albumin may be any mammalian serum albumin known to those of skill in the art, including but not limited to mouse, rat, rabbit, guinea pig, dog, cat, sheep, bovine, ovine, equine, or human albumin. In some embodiments, the albumin is human serum albumin (HSA).

[0093] In some cases, the albumin can be obtained from serum or a genomic source. In other cases, the albumin can comprise recombinant albumin. The recombinant albumin may be any mammalian albumin known to those of skill in the art, including but not limited to mouse, rat, rabbit, guinea pig, dog, cat, sheep, bovine, ovine, equine, or human albumin. In some cases, the recombinant albumin is recombinant human albumin, in particular, recombinant human serum albumin (rHSA).

[0094] Human serum albumin (HSA) is responsible for a significant proportion of the osmotic pressure of serum and also functions as a carrier of endogenous and exogenous ligands. In its mature form, HSA is a non-glycosylated monomeric protein of 585 amino acids, corresponding to a molecular weight of about 66 kDa. Its globular structure is maintained by 17 disulfide bridges which create a sequential series of 9 double loops.

[0095] In some embodiments, the composition can include polymerized albumin, wherein the composition includes less than 5% by weight low molecular weight albumin species having a molecular weight less than 500 kDa and less than 5% by weight high molecular weight albumin species.

[0096] In some embodiments, the composition includes less than 1% by weight low molecular weight albumin species having a molecular weight less than 500 kDa and less than 1% by weight high molecular weight albumin species, such as less than 0.1% by weight low molecular weight albumin species having a molecular weight less than 500 kDa and less than 0.1% by weight high molecular weight albumin species. In some embodiments, the low molecular weight albumin species include unreacted albumin, albumin oligomers, or combinations thereof.

[0097] In some embodiments, the high molecular weight albumin species comprise albumin species that are retained on a filtration membrane having a pore size of 0.2 pm.

[0098] In some embodiments, the high molecular weight albumin species comprise albumin species having a molecular weight greater than 10,000 kDa.

[0099] In some embodiments, the high molecular weight albumin species comprise albumin species having a molecular weight greater than 500 kDa. In some embodiments, the composition comprises less than 5% by weight albumin species having a molecular weight less than 100 kDa; and less than 5% by weight albumin species that are retained on a filtration membrane having a pore size of 0.2 pm.

[0100] In some embodiments, the composition comprises less than 5% by weight albumin species having a molecular weight less than 100 kDa; and less than 5% by weight albumin species having a molecular weight greater than 10,000 kDa.

[0101] In some embodiments, the composition comprises less than 5% by weight albumin species having a molecular weight less than 100 kDa; and less than 5% by weight albumin species having a molecular weight greater than 500 kDa.

[0102] In some embodiments, the composition comprises less than 5% by weight albumin species having a molecular weight less than 500 kDa; and less than 5% by weight albumin species that are retained on a filtration membrane having a pore size of 0.2 pm.

[0103] In some embodiments, the composition comprises less than 1% by weight albumin species having a molecular weight less than 100 kDa; and less than 1% by weight albumin species that are retained on a filtration membrane having a pore size of 0.2 pm.

[0104] In some embodiments, the composition comprises less than 1% by weight albumin species having a molecular weight less than 100 kDa; and less than 1% by weight albumin species having a molecular weight greater than 10,000 kDa.

[0105] In some embodiments, the composition comprises less than 1% by weight albumin species having a molecular weight less than 100 kDa; and less than 1% by weight albumin species having a molecular weight greater than 500 kDa.

[0106] In some embodiments, the composition comprises less than 1% by weight albumin species having a molecular weight less than 500 kDa; and less than 1% by weight albumin species that are retained on a filtration membrane having a pore size of 0.2 pm.

[0107] In some embodiments, the polymerized albumin is prepared by a process that includes: polymerizing albumin; filtering the polymerized albumin by ultrafiltration against a first filtration membrane having a pore size that separates the low molecular weight albumin species from the polymerized albumin; and filtering the polymerized albumin by ultrafiltration against a second filtration membrane having a pore size that separates the high molecular weight albumin species from the polymerized albumin.

[0108] As used herein, “filtration membrane” refers to microporous barriers of, for example, polymeric, ceramic, or metallic materials which are used to separate dissolved materials (solutes), colloids, or find particular from solutions. A filtration membrane can be rated for retaining solutes have a specific molecular weight range from the molecular weight of one component in the solution to another component in the solution. By way of example, a filtration membrane can be rated for retaining polymerized albumin with a molecular weight above that of a low molecular weight albumin species (e.g., such as a membrane rated for retaining solutes have a molecular weight above 500 kDa). A filtration membrane can also be rated based on its pore size (e.g., a pore size of 0.2 pm).

[0109] In some embodiments, the first filtration membrane can be rated for retaining solutes having a molecular weight greater than the molecular weight of the low molecular weight albumin species, thereby forming a retentate fraction including the polymerized albumin and a permeate fraction including the low molecular weight albumin species. As used herein, a “retentate fraction” refers to the fraction of solution that is unable to pass through the filtration membrane. In some embodiments, the retentate fraction can include polymerized albumin. As used herein, a “permeate fraction” refers to the fraction of solution that permeates the filtration membrane.

[0110] In some embodiments, ultrafiltration can include tangential -flow filtration. Tangential- flow filtration refers to a process in which the fluid mixture containing the components to be separated by filtration is recirculated at high velocities tangential to the plane of the filtration membrane to reduce fouling of the filter. In such filtrations a pressure differential is applied along the length of the filtration membrane to cause the fluid and filterable solutes to flowthrough the membrane (e.g., filter). This filtration is suitably conducted as a batch process as well as a continuous-flow process. For example, the solution may be passed repeatedly over the membrane while that fluid which passes through the filter is continually drawn off into a separate unit or the solution is passed once over the membrane and the fluid passing through the filter is continually processed downstream.

[0111] In some embodiments, the first filtration membrane is rated for retaining solutes having a molecular weight greater than 100 kDa, thereby forming a retentate fraction comprising the polymerized albumin and a permeate fraction comprising low molecular weight albumin species having a molecular weight less than 100 kDa. In some embodiments, the second filtration membrane is rated for retaining solutes having a molecular weight greater than 500 kDa, thereby forming a retentate fraction comprising the high molecular weight albumin species and a permeate fraction comprising the polymerized albumin. In other embodiments, the second filtration membrane has a pore size of 0.2 pm, thereby forming a retentate fraction comprising the high molecular weight albumin species and a permeate fraction comprising the polymerized albumin. In some embodiments, the first filtration membrane is rated for retaining solutes having a molecular weight greater than 500 kDa, thereby forming a retentate fraction comprising the polymerized albumin and a permeate fraction comprising low molecular weight albumin species having a molecular weight less than 500 kDa. In some embodiments, the second filtration membrane has a pore size of 0.2 pm, thereby forming a retentate fraction comprising the high molecular weight albumin species and a permeate fraction comprising the polymerized albumin.

[0112] In some embodiments, the polymerized albumin can have a cross-linker to albumin molar ratio, referred to herein as the cross-link density, of at least 10:1, (e.g., at least 20: 1, at least 30:1, at least 40:1, at least 50:1, at least 60:1, at least 70:1, at least 80:1, at least 90:1). In some embodiments, the polymerized albumin can have a cross-linker to albumin molar ratio, referred to herein as the cross-link density, of 100: 1 or less, (e.g., 90: 1 or less, 80: 1 or less, 70: 1 or less, 60: 1 or less, 50: 1 or less, 40: 1 or less, 30: 1 or less, 20: 1 or less), and may optionally range between any of these cross-linking densities.

[0113] The polymerized albumin can have a cross-linker to albumin molar ratio, referred to herein as the cross-link density, ranging from any of the minimum values described above to any of the maximum values described above. For example, the cross-linking density can be in a range from 10:1 to 100:1 (e.g., from 10:1 to 20:1, from 10:1 to 30:1, from 10:1 to 40:1, from 10:1 to 50:1, from 10:1 to 60:1, from 10:1 to 70:1, from 10:1 to 80:1, from 10:1 to 90:1, from

[0114] 20:1 to 30:1, from 20:1 to 40:1, from 20:1 to 50:1, from 20:1 to 60:1, from 20:1 to 70:1, from

[0115] 20:1 to 80:1, from 20:1 to 90:1, from 20:1 to 100:1, from 30:1 to 40:1, from 30:1 to 50:1, from 30:1 to 60:1, from 30:1 to 70:1, from 30:1 to 80:1, from 30:1 to 90:1, from 30:1 to 100:1, from

[0116] 40:1 to 50:1, from 40:1 to 60:1, from 40:1 to 70:1, from 40:1 to 80:1, from 40:1 to 90:1, from

[0117] 40:1 to 100:1, from 50:1 to 60:1, from 50:1 to 70:1, from 50:1 to 80:1, from 50:1 to 90:1, from 50:1 to 100:1, 60:1 to 70:1, from 60:1 to 80:1, from 60:1 to 90:1, from 60:1 to 100:1, from 70:1 to 80:1, from 70:1 to 90:1, from 70:1 to 100:1, from 80:1 to 90:1, from 80:1 to 100:1, from 90:1 to 100:1). The molecular weight and / or cross-link density of the polymerized albumin compositions affect their biophysical characteristics, which directly determine viscosity and colloid osmotic pressure. As shown in the examples below, high MW polymerized albumin compositions having higher cross-link densities generally have improved biophysical characteristics relative to native albumin and dextran.

[0118] The polymerized albumin compositions have a higher viscosity than monomeric albumin compositions, when formulated at the same protein concentration. In one embodiment, the viscosity of the polymerized albumin composition is 1.1 times greater than the viscosity of the monomeric albumin composition having the same concentration (e.g., 2 times greater, 3 times greater, 4 times greater, 5 times greater, 6 times greater, 7 times greater, 8 times greater, 9 times greater, or 10 times greater).

[0119] In some embodiments, the polymerized albumin compositions have a lower COP than monomeric compositions at the same concentration level. In one embodiment, the COP of the polymerized albumin composition can be i the COP of monomeric albumin (e.g., 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9, 1 / 10, 1 / 15, 1 / 20, 1 / 25, 1 / 30, 1 / 35, 1 / 40, 1 / 45, or 1 / 50 the COP of monomeric albumin). In another embodiment, the COP of the polymerized albumin composition is about 1 / 10 the COP of monomeric albumin. In another embodiment, the COP is about 1 / 50 the COP of monomeric albumin.

[0120] In some embodiments, the polymerized albumin may have a molecular weight of at least 100 kDa and a cross-link density of at least 10: 1. In some embodiments, the polymerized albumin may have a molecular weight of at least 200 kDa and a cross-link density of at least 10: 1. In some embodiments, the polymerized albumin may have a molecular weight of at least 300 kDa and a cross-link density of at least 10: 1. In some embodiments, the polymerized albumin may have a molecular weight of at least 400 kDa and a cross-link density of at least 10: 1. In some embodiments, the polymerized albumin may have a molecular weight of at least 500 kDa and a cross-link density of at least 10: 1. In some embodiments, the polymerized albumin may have a molecular weight of at least 100 kDa and a cross-link density of at least 25 : 1. In some embodiments, the polymerized albumin may have a molecular weight of at least 200 kDa and a cross-link density of at least 25: 1. In some embodiments, the polymerized albumin may have a molecular weight of at least 300 kDa and a cross-link density of at least 25 : 1. In some embodiments, the polymerized albumin may have a molecular weight of at least 400 kDa and a cross-link density of at least 25: 1. In some embodiments, the polymerized albumin may have a molecular weight of at least 500 kDa and a cross-link density of at least 25: 1. In some embodiments, the polymerized albumin may have a molecular weight of at least 100 kDa and a cross-link density of at least 50: 1. In some embodiments, the polymerized albumin may have a molecular weight of at least 200 kDa and a cross-link density of at least about 50: 1. In some embodiments, the polymerized albumin may have a molecular weight of at least 300 kDa and a cross-link density of at least 50: 1. In some embodiments, the polymerized albumin may have a molecular weight of at least 400 kDa and a cross-link density of at least 50: 1. In some embodiments, the polymerized albumin may have a molecular weight of at least 500 kDa and a cross-link density of at least 50: 1. In some embodiments, the polymerized albumin may have a molecular weight of at least 100 kDa and a cross-link density of at least 75 : 1. In some embodiments, the polymerized albumin may have a molecular weight of at least 200 kDa and a cross-link density of at least 75: 1. In some embodiments, the polymerized albumin may have a molecular weight of at least 300 kDa and a cross-link density of at least 75 : 1. In some embodiments, the polymerized albumin may have a molecular weight of at least 400 kDa and a cross-link density of at least 75: 1. In some embodiments, the polymerized albumin may have a molecular weight of at least 500 kDa and a cross-link density of at least 75 : 1. In some embodiments, the polymerized albumin may have a molecular weight of at least 100 kDa and a cross-link density of at least 100: 1. In some embodiments, the polymerized albumin may have a molecular weight of at least 200 kDa and a cross-link density of at least 100: 1. In some embodiments, the polymerized albumin may have a molecular weight of at least about 300 kDa and a cross-link density of at least 100: 1. In some embodiments, the polymerized albumin may have a molecular weight of at least 400 kDa and a cross-link density of at least 100: 1. In some embodiments, the polymerized albumin may have a molecular weight of at least 500 kDa and a cross-link density of at least 100: 1.

[0121] In some embodiments, the polymerized albumin can be made by polymerizing monomeric albumin with a cross-linker, quenching the polymerization reaction with a reducing agent, and collecting the polymerized albumin having the desired molecular weight.

[0122] As discussed above, suitable monomeric albumin can come from any source such as human serum albumin isolated from human serum using known techniques or recombinant human serum albumin. The monomeric albumin is diluted or concentrated to the desired level, such as to 25 mg / mL with a suitable buffer. The polymerization reaction is initiated by the addition of a cross-linker, such as a 70% glutaraldehyde solution, to the polymerized albumin solution at the desired molar ratio of cross-linker to albumin: such as at least 10: 1, at least 50: 1, and at least 100: 1. The cross-linking density of the resulting polymerized albumin composition may be controlled by controlling this molar ratio or by controlling the parameters of the polymerization reaction, such as the duration and temperature of the reaction. The cross-link density of a polymerized albumin composition can be confirmed by separating polymerized albumin from any free cross-linker after the polymerization reaction and quantifying the amount of free cross-linker compared to the initial amount of cross-linker used in the reaction. The difference between the two quantities would be equivalent to the amount of cross-linker that is cross-linked to the protein. Glutaraldehyde, like many cross-linkers, reacts with lysine, histidine, tyrosine, arginine, and primary amine groups, forming both intra and intermolecular cross-links within albumin and between neighboring albumin molecules in solution. Therefore, cross-linked albumin compositions can include polymers of various molecular weights. Suitable cross-linkers in addition to glutaraldehyde can include succindialdehyde, activated forms of polyoxyethylene and dextran, a-hydroxy aldehydes, such as glycolaldehyde, N-maleimido-6-aminocaproyl-(2'-nitro,4'-sulfonic acid)-phenyl ester, m-maleimidobenzoic acid- N-hydroxy succinimide ester, succinimidyl 4-(N-maleimidomethyl)cyclohexane-l -carboxylate, sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-l -carboxylate, m-maleimidobenzoyl-N- hydroxysuccinimide ester, m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester, N- succinimidyl(4-iodoacetyl)aminobenzoate, sulfosuccinimidyl(4-iodoacetyl)aminobenzoate, succinimidyl 4-(p-maleimidophenyl)butyrate, sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate, l-ethyl-3-(3-dimethylarninopropyl)carbodiimide hydrochloride, N,N'-phenylene dimal eimide, and compounds belonging to the bisimidate class, the acyl diazide class or the aryl dihalide class, and combinations thereof.

[0123] The albumin is allowed to polymerize with the cross-linker for a suitable period of time to obtain polymerized albumin having the desired MW. For example, the polymerization reaction may be incubated at about 37° C. for between 1 and 4 hours. The polymerization reaction is then quenched with a molar excess of reducing agent, preferably a strong reducing agent that is capable of reducing the Schiff bases in the polymerized albumin and any remaining free aldehyde groups on the cross-linker. For example, the reaction may be quenched by incubating the reaction mixture with a 1 M sodium borohydride solution for 30 min at 37° C. Quenching the Schiff bases in the polymerized albumin stabilizes the polymer and prevents the hydrolysis of polymerized albumin back to monomeric albumin, which could extravasate and cause detrimental side effects. Moreover, reducing the aldehyde group on the cross-linker completely quenches the polymerization reaction. An exemplary strong reducing agent capable for use in embodiments of the invention is sodium borohydride, however it is understood that other reducing agents may be useful as well.

[0124] The MW distribution of the polymerized albumin in the quenched reaction mixture will be affected by the conditions under which the polymerization reaction is conducted, such as duration and temperature of the incubation along with the cross-linker to albumin molar ratio. To control for variables in the polymerization reaction that might result in polymerized albumin having a MW outside of the desired range, the process further includes the step of collecting polymerized albumin having the desired MW range. The collecting step may include separating or purifying polymerized albumin having the desired MW range or making the polymerized albumin free from undesirable elements such as albumin having a MW outside of the desired range. For example, the polymerized albumin solution may be clarified such as by being passed through a glass chromatography column packed with glass wool to remove large particles. The clarified polymerized albumin solution is then separated into distinct molecular mass fractions using known separation methods such as passing the clarified polymerized albumin solution through a tangential flow filtration (TFF) hollow fiber (HF) cartridge selected to collect polymerized albumin having the desired MW. For example, fractionation of the polymerized albumin composition with a 100 kDa TFF HF cartridge (Spectrum Labs, Rancho Dominguez, Calif.) will result in the retentate containing polymerized albumin molecules that are at least 100 kDa or larger and that fall within the desired MW in one embodiment. In that example, the filtrate will mostly contain polymerized albumin molecules that are smaller than 100 kDa, i.e., molecules that are smaller than the desired MW. The MW of the polymerized albumin can be controlled by passing the clarified polymerized albumin solution through TFF HF cartridges having different pore sizes selective for the desired MW.

[0125] The polymerized albumin solution may then be subjected to as many cycles of diafiltration with an appropriate buffer as needed in order to remove impurities having a MW outside of the desired range. The polymerized albumin solution may also buffer exchanged to remove impurities such as unpolymerized cross-linkers and quenching agents which may be cytotoxic. After separation of the desired fraction, the filtrate may subsequently be concentrated such as with a 100 kDa TFF HF cartridge (Spectrum Labs). The MW distribution of the polymerized albumin may be confirmed by known methods such as SDS-PAGE analysis or size exclusion chromatography coupled with multi-angle static light scattering.

[0126] In some embodiments, the polymerized albumin is covalently modified with a polymer. In some embodiments, the polymer comprises a polyalkylene oxide, such as polyethylene glycol (PEG). In other embodiments, the polymer comprises a zwitterionic polymer. In some embodiments, the polymer has a molecular weight of from 200 Da to 1,000,000 Da, such as from 1,000 Da to 10,000 Da, or from 3,000 Da to 6,000 Da, or about 5,000 Da.

[0127] In some embodiments, the polymerized albumin is covalently modified with the polymer in a process that comprises: conjugating one or more polymers to the polymerized albumin to form a polymer-modified polymerized albumin; filtering the polymer-modified polymerized albumin by ultrafiltration against a first filtration membrane having a pore size that separates low molecular weight contaminants from the polymer-modified polymerized albumin; and filtering the polymer-modified polymerized albumin by ultrafiltration against a second filtration membrane having a pore size that separates the high molecular weight contaminants from the polymer-modified polymerized albumin. In some embodiments, the ultrafiltration comprises tangential-flow filtration. In some embodiments, the first filtration membrane is rated for retaining solutes having a molecular weight greater than 100 kDa, thereby forming a retentate fraction comprising the polymer-modified polymerized albumin and a permeate fraction comprising low molecular weight contaminants having a molecular weight less than 100 kDa. In some embodiments, the second filtration membrane is rated for retaining solutes having a molecular weight greater than 500 kDa, thereby forming a retentate fraction comprising the high molecular weight contaminants and a permeate fraction comprising the polymer-modified polymerized albumin. In other embodiments, the second filtration membrane has a pore size of 0.2 pm, thereby forming a retentate fraction comprising the high molecular weight contaminants and a permeate fraction comprising the polymer-modified polymerized albumin.

[0128] In some embodiments, the first filtration membrane is rated for retaining solutes having a molecular weight greater than 500 kDa, thereby forming a retentate fraction comprising the polymer-modified polymerized albumin and a permeate fraction comprising low molecular weight contaminants having a molecular weight less than 500 kDa. In some embodiments, the second filtration membrane has a pore size of 0.2 pm, thereby forming a retentate fraction comprising the high molecular weight albumin species and a permeate fraction comprising the polymerized albumin.

[0129] In certain embodiments, the polymerized albumin is covalently modified with a polyethylene glycol (PEG) polymer. In some embodiments, the PEG polymer can have a molecular weight of from 200 Da to 20,000 Da, such as from 1,000 Da to 10,000 Da, or from 3,000 Da to 6,000 Da, or about 5,000 Da.

[0130] In some embodiments, the polymerized albumin is covalently modified with the PEG polymer in a process that includes: conjugating one or more PEG polymers to the polymerized albumin to form PEGylated polymerized albumin; filtering the PEGylated polymerized albumin by ultrafiltration against a first filtration membrane having a pore size that separates low molecular weight contaminants from the PEGylated polymerized albumin; and filtering the PEGylated polymerized albumin by ultrafiltration against a second filtration membrane having a pore size that separates the high molecular weight contaminants from the PEGylated polymerized albumin.

[0131] In some embodiments, conjugating the one or more polyethylene glycol (PEG) polymers to the polymerized albumin to form the PEGylated polymerized albumin can comprise contacting the polymerized albumin with a derivatized PEG under conditions permitting formation of a covalent bond between the PEG and the polymerized albumin so as to form the PEGylated polymerized albumin. In some examples, the derivatized PEG can comprise succinimidyl-PEG, cyanuric chloride-PEG, or maleimide-PEG. In certain examples, conjugating the one or more polyethylene glycol (PEG) polymers to the polymerized albumin to form the PEGylated polymerized albumin can further comprise contacting the PolyHSA with a thiolation reagent, such as 2-iminothiolane hydrochloride, to introduce thiol moieties that react with the derivatized PEG.

[0132] Methods of Use

[0133] Described herein are methods of treating hypercytokinemia in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a composition described herein to reduce circulating cytokine levels by at least 5% compared to an untreated subject (e.g., at least 10%, at least 15%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, or at least 65%). In some embodiments, the method comprising administering to the subject a therapeutically effective amount of a composition described herein to reduce circulating cytokine levels by 70% or less compared to an untreated subject, (e.g., 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, or 25% or less, or 20% or less, or 15% or less, or 10% or less).

[0134] The method comprising administering to the subject a therapeutically effective amount of a composition described herein to reduce circulating cytokine levels can range from any of the minimum values described above to any of the maximum values described above. For example, the method comprising administering to the subject a therapeutically effective amount of a composition described herein to reduce circulating cytokine levels can range from 5% to 70% compared to an untreated subject, (e.g., from 5% to 60%, from 5% to 50%, from 5% to 40%, 5% to 30%, from 5% to 20%, from 5% to 10%, from 10% to 70%, from 10% to 60%, from 10% to 50%, from 10% to 40%, 10% to 30%, from 10% to 20%, 20% to 60%, from 20% to 50%, from 20% to 40%, 20% to 30%, from 30% to 70%, from 30% to 60%, from 30% to 50%, from 30% to 40%, from 40% to 70%, from 40% to 60%, from 40% to 50%, from 50% to 70%, from 50% to 60%, or from 60% to 70%).

[0135] The hypercytokinemia can be associated with an infectious or non-infectious etiology. For example, in some embodiments, the hypercytokinemia can be induced by an infectious agent such as influenza (e.g., H1N1 influenza or H5N1 influenza), coronavirus infection (e.g., avian coronavirus (IBV), porcine coronavirus HKU15 (PorCoV HKU15), Porcine epidemic diarrhea virus (PEDV), HCoV-229E, HCoV-OC43, HCoV-HKUl, HCoV-NL63, SARS-CoV, SARS- CoV-2, or MERS-CoV), Influenza B, Parainfluenza virus, Ebola, Epstein-Barr virus, cytomegalovirus, or group A streptococcus. The hypercytokinemia can also be associated with a non-infectious condition such as graft-versus-host disease.

[0136] Also described herein are methods of preventing hypercytokinemia in a subject comprising administering to the subject a therapeutically effective amount of a composition described herein to reduce or prevent an increase circulating cytokine levels. In some embodiments, the subject is infected with or has been exposed to an infectious agent such as influenza (e.g., H1N1 influenza or H5N1 influenza), coronavirus infection (e.g., avian coronavirus (IBV), porcine coronavirus HKU15 (PorCoV HKU15), Porcine epidemic diarrhea virus (PEDV), HCoV-229E, HCoV-OC43, HCoV-HKUl, HCoV-NL63, SARS-CoV, SARS- CoV-2, or MERS-CoV), Influenza B, Parainfluenza virus, Ebola, Epstein-Barr virus, cytomegalovirus, or group A streptococcus. In some embodiments, the subject has received or will receive transplanted cells, transplanted tissue, a transplanted organ, or any combination thereof. In some embodiments, the transplanted cells, transplanted tissue, a transplanted organ, or any combination thereof comprise an allograft or a xenograft.

[0137] Also described herein are methods of preventing, protecting and / or treating endothelial dysfunction comprising administering to the subject a therapeutically effective amount of a composition described herein to reduce circulating levels of a biomarker for endothelial dysfunction in the subject.

[0138] In some embodiments, described herein are methods of treating endothelial dysfunction in a subject comprising administering to the subject a therapeutically effective amount of a composition described herein to reduce circulating levels of a biomarker for endothelial dysfunction in the subject. In some embodiments, described herein are methods of treating endothelial dysfunction in a subject comprising administering to the subject a therapeutically effective amount of a composition described herein to reduce or prevent an increase in circulating levels of a biomarker for endothelial dysfunction in the subject.

[0139] In some embodiments, the composition described herein can be administered in an effective amount to prevent circulating levels of the biomarker for endothelial dysfunction rising above normal levels for subjects without endothelial dysfunction. In some embodiments, circulating biomarkers for endothelial dysfunction can include but are not limited to angiopoietin-1, angiopoietin-2, syndecan-1, von Willebrand factor (vWF), thrombomodulin, thrombospondin-2, circulating endothelial cells (CEC) and circulating endothelial progenitor cells (or in general CEC expressing the membrane glycoprotein CD146), E-selectin (selectin family expressed on the surface of endothelial cells), ICAM-1 and VCAM-1 (endothelial ligands for leukocytes and platelets), and endothelial microparticles (EMP). In some embodiments, the circulating biomarkers for endothelial dysfunction van be syndecan-1. Circulating biomarkers for endothelial dysfunction can be measured from a blood sample.

[0140] In some embodiments, the methods of preventing, protecting and / or treating endothelial dysfunction can include administering to the subject a therapeutically effective amount of a composition described herein to reduce endothelial barrier permeability by at least at least 5%, (e.g., at least 10%, at least 20%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, or at least 65%). In some embodiments, the methods of preventing, protecting and / or treating endothelial dysfunction can include administering to the subject a therapeutically effective amount of a composition described herein to reduce endothelial barrier permeability by 70% or less, (e.g., 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 20% or less, or 10% or less). The methods of preventing, protecting and / or treating endothelial dysfunction can include administering to the subject a therapeutically effective amount of a composition described herein to reduce endothelial barrier permeability ranging from any of the minimum values described above to any of the maximum values described above. For example, the methods of preventing, protecting and / or treating endothelial dysfunction can include administering to the subject a therapeutically effective amount of a composition described herein to reduce endothelial barrier permeability can range from 5% to 70%, (e.g., from 5% to 60%, from 5% to 50%, from 5% to 40%, 5% to 30%, from 5% to 20%, from 5% to 10%, from 10% to 70%, from 10% to 60%, from 10% to 50%, from 10% to 40%, 10% to 30%, from 10% to 20%, 20% to 60%, from 20% to 50%, from 20% to 40%, 20% to 30%, from 30% to 70%, from 30% to 60%, from 30% to 50%, from 30% to 40%, from 40% to 70%, from 40% to 60%, from 40% to 50%, from 50% to 70%, from 50% to 60%, or from 60% to 70%).

[0141] In some embodiments, the endothelial barrier permeability is reduced compared to the endothelial barrier permeability in a subject not treated with the composition described herein. In some embodiments, the endothelial barrier permeability is reduced compared to the endothelial barrier permeability in the subject prior to treatment with the composition described herein. In some embodiments, the composition prevents the endothelial barrier permeability.

[0142] In some embodiments, the methods of preventing, protecting and / or treating endothelial dysfunction can include administering to the subject a therapeutically effective amount of a composition described herein to protect endothelial tissue from damage. In some embodiments, the methods of preventing, protecting and / or treating endothelial dysfunction can include administering to the subject a therapeutically effective amount of a composition described herein to reduce inflammatory immune response by at least 5% (e.g., at least 10%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, or at least 65%). In some embodiments, the methods of preventing, protecting and / or treating endothelial dysfunction can include administering to the subject a therapeutically effective amount of a composition described herein to reduce inflammatory immune response by 70% or less (e.g., 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, or 25% or less, 15% or less, or 10% or less).

[0143] The methods of preventing, protecting and / or treating endothelial dysfunction can include administering to the subject a therapeutically effective amount of a composition described herein to reduce inflammatory immune response can range from any of the minimum values described above to any of the maximum values described above. For example, the methods of preventing, protecting and / or treating endothelial dysfunction can include administering to the subject a therapeutically effective amount of a composition described herein to reduce inflammatory immune response can range from 5% to 70%, (e.g., from 5% to 60%, from 5% to 50%, from 5% to 40%, 5% to 30%, from 5% to 20%, from 5% to 10%, from 10% to 70%, from 10% to 60%, from 10% to 50%, from 10% to 40%, 10% to 30%, from 10% to 20%, 20% to 60%, from 20% to 50%, from 20% to 40%, 20% to 30%, from 30% to 70%, from 30% to 60%, from 30% to 50%, from 30% to 40%, from 40% to 70%, from 40% to 60%, from 40% to 50%, from 50% to 70%, from 50% to 60%, or from 60% to 70%). In some embodiments, the inflammatory immune response is reduced compared to the inflammatory immune response in a subject not treated with the composition described herein. In some embodiments, the inflammatory immune response is reduced compared to the inflammatory immune response in the subject prior to treatment with the composition described herein. In some embodiments, the composition prevents an increase in circulating biomarkers for endothelial dysfunction, which can be measured from a blood sample, comprise angiopoietin-1, angiopoietin-2, syndecan-1, vWF, thrombomodulin, thrombospondin-2, CEC, E-selectin, ICAM-1 and VCAM-1, and / or EMP.

[0144] In some embodiments, the methods of preventing, protecting and / or treating endothelial dysfunction can include administering to the subject a therapeutically effective amount of a composition described herein to reduce the number of leukocytes adhered to an endothelial tissue by at least 5%, (e.g., at least 10%, at least 15%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%). In some embodiments, the methods of preventing, protecting and / or treating endothelial dysfunction can include administering to the subject a therapeutically effective amount of a composition described herein to reduce the number of leukocytes adhered to an endothelial tissue by 80% or less, (e.g, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, or 10% or less).

[0145] The methods of preventing, protecting and / or treating endothelial dysfunction can include administering to the subject a therapeutically effective amount of a composition described herein to reduce the number of leukocytes adhered to an endothelial tissue can range from any of the minimum values described above to any of the maximum values described above. For example, the methods of preventing, protecting and / or treating endothelial dysfunction can include administering to the subject a therapeutically effective amount of a composition described herein to reduce the number of leukocytes adhered to an endothelial tissue can range from 5% to 80%, (e.g., from 5% to 70%, from 5% to 60%, from 5% to 50%, from 5% to 40%, 5% to 30%, from 5% to 20% from 5% to 10%, from 10% to 80%, from 10% to 70%, from 10% to 60%, from 10% to 50%, from 10% to 40%, from 10% to 30%, from 10% to 20% from 20% to 80%, from 20% to 70%, from 20% to 60%, from 20% to 50%, from 20% to 40%, 20% to 30%, from 30% to 80%, from 30% to 70%, from 30% to 60%, from 30% to 50%, from 30% to 40%, from 40% to 70%, from 40% to 60%, from 40% to 50%, from 50% to 80%, from 50% to 70%, from 50% to 60%, from 60% to 80%, from 60% to 70%, or from 70% to 80%). In some embodiments, the number of leukocytes adhered to an endothelial tissue is reduced compared to the number of leukocytes adhered to an endothelial tissue in a subject not treated with the composition described herein. In some embodiments, the number of leukocytes adhered to an endothelial tissue is reduced compared to the number of leukocytes adhered to an endothelial tissue in the subject prior to treatment with the composition described herein. In some embodiments, the composition prevents an increase in number of leukocytes adhered to an endothelial tissue. Changes in leukocyte activation can be measured from circulating biomarkers for endothelial surface selectins including E-selectin, ICAM-1 and VCAM-1.

[0146] In some embodiments, the preventing, protecting and / or treating endothelial dysfunction can include administering to the subject a therapeutically effective amount of a composition described herein to improve vascular integrity by at least at least 5%, (e.g., at least 10%, at least 20%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, or at least 65%). In some embodiments, the preventing, protecting and / or treating endothelial dysfunction can include administering to the subject a therapeutically effective amount of a composition described herein to improve vascular integrity by 70% or less, (e.g., 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 20% or less, or 10% or less). The preventing, protecting and / or treating endothelial dysfunction can include administering to the subject a therapeutically effective amount of a composition described herein to improve vascular integrity ranging from any of the minimum values described above to any of the maximum values described above. For example, the preventing, protecting and / or treating endothelial dysfunction can include administering to the subject a therapeutically effective amount of a composition described herein to improve vascular integrity can range from 5% to 70%, (e.g., from 5% to 60%, from 5% to 50%, from 5% to 40%, 5% to 30%, from 5% to 20%, from 5% to 10%, from 10% to 70%, from 10% to 60%, from 10% to 50%, from 10% to 40%, 10% to 30%, from 10% to 20%, 20% to 60%, from 20% to 50%, from 20% to 40%, 20% to 30%, from 30% to 70%, from 30% to 60%, from 30% to 50%, from 30% to 40%, from 40% to 70%, from 40% to 60%, from 40% to 50%, from 50% to 70%, from 50% to 60%, or from 60% to 70%). In some embodiments, the vascular integrity is improved compared to the vascular integrity in a subject not treated with the composition described herein. In some embodiments, the vascular integrity is improved compared to the improve vascular integrity in the subject prior to treatment with the composition described herein. Improvements in vascular integrity can be measured by a reduction or normalization of circulating biomarkers for endothelial dysfunction, which can be measured from a blood sample, comprise angiopoietin-1, angiopoietin-2, syndecan-1, vWF, thrombomodulin, thrombospondin-2, CEC, E-selectin, ICAM-1 and VCAM- 1, and / or EMP.

[0147] In some embodiments, the subject can have a normal pressure of circulating blood on the walls of blood vessels (“normal blood pressure”) based on the subjects age, gender, posture, and exercise state. In some embodiments, the subject can have a low pressure of circulating blood on the walls of blood vessels (“low blood pressure”). Normal blood pressure levels are known in the art and may vary slightly depending on the subject’s age, gender, posture, and exercise state.

[0148] In some embodiments, the compositions described herein can be administered in an effective amount to reduce extravasation through the endothelium, resulting in the maintenance of intravascular oncotic pressure. In some embodiments, the administration of compositions described herein can stabilize intravascular oncotic pressure for a period of at least 24 hours.

[0149] In some embodiments, the compositions described herein can also be useful to treat clinical conditions such as hypercytokinemia, inflammatory immune response, endothelial dysfunction, multiorgan dysfunction syndrome, endotoxemia, sepsis or combinations thereof. The compositions described herein could be used to treat vascular leakage due to inflammation and fibrosis such as diabetes, chronic inflammation, brain edema, arthritis, uvietis, macular edema, cancer, hyperglycemia, a kidney inflammatory disease, a disorder resulting in kidney fibrosis, a disorder of the kidney resulting in proteinuria, sepsis, or combinations thereof. PolyHSA’s ability to preserve the glycocalyx may also indicate its use as an agent to increase plasma viscosity (and thus endothelial shear stress) following ischemia, such as after surgery, stroke, myocardial infarction, or extended tourniquet application, or during states of hypercoagulability, in order to prevent the microvascular dysfunction that frequently occurs due to ischemia injury.

[0150] In some embodiments, the conditions mentioned may be caused by an infectious agent such as influenza (e.g., H1N1 influenza or H5N1 influenza), coronavirus infection (e.g., avian coronavirus (IBV), porcine coronavirus HKU15 (PorCoV HKU15), Porcine epidemic diarrhea virus (PEDV), HCoV-229E, HCoV-OC43, HCoV-HKUl, HCoV-NL63, SARS-CoV, SARS- CoV-2, or MERS-CoV), Influenza B, Parainfluenza virus, Ebola, Epstein-Barr virus, cytomegalovirus, or group A streptococcus.

[0151] Methods of Administration

[0152] The compositions as used in the methods described herein can be administered by any suitable method and technique presently or prospectively known to those skilled in the art. For example, the active components described herein can be formulated in a physiologically- or pharmaceutically acceptable form. In some embodiments, the composition described herein can be administered via infusion or exchange transfusion into the circulatory system of a subject, such as intravenous or intraarterial through a catheter.

[0153] In some embodiments, the composition described herein can also be mixed with blood compositions, which includes whole blood, red blood cells, plasma, blood fractions, crystalloid solutions, or plasma expanders (PEs), or any combination thereof prior to infusion or exchange transfusion into the subject.

[0154] Compositions, as described herein, comprising an active compound (e.g., PolyHSA and / or PEGylated PolyHSA) and an excipient of some sort may be useful in a variety of medical and non-medical applications.

[0155] “Excipients” include any and all solvents, diluents or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. General considerations in formulation and / or manufacture can be found, for example, in Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980), and Remington: The Science and Practice of Pharmacy, 21st Edition (Lippincott Williams & Wilkins, 2005). Exemplary excipients include, but are not limited to, any non-toxic, inert solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Some examples of materials which can serve as excipients include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; detergents such as Tween 80; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator. As would be appreciated by one of skill in this art, the excipients may be chosen based on what the composition is useful for. For example, with a pharmaceutical composition or cosmetic composition, the choice of the excipient will depend on the route of administration, the agent being delivered, time course of delivery of the agent, etc., and can be administered to humans and / or to animals, orally, rectally, parenterally, intraci stemally, intravaginally, intranasally, intraperitoneally, topically (as by powders, creams, ointments, or drops), buccally, or as an oral or nasal spray. In some embodiments, the active compounds disclosed herein are administered topically.

[0156] Exemplary diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc., and combinations thereof.

[0157] Exemplary granulating and / or dispersing agents include potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross- linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, etc., and combinations thereof.

[0158] Exemplary surface active agents and / or emulsifiers include natural emulsifiers (e.g. acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g. bentonite [aluminum silicate] and Veegum [magnesium aluminum silicate]), long chain amino acid derivatives, high molecular weight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxy vinyl polymer), carrageenan, cellulosic derivatives (e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylene sorbitan monolaurate [Tween 20], polyoxyethylene sorbitan [Tween 60], polyoxyethylene sorbitan monooleate [Tween 80], sorbitan monopalmitate [Span 40], sorbitan monostearate [Span 60], sorbitan tristearate [Span 65], glyceryl monooleate, sorbitan monooleate [Span 80]), polyoxyethylene esters (e.g. polyoxyethylene monostearate [Myrj 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and Solutol), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g. Cremophor), polyoxyethylene ethers, (e.g. polyoxyethylene lauryl ether [Brij 30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic F 68, Pol oxamer 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, etc. and / or combinations thereof. Exemplary binding agents include starch (e.g. cornstarch and starch paste), gelatin, sugars (e.g. sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol, etc.), natural and synthetic gums (e.g. acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum), and larch arabogalactan), alginates, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylates, waxes, water, alcohol, etc., and / or combinations thereof.

[0159] Exemplary preservatives include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and other preservatives. Exemplary antioxidants include alpha tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxy anisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite.

[0160] Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA) and salts and hydrates thereof (e.g., sodium edetate, disodium edetate, trisodium edetate, calcium disodium edetate, dipotassium edetate, and the like), citric acid and salts and hydrates thereof (e.g., citric acid monohydrate), fumaric acid and salts and hydrates thereof, malic acid and salts and hydrates thereof, phosphoric acid and salts and hydrates thereof, and tartaric acid and salts and hydrates thereof. Exemplary antimicrobial preservatives include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.

[0161] Exemplary antifungal preservatives include butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid.

[0162] Exemplary alcohol preservatives include ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and phenylethyl alcohol.

[0163] Exemplary acidic preservatives include vitamin A, vitamin C, vitamin E, beta- carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid. Other preservatives include tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, Glydant Plus, Phenonip, methylparaben, Germall 115, Germaben II, NeoIone, Kathon, and Euxyl. In certain embodiments, the preservative is an antioxidant. In other embodiments, the preservative is a chelating agent.

[0164] Exemplary buffering agents include citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen- free water, isotonic saline, Ringer's solution, ethyl alcohol, etc., and combinations thereof.

[0165] Exemplary lubricating agents include magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, etc., and combinations thereof.

[0166] Exemplary natural oils include almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, chamomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, and wheat germ oils. Exemplary synthetic oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyl dodecanol, oleyl alcohol, silicone oil, and combinations thereof.

[0167] Additionally, the composition may further comprise a polymer. Exemplary polymers contemplated herein include, but are not limited to, cellulosic polymers and copolymers, for example, cellulose ethers such as methylcellulose (MC), hydroxyethylcellulose (ELEC), hydroxypropyl cellulose (HPC), hydroxypropyl methyl cellulose (HPMC), methylhydroxyethylcellulose (MHEC), methylhydroxypropylcellulose (MHPC), carboxymethyl cellulose (CMC) and its various salts, including, e.g., the sodium salt, hydroxy ethylcarboxymethylcellulose (HECMC) and its various salts, carboxymethylhydroxyethylcellulose (CMHEC) and its various salts, other polysaccharides and polysaccharide derivatives such as starch, dextran, dextran derivatives, chitosan, and alginic acid and its various salts, carageenan, various gums, including xanthan gum, guar gum, gum arabic, gum karaya, gum ghatti, konjac and gum tragacanth, glycosaminoglycans and proteoglycans such as hyaluronic acid and its salts, proteins such as gelatin, collagen, albumin, and fibrin, other polymers, for example, polyhydroxyacids such as polylactide, polyglycolide, polyl(lactide-co- glycolide) and poly(. epsilon. -caprolactone-co-glycolide)-, carboxyvinyl polymers and their salts (e.g., carbomer), polyvinylpyrrolidone (PVP), polyacrylic acid and its salts, polyacrylamide, polyacrylic acid / acrylamide copolymer, polyalkylene oxides such as polyethylene oxide, polypropylene oxide, poly(ethylene oxide- propylene oxide), and a Pluronic polymer, polyoxy ethylene (polyethylene glycol), polyanhydrides, polyvinylalchol, polyethyleneamine and polypyrridine, polyethylene glycol (PEG) polymers, such as PEGylated lipids (e.g., PEG- stearate, l,2-Distearoyl-sn-glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)- 1000], l,2-Distearoyl-sn-glycero-3-Phosphoethanolamine-N-[Methoxy(Poly ethylene glycol)- 2000], and l,2-Distearoyl-sn-glycero-3-Phosphoethanolamine-N-[Methoxy(Poly ethylene glycol)-5000]), copolymers and salts thereof.

[0168] Additionally, the composition may further comprise an emulsifying agent. Exemplary emulsifying agents include, but are not limited to, a polyethylene glycol (PEG), a polypropylene glycol, a polyvinyl alcohol, a poly-N-vinyl pyrrolidone and copolymers thereof, poloxamer nonionic surfactants, neutral water-soluble polysaccharides (e.g., dextran, Ficoll, celluloses), non-cationic poly(meth)acrylates, non-cationic polyacrylates, such as poly (meth) acrylic acid, and esters amide and hydroxy alkyl amides thereof, natural emulsifiers (e.g. acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g. bentonite [aluminum silicate] and Veegum [magnesium aluminum silicate]), long chain amino acid derivatives, high molecular weight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxy vinyl polymer), carrageenan, cellulosic derivatives (e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylene sorbitan monolaurate [Tween 20], polyoxyethylene sorbitan [Tween 60], polyoxyethylene sorbitan monooleate [Tween 80], sorbitan monopalmitate [Span 40], sorbitan monostearate [Span 60], sorbitan tristearate [Span 65], glyceryl monooleate, sorbitan monooleate [Span 80]), polyoxyethylene esters (e.g. polyoxyethylene monostearate [Myrj 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and Solutol), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g. Cremophor), polyoxyethylene ethers, (e.g. polyoxyethylene lauryl ether [Brij 30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic F 68, Poloxamer 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, etc. and / or combinations thereof. In certain embodiments, the emulsifying agent is cholesterol.

[0169] Liquid compositions include emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compound, the liquid composition may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0170] Injectable compositions, for example, injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be an injectable solution, suspension, or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3 -butanediol. Among the acceptable vehicles and solvents for pharmaceutical compositions that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. Any bland fixed oil can be employed including synthetic mono- or di glycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. In certain embodiments, the particles are suspended in a carrier fluid comprising 1% (w / v) sodium carboxymethyl cellulose and 0.1% (v / v) Tween 80. The injectable composition can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0171] In some embodiments, the composition described herein is administered via infusion or exchange transfusion. In some embodiments, the composition described herein is administered via infusion. In some embodiments, the composition described herein is administered via exchange transfusion

[0172] In some embodiments, when the composition described herein is administered via infusion the volume varies depending on the condition in the subject. In some embodiments, the infusion includes infusion of a volume of a composition described herein, and wherein the volume includes at least 10% of the subject’s total blood volume (e.g., at least 15%, at least 20%, at least 25%). In some embodiments, the infusion includes infusion of a volume of the composition described herein, and wherein the volume includes 30% or less of the subject’s total blood volume (e.g., 25% or less, 20% or less, or 15% or less).

[0173] The infusion includes infusion of a volume of a composition described herein, and wherein the volume includes a range from any of the minimum values described above to any of the maximum values described above. For example, in some embodiments, the infusion includes infusion of a volume of a composition described herein, and wherein the volume includes a range from 10% to 30% of the subject’s total blood volume (e.g., from 10% to 25%, from 10% to 20%, from 10% to 15%, from 15% to 20%, from 15% to 25%, from 15% to 30%, from 20% to 25%, from 20% to 30%, or from 25% to 30%). In some embodiments, the infusion includes infusion of a volume of a composition described herein, and wherein the volume includes from 10% to 30% of the subject’s total blood volume.

[0174] In some embodiments, when the composition described herein is administered via exchange transfusion the exchanged transfusion volume varies depending on the condition in the subject. In some embodiments, the exchange transfusion includes exchange transfusion of at least 5% of the subject’s total blood volume with a composition described herein (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45%). In some embodiments, the exchange transfusion includes exchange transfusion of 90% or less of the subject’s total blood volume with a composition described herein (e.g., 90% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less). In some embodiments, the exchange transfusion includes exchange transfusion of 50% or less of the subject’s total blood volume with a composition described herein.

[0175] The exchange transfusion includes exchange transfusion ranging from any of the minimum values described above to any of the maximum values described above. For example, in some embodiments, the exchange transfusion includes exchange transfusion ranging from 5% to 90% of the subject’s total blood volume with a composition described herein (e.g, from 5% to 45%, from 5% to 40%, from 5% to 35%, from 5% to 30%, from 5% to 25%, from 5% to 20%, from 5% to 15%, from 5% to 10%, from 10% to 50%, from 10% to 45%, from 10% to 40%, from 10% to 35%, from 10% to 30%, from 10% to 25%, from 10% to 20%, from 10% to 15%, from 15% to 20%, from 15% to 25%, from 15% to 30%, from 15% to 35%, from 15% to 40%, from 15% to 45%, from 15% to 50%, from 20% to 25%, from 20% to 30%, from 25% to 30%, from 25% to 35%, from 25% to 40%, from 25% to 45%, from 25% to 50%, from 30% to 35%, from 30% to 40% from 30% to 45%, from 30% to 50%, from 35% to 40%, from 35% to 45%, from 35% to 50%, from 40% to 45%, from 40% to 50%, from 45% to 50%, or from 50% to 90%). In some embodiments, the exchange transfusion includes exchange transfusion of from 5% to 90% of the subject’s total blood volume with a composition described herein.

[0176] Administration of the compositions can be a single administration, or at continuous and distinct intervals as can be readily determined by a person skilled in the art.

[0177] The composition may be administered in such amounts, time, and route deemed necessary in order to achieve the desired result. The exact amount of the active ingredient (e.g., PolyHSA and / or PEGylated PolyHSA) will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the infection, the particular active ingredient, its mode of administration, its mode of activity, and the like. The active ingredient, whether the active compound itself, or the active compound in combination with an agent, is preferably formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the active ingredient will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the active ingredient employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific active ingredient employed; the duration of the treatment; drugs used in combination or coincidental with the specific active ingredient employed; and like factors well known in the medical arts.

[0178] The exact amount of an active ingredient required to achieve a therapeutically or prophylactically effective amount will vary from subject to subject, depending on species, age, and general condition of a subject, severity of the side effects or disorder, identity of the particular compound(s), mode of administration, and the like. The amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult.

[0179] Useful dosages of the active agents and pharmaceutical compositions disclosed herein can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art.

[0180] The dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms or disorder are affected. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any counterindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days.

[0181] In some embodiments, the compound as used in the methods described herein may be administered in combination or alternation with one or more additional active agents. Representative examples additional active agents include antimicrobial agents (including antibiotics, antiviral agents and anti-fungal agents), anti-inflammatory agents (including steroids and non-steroidal anti-inflammatory agents), anti-coagulant agents, immunomodulatory agents, anticytokine, antiplatelet agents, and antiseptic agents.

[0182] Representative examples of antibiotics include amikacin, amoxicillin, ampicillin, atovaquone, azithromycin, aztreonam, bacitracin, carbenicillin, cefadroxil, cefazolin, cefdinir, cefditoren, cefepime, cefiderocol, cefoperazone, cefotetan, cefoxitin, cefotaxime, cefpodoxime, cefprozil, ceftaroline, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, chloramphenicol, colistimethate, cefuroxime, cephalexin, cephradine, cilastatin, cinoxacin, ciprofloxacin, clarithromycin, clindamycin, dalbavancin, dalfopristin, daptomycin, demeclocycline, dicloxacillin, doripenem, doxycycline, eravacycline, ertapenem, erythromycin, fidaxomicin, fosfomycin, gatifloxacin, gemifloxacin, gentamicin, imipenem, lefamulin, lincomycin, linezolid, lomefloxacin, loracarbef, meropenem, metronidazole, minocycline, moxifloxacin, nafcillin, nalidixic acid, neomycin, norfloxacin, ofloxacin, omadacycline, oritavancin, oxacillin, oxytetracycline, paromomycin, penicillin, pentamidine, piperacillin, plazomicin, quinupristin, rifaximin, sarecycline, secnidazole, sparfloxacin, spectinomycin, sulfamethoxazole, sulfisoxazole, tedizolid, telavancin, telithromycin, ticarcillin, tigecycline, tobramycin, trimethoprim, trovafloxacin, and vancomycin.

[0183] Representative examples of antiviral agents include, but are not limited to, abacavir, acyclovir, adefovir, amantadine, amprenavir, atazanavir, balavir, baloxavir marboxil, boceprevir, cidofovir, cobicistat, daclatasvir, darunavir, delavirdine, didanosine, docasanol, dolutegravir, doravirine, ecoliever, edoxudine, efavirenz, elvitegravir, emtricitabine, enfuvirtide, entecavir, etravirine, famciclovir, fomivirsen, fosamprenavir, forscamet, fosnonet, famciclovir, favipravir, fomivirsen, foscavir, ganciclovir, ibacitabine, idoxuridine, indinavir, inosine, inosine pranobex, interferon type I, interferon type II, interferon type III, lamivudine, letermovir, letermovir, lopinavir, loviride, maraviroc, methisazone, moroxydine, nelfinavir, nevirapine, nitazoxanide, oseltamivir, peginterferon alfa-2a, peginterferon alfa-2b, penciclovir, peramivir, pleconaril, podophyllotoxin, pyramidine, raltegravir, remdesevir, ribavirin, rilpivirine, rimantadine, rintatolimod, ritonavir, saquinavir, simeprevir, sofosbuvir, stavudine, tarabivirin, telaprevir, telbivudine, tenofovir alafenamide, tenofovir disoproxil, tenofovir, tipranavir, trifluridine, trizivir, tromantadine, umifenovir, valaciclovir, valganciclovir, vidarabine, zalcitabine, zanamivir, and zidovudine.

[0184] Representative examples of anticoagulant agents include, but are not limited to, heparin, warfarin, rivaroxaban, dabigatran, apixaban, edoxaban, enoxaparin, and fondaparinux.

[0185] Representative examples of antiplatelet agents include, but are not limited to, clopidogrel, ticagrelor, prasugrel, dipyridamole, dipyridamole / aspirin, ticlopidine, and eptifibatide.

[0186] Representative examples of antifungal agents include, but are not limited to, voriconazole, itraconazole, posaconazole, fluconazole, ketoconazole, clotrimazole, isavuconazonium, miconazole, caspofungin, anidulafungin, micafungin, griseofulvin, terbinafine, flucytosine, terbinafine, nystatin, and amphotericin b.

[0187] Representative examples of steroidal anti-inflammatory agents include, but are not limited to, hydrocortisone, dexamethasone, prednisolone, prednisone, triamcinolone, methylprednisolone, budesonide, betamethasone, cortisone, and deflazacort. Representative examples of non-steroidal anti-inflammatory drugs include ibuprofen, naproxen, ketoprofen, tolmetin, etodolac, fenoprofen, flurbiprofen, diclofenac, piroxicam, indomethacin, sulindax, meloxicam, nabumetone, oxaprozin, mefenamic acid, and diflunisal.

[0188] Other examples of additional active agents include chloroquine, hydrochloroquine, Vitamin D, and Vitamin C.

[0189] A number of embodiments of the disclosure have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

[0190] By way of non-limiting illustration, examples of certain embodiments of the present disclosure are given below.

[0191] EXAMPLES

[0192] The following examples are set forth below to illustrate the methods and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention, which are apparent to one skilled in the art. Efforts have been made to ensure accuracy with respect to numbers e.g., amounts, temperature, etc.) but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric. There are numerous variations and combinations of reaction conditions, e.g., component concentrations, temperatures, pressures and other reaction ranges and conditions that can be used to optimize the product purity and yield obtained from the described process. Only reasonable and routine experimentation will be required to optimize such process conditions.

[0193] Example 1. Tangential Flow Filtration Facilitated Fractionation of Polymerized Human Serum Albumin: Insights into the Effects of Molecular Size on Biophysical Properties

[0194] Overview

[0195] Human serum albumin (HSA) is currently used as a plasma expander (PE) to increase blood volume during hypovolemic conditions (i.e., blood loss), but is suboptimal for treating patients suffering from septic shock and bums due to the enhanced permeability of the endothelium and subsequent extravasation of HSA from the blood volume into the tissue space which leads to tissue edema and tissue deposition of toxic metabolites bound to HSA. In order to expand HSA’s applicability towards treating patients with compromised endothelial permeability and to avoid HSA extravasation from the vascular space into the tissue space, HSA can be polymerized to increase its molecular size thus compartmentalizing the polymerized HSA (PolyHSA) molecules in the vascular space. In this example, PolyHSA was synthesized at two cross-link densities (i.e., molar ratio of glutaraldehyde to HSA) and subsequently fractionated via tangential flow filtration into two narrower brackets: bracket A comprising PolyHSA molecules ranging from 500 kDa - 0.2 pm and bracket B comprising PolyHSA molecules ranging from 50 - 500 kDa. PolyHSA within the same size bracket at the 2 different cross-link densities exhibited similar solution osmolality, but differed with respect to their hydrodynamic diameter, zeta potential, and viscosity. Additionally at the same cross-link density, the PolyHSA A bracket exhibited higher viscosity, more negative zeta potential, and larger hydrodynamic diameter compared to the PolyHSA B bracket. Interestingly, PolyHSA 43 : 1 B, PolyHSA 60: 1 A and PolyHSA 60: 1 B brackets exhibited similar colloid osmotic pressure to HSA, which is indicative of their potential to serve as PEs. Future work will assess the in vivo response towards transfusion of these PolyHSA brackets in animal models with normal and impaired endothelial function. Introduction

[0196] To avoid complications associated with HSA administration to patients with compromised endothelial function, the MW / size of HSA can be increased via polymerization to avoid HSA extravasation into the tissue space. For example, a crosslinker, such as glutaraldehyde, can be used to polymerize HSA into polymerized HSA (PolyHSA), which dually increases the molecular diameter of HSA and increases solution viscosity.

[0197] In this Example, PolyHSA was synthesized and bracketed between 500 kDa and 0.2 pm. We tuned the size distribution of PolyHSA molecules via tangential flow filtration (TFF) facilitated fractionation of PolyHSA into two size brackets and evaluated the biophysical properties of the fractions. The first PolyHSA bracket was fractionated between 500 kDa and 0.2 pm (bracket A), while the second bracket was fractionated between 50 kDa and 500 kDa (bracket B). The hydrodynamic diameter, zeta potential, osmolality, viscosity, and COP of the fractionated PolyHSA products were analyzed to determine the in vitro potential of PolyHSA as a PE. Future work will assess the in vivo response towards transfusion of these PolyHSA brackets in animal models with normal and impaired endothelial function.

[0198] Materials and Methods

[0199] Materials: Glutaraldehyde (CsHsCh) (70%), sodium chloride (NaCl), potassium chloride (KC1), sodium hydroxide (NaOH), calcium chloride (CaCh-2H2O), sodium lactate (NaCsHsCh), N-acetyl-L-cysteine (C5H9NO3S), sodium cyanoborohydride (NaCNBHi), sodium phosphate dibasic (Na2HPO4), and sodium phosphate monobasic (Na^PCh) were purchased from Sigma Aldrich (St. Louis, MO). Polyethersulfone (PES) hollow fiber TFF modules with a pore size of 0.2 pm and polysulfone (PS) TFF modules with pore sizes of 500 kDa and 50 kDa were purchased from Repligen (Rancho Dominguez, CA). HSA (AlbuRx® 25, CSL Behring, King of Prussia, PA) was purchased from The Ohio State Outpatient Pharmacy (Columbus, Ohio).

[0200] PolyHSA Synthesis: HSA was diluted to 25 mg / mL in phosphate buffered saline (PBS) to achieve a final volume of 1.5 L in a 2 L reactor. Glutaraldehyde (GA) was added to the HSA reaction vessel at a rate of 2 mL / min to achieve GA:HSA molar ratios of 43 : 1 and 60: 1. The polymerization reaction was allowed to proceed at 37 °C for 3 hours and was quenched with sodium cyanoborohydride at a 3: 1 molar ratio of sodium cyanoborohydride:glutaraldehyde for 30 min. The reaction vessel was then placed in a 4 °C refrigerator overnight. Figure 1 A shows the reaction setup and procedural timeline. The PolyHSA was initially filtered through a 0.2 pm PES TFF module, and underwent 10 diafiltrations against a 500 kDa PS TFF module with a modified lactated Ringer’s solution as described in the literature15. The 500 kDa TFF permeate was then subjected to 10 diafiltrations on a 50 kDa PS TFF module with the modified lactated Ringer’s solution. The two PolyHSA fractions polymerized at the same cross-link density were concentrated to above 100 mg / mL and stored at -80 °C. Figure IB shows the TFF separation train. Bracket A consisted of PolyHSA molecules between 500 kDa and 0.2 pm, while bracket B consisted of PolyHSA molecules between 50 - 500 kDa (Figure IB). PolyHSA at each GA:HSA molar ratio was produced in triplicate.

[0201] PolyHSA Concentration: The PolyHSA mass concentration from a known volume of solution was quantified via its’ dry mass using a Labconco Freeze dryer (Kansas City, MO). Samples were initially frozen to be placed under vacuum. The frozen water from the PolyHSA solution was then removed via sublimation to enable dry mass determination of PolyHSA at the end of the freeze-drying process (48 hours).

[0202] Size Exclusion Chromatography Elution Profile, MW Distribution and Hydrodynamic Diameter: The elution profile of HSA and PolyHSA brackets were measured by size exclusion high-performance liquid chromatography (SEC-HPLC), while the MW distribution of low MW species was assessed via SDS-PAGE, and the hydrodynamic diameter was measured via dynamic light scattering (DLS). SEC-HPLC was performed on a SEC- 1000 column (ThermoFisher Scientific, Walthman, MA) attached to an Ultimate 3000 HPLC / UHPLC system (ThermoFisher Scientific, Walthman, MA). The absorbance at 280 nm was used to measure total protein. For SDS-PAGE, 2 pg of each sample was mixed in a 1 : 1 mass ratio with tris-glycine SDS buffer and incubated at 85 °C for 20 minutes. The mixture was then run on a polyacrylamide gel with 12% resolving gel and 4% stacking gel at 225 V (40 min). The gel was stained with Coomassie blue (1 hour) and was destained (3x; 1 hour) with 30% methanol and 10% acetic acid. The hydrodynamic diameter was measured using a Zetasizer Nano DLS spectrometer (Malvern Instruments, Worcestershire, UK).

[0203] Zeta Potential: The zeta ( ) potential of HSA and PolyHSA fractions was measured using a Zeta Potential Analyzer (ZetaPALS, Brookhaven, Holtsville, NY).

[0204] Osmolality: The osmolality of HSA and PolyHSA solutions at a total protein concentration of 100 mg / mL was measured using an osmometer based on the freezing point depression method (OSMOMAT 030, Berlin, Germany).

[0205] Viscosity: The viscosity of HSA and PolyHSA solutions at a total protein concentration of 100 mg / mL was measured using a Brookfield DV3T rheometer with a CP-40 spindle (Brookfield, Middleboro, MA) at a shear rate of 160 s'1. The material was allowed to equilibrate at 37 °C for five minutes before the measurement was taken. Colloid Oncotic Pressure (COP):

[0206] The COP of HSA and PolyHSA solutions was measured at 100 mg / mL using a Wescor 4420 Colloid Osmometer (Wescor, Logan, UT).

[0207] Circular Dichroism: The secondary structure of HSA and PolyHSA was assessed via circular dichroism (CD) on a JASCO J- 1500 CD spectrometer (JASCO, Easton, MD). Samples were prepared at 3 pM, and measured between 200-250 nm for changes in secondary structure at 25 °C. Protein secondary structure, i.e., a-helical content, was assessed at the 222 nm minima prior to and after protein polymerization.

[0208] Data Analysis: In this Example, all statistical analysis was performed using the Tukey HSD test. Statistical significance was established if p < 0.05. Additionally, all results were presented as the mean ± standard deviation, and all statistical analyses were performed using JMP Pro v 16 (Cary, NC). Comparisons were made between (1) unmodified HSA and all PolyHSA brackets; (2) the same PolyHSA bracket at the 2 different cross-link densities; and (3) the two PolyHSA brackets synthesized at the same cross-link density.

[0209] Results and Discussion

[0210] The biophysical properties of HSA and PolyHSA solutions are summarized in Table 1.

[0211] Table 1. Biophysical properties of HSA and PolyHSA solutions. Viscosity, COP and osmolality were measured at a total protein concentration of 100 mg / mL.

[0212] Hydrodynamic Viscosity Zeta Potential Osmolality COP

[0213] Fraction Diameter (nm) (cP) (mV) (osmol / kg) (mm Hg)

[0214] HSA 5.03 ± 0.15 1.37 ± 0.02 -17.77± 0.15 0.293 ±0.002 41.00 ± 1.00

[0215] 43: 1 A 21.20 ± 0.20 8.95 ± 0.33 -26.08 ± 0.53 0.290 ± 0.005 34.00 ± 1.00

[0216] 43: 1 B 9.87 ± 1.11 5.78 ± 0.17 -21.27 ± 1.62 0.296 ± 0.013 39.67 ± 1.15

[0217] 60: 1 A 29.07± 4.21 9.32 ± 0.80 -28.97 ± 2.48 0.280± 0.010 38.33 ± 3.05

[0218] 60: 1 B 12.00 ± 2.05 5.66 ± 0.50 -19.68 ± 2.60 0.280 ± 0.004 40.00 ± 1.00

[0219] SEC-HPLC: The elution profile of HSA and PolyHSA brackets was analyzed using SEC-HPLC and the results are shown in Figures 2A-2C. HSA was used as a control, and eluted at 9.48 min. All four PolyHSA fractions eluted at earlier times, which was indicative of an increase in HSA MW after polymerization. The presence of unpolymerized HSA in fraction B was observed and is expected, since the lower MW cut-off (MWCO) of the TFF module used to prepare fraction B was 50 kDa, which retains free HSA (MW ~ 67 kDa). PolyHSA 43 : 1 A had an elution time of 8.17 minutes, while PolyHSA 60: 1 A eluted at 7.92 minutes. For the same A fraction (500 kDa-0.2 pm), the MW of the PolyHSA product increased with increasing GA:HSA molar ratio as is evident with the decreased SEC elution time.

[0220] On the other hand, fraction B (50-500 kDa) at both GA:HSA molar ratios displayed two elution peaks corresponding to the presence of two MW distributions. The two observed elution peaks for PolyHSA 43 : 1 B occurred at 9.05 and 8.76 minutes, while PolyHSA 60: 1 B eluted at 8.98 and 8.64 minutes. It is important to note that unpolymerized HSA is retained in the B fraction, which contributes to the wide peaks. Specifically, PolyHSA 43 : 1 B has a significantly higher amount of free HSA compared to PolyHSA 60: 1 B.

[0221] It is important to note that the A and B fractions of PolyHSA 43 : 1 and PolyHSA 60: 1 contain a minor peak eluting at 13.33 mins. This peak is attributed to the stabilizing amino acid, tryptophan, that was formulated into the manufactured HSA. Tryptophan absorbs at the same wavelength as HSA (280 nm), which consequently limited our concentration quantification via UV-visible spectrometry. Therefore, the dry mass approach described in the methods section was used to calculate the final PolyHSA concentration in each bracket. A vial of HSA containing tryptophan was buffer exchanged with PBS using a 50 kDa TFF module for 10 diacycles to remove tryptophan from the HSA solution. The original HSA solution containing tryptophan and the washed HSA were both analyzed via SEC-HPLC to ensure tryptophan removal as shown in Figure 2C. Therefore, the amino acid peak corresponding to tryptophan dramatically decreased in magnitude and was indicative of successful amino acid removal after TFF buffer exchange.

[0222] It is notable that the HSA precursor for PolyHSA synthesis was the manufacturer’s stock solution rather than TFF buffer exchanged HSA. Therefore, tryptophan is not being removed prior to polymerization, but is removed after HSA polymerization via the buffer exchange procedure, which consists of 10 diacycles. As a result, a minor peak was observed in Figures 2A and 2B for the PolyHSA brackets.

[0223] SDS-PAGE: As further confirmation of HSA polymerization, HSA and PolyHSA were analyzed via SDS-PAGE. Figure 3 clearly indicates polymerization did indeed occur due to the smearing of the PolyHSA lanes with PolyHSA species larger than 67 kDa. The HSA control has a strong band above 55 kDa, which is close to its actual MW (~ 67 kDa). HSA is clearly being retained in the PolyHSA B fractions, which corroborates the SEC-HPLC data. In addition, PolyHSA molecules in fraction A are visibly larger than PolyHSA molecules in fraction B, when synthesized at the same molar ratio of glutaraldehyde to HSA. DLS: DLS measurement of the hydrodynamic diameter reinforces the MW distributions from SEC-HPLC and SDS-PAGE analysis. The measurements confirmed a statistically significant increase in the HSA hydrodynamic diameter from 5.03 ± 0.15 nm to 29.07 ± 4.21 nm (p < 0.05) and 12.00 ± 2.05 nm (p < 0.05) for PolyHSA 60: 1 A and PolyHSA 60: 1 B, respectively (Figure 4). Similarly, PolyHSA 43: 1 A displayed a statistically significant increased hydrodynamic diameter of 21.20 ± 0.20 nm compared to HSA (p < 0.05). PolyHSA 43 : 1 A and PolyHSA 60: 1 A are statistically significant in their diameter (p < 0.05). At both fixed GA:HSA molar ratios, the A fraction was statistically significantly larger than the B fraction (p < 0.05). Additionally, the poly dispersity index of all PolyHSA solutions was less than 0.2. These results are overall consistent with SEC-HPLC and SDS-PAGE analysis.

[0224] Zeta Potential: Insights into the potential colloidal stability of PolyHSA were obtained by measuring the zeta potential of PolyHSA in comparison to HSA as a control. The zeta potential results are shown in Figure 5. HSA has a zeta potential of -17.77 ± 0.15 mV, and polymerization statistically decreased the zeta potential to -28.97 ± 2.48 mV (p < 0.05) and - 26.08 ± 0.53 mV (p < 0.05) for PolyHSA 60: 1 A and PolyHSA 43: 1 A, respectively. The PolyHSA B fraction exists between the HSA control and the higher MW PolyHSA A fraction: - 19.68 ± 2.60 mV and -21.27 ± 1.62 mV for PolyHSA 60: 1 B and PolyHSA 43: 1 B, respectively. However, there was no statistical difference in the zeta potential between either PolyHSA B fraction or the HSA control (p < 0.05). On the contrary, at a fixed glutaraldehyde:HSA molar ratio, the A and B fractions at the same GA:HSA molar ratio are significantly different from one another (p < 0.05). It is important to note that the zeta potential is independent of molecular size and is dependent on the surface charge distribution. Since HSA has a hydrophilic exterior and a hydrophobic interior, we can infer that polymerizing HSA results in a more polar surface, and thus results in a more negative zeta potential due to the altered topology and increased surface charge. Furthermore, highly charged polymerized proteins prevent aggregation due to electric repulsion and thus make a colloidally stable product for potential biomedical applications.

[0225] Viscosity: Increasing the GA:HSA molar ratio results in a subsequent increase in PolyHSA molecular size, which has a significant effect on the viscosity of the PolyHSA solution. This is clearly observed in the results shown in Figure 6. As a result of polymerizing the HSA, the PolyHSA solution viscosity significantly increased compared to HSA at a fixed total protein concentration of 100 mg / mL for all PolyHSA brackets (p < 0.05). At the same GA:HSA molar ratio, the viscosity of PolyHSA fraction A was significantly higher than PolyHSA fraction B (p < 0.05). There was no significant difference in viscosity between the same PolyHSA fractions synthesized at different GA:HSA molar ratios. This is expected and can be explained by the increase in molecular interactions and lower dynamics on the molecular scale that results in a more viscous material. The viscosity of the HSA was measured to be 1.37 ± 0.02 cP. The viscosity of the PolyHSA fraction A at both GA:HSA molar ratios ranged between 8-10 cP, and the range for PolyHSA fraction B at both GA:HSA molar ratios was between 5-6 cP. This trend aligns with previous viscosity measurements on PolyHSA solutions.

[0226] Osmolality: The osmolality of the PolyHSA product was measured, and the results summarized in Figure 7. Upon polymerization, the osmolality of all PolyHSA fractions remained unchanged compared to that of unmodified HSA (p < 0.05).

[0227] Colloid Oncotic Pressure (COP): The COP was measured for all PolyHSA brackets as well as unmodified HSA, and the results are shown in Figure 8. The COP of HSA is statistically higher compared to the PolyHSA 43 : 1 A bracket (p < 0.05), and that same bracket is significantly lower compared to the PolyHSA 43: 1 B bracket (p < 0.05). The lack of statistical significance in COP between HSA and PolyHSA 43 : 1 B, PolyHSA 60: 1 A and PolyHSA 60: 1 B brackets is indicative of the ability of those PolyHSA brackets to serve as PEs.

[0228] Circular Dichroism: Changes in PolyHSA secondary structure (i.e., a-helical content) were assessed via circular dichroism spectroscopy. The characteristic signature of a helices includes two minima at 208 nm and 222 nm. Figure 9 shows the circular dichroism spectra of the PolyHSA brackets and unmodified HSA for comparison. While the spectral change is minor in the PolyHSA secondary structure compared to HSA, it is important to observe the directionality of change. Upon polymerization, the minima in the ellipticity decreased for all PolyHSA brackets. This is indicative of an increase in a-helical content, and thus indicates a more stable secondary structure conformation for PolyHSA at both cross-linking densities and sized brackets in comparison to unmodified HSA.

[0229] Conclusion

[0230] HSA was polymerized at two different GA:HSA molar ratios, and the PolyHSA product was separated into two different size fractions for subsequent characterization of their biophysical properties. For the 43: 1 GA:HSA molar ratio PolyHSA brackets, there was an increase in MW, hydrodynamic diameter, viscosity, along with a more negative zeta potential compared to HSA. The PolyHSA 43 : 1 A bracket exhibited a statistically significant lower COP than HSA and the PolyHSA 43 : 1 B bracket. Increasing the GA:HS A molar ratio from 43 : 1 to 60: 1 yielded similar trends. Additionally, PolyHSA 60: 1 A and PolyHSA 60: 1 B brackets exhibited similar COP compared to HSA. At a fixed GA:HSA molar ratio there was a significant difference in MW, size, viscosity, and zeta potential between the PolyHSA A and B brackets. Therefore in summary, PolyHSA 43 : 1 B, PolyHSA 60: 1 A and PolyHSA 60: 1 B brackets can potentially serve as PEs, since they exhibited similar COPs compared to HSA. In future studies, the ability of the PolyHSA fractions to resuscitate animals during hypovolemic conditions will be evaluated.

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[0249] 18. Absolom, D.; Zingg, W .; Neumann, A., Protein adsorption to polymer particles: role of surface properties. Journal of biomedical materials research 1987, 21 (2), 161-171.

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[0254] 23. Anraku, M.; Tsurusaki, Y.; Watanabe, H.; Maruyama, T.; Kragh-Hansen, U.; Otagiri, M., Stabilizing mechanisms in commercial albumin preparations: octanoate and N-acetyl-L- tryptophanate protect human serum albumin against heat and oxidative stress. Biochimica et Biophysica Acta (BB A) -Proteins and Proteomics 2004, 1702 (1), 9-17.

[0255] 24. Anthis, N. J.; Clore, G. M., Sequence-specific determination of protein and peptide concentrations by absorbance at 205 nm. Protein Sci 2013, 22 (6), 851-8.

[0256] 25. Mohammad-Beigi, H.; Shojaosadati, S. A.; Morshedi, D.; Mirzazadeh, N.; Arpanaei, A. The Effects of Organic Solvents on the Physicochemical Properties of Human Serum Albumin Nanoparticles. Iran J Biotechnol 2016, 14 (1), 45-50. 26. Li, Q.; Wang, Z.; Dai, C.; Wang, Y.; Chen, W.; Ju, X.; Yuan, J.; He, R., Physical stability and microstructure of rapeseed protein isolate / gum Arabic stabilized emulsions at alkaline pH. Food Hydrocolloids 2019, 88, 50-57.

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[0260] 30. Greenfield, N. J. Using circular dichroism collected as a function of temperature to determine the thermodynamics of protein unfolding and binding interactions. Nat Protoc 2006, 1 (6), 2527-2535.

[0261] Example 2. Tangential Flow Filtration Facilitated Fractionation and PEGylation of Polymerized Human Serum Albumin and Their Biophysical Properties

[0262] Overview

[0263] Plasma expanders (PEs) are frequently used to restore blood volume during hypovolemic conditions. Human serum albumin (HSA) represents the gold standard PE, due to its native ability to restore blood colloid osmotic pressure (COP). However, HSA is prone to tissue extravasation when used to treat pathophysiological conditions that increase endothelial permeability. Under these conditions, extravasation of HSA from the circulation into the tissue space disrupts the COP balance and drives fluid transport from the vascular space into the tissue space resulting in edema and exposing the tissues to HSA-bound metabolites. Therefore, to enable HSA use under clinical conditions characterized by endothelial dysfunction, the molecular diameter of HSA has previously been increased via chemical polymerization to limit / prevent protein extravasation into the tissue space, and thus retain the polymerized HSA (PHSA) in the vascular space. In this study, the molecular diameter and COP of PHSA was further optimized via polyethylene glycol (PEG) surface conjugation. Previously synthesized and size fractionated PHSA molecules were utilized as the starting material for subsequent PEGylation. PHSA was synthesized at two distinct molar ratios of glutaraldehyde to HSA — 43 : 1 and 60: 1. Each PHSA product was then fractionated via tangential flow filtration (TFF) to yield an A bracket ranging from 500 kDa- 0.2 pm and a B bracket ranging from 50- 500 kDa. Subsequently, each resulting PHSA bracket was then PEGylated with monofunctional 5 kDa maleimide PEG to yield PEGylated PHSA (PPHSA). All PPHSA solutions exhibited increased MW, hydrodynamic diameter, zeta potential, osmolality, and colloid osmotic pressure compared to the parent PHSA (non-PEGylated material). Future work will assess the in vivo response of transfused PPHSA brackets in animal models with normal and impaired endothelial function

[0264] Introduction

[0265] A specific niche of hypovolemic patients experience endothelial dysfunction, which affects their ability to retain human serum albumin (HSA) and fluid in the vascular space. These patients typically suffer from septic shock, ischemia-reperfusion injury and burns, which deteriorates the integrity of the endothelium. In the absence of endothelial dysfunction, hypovolemia is efficiently treated via transfusion of a plasma expander (PE). The most common PE is HSA, which is highly effective at restoring lost blood volume and colloid osmotic pressure (COP). The COP difference between the blood volume and the tissue space determines fluid transport into either compartment. However, under states of endothelial dysfunction, transfusion of HSA promotes its transport into the tissue space, which results in the movement of fluid into the tissue space (edema) via the COP difference between the vascular and tissue spaces.

[0266] Thus, to limit / prevent tissue extravasation of HSA, one strategy is to increase its’ molecular diameter via chemical polymerization in order to prevent its’ filtration through the blood vessel wall. In previous work, polymerized HSA (PHSA) was synthesized via polymerization with the chemical crosslinker glutaraldehyde to increase HSA molecular diameter. Therefore, to further optimize PHSA as a PE, prior work has shown that PEGylation of proteins represents a viable approach for increasing solution COP.

[0267] Therefore, this Example focuses on the synthesis and biophysical characterization of PEGylated PHSA (PPHSA) as a potential PE. To prepare PHSA for PEGylation, the lysine residues on the surface of the PHSA molecule were converted to thiol groups. The thiolated PolyHSA was then allowed to react with monofunctional 5 kDa maleimide-PEG to yield PPHSA.

[0268] This current extends the results from Example 1 — in which PHSA was synthesized at two different glutaraldehyde to HSA molar ratios (43 : 1 and 60: 1) and fractionated the PHSA into two distinct fractions (bracket A: 500 kDa - 0.2 pm; bracket B: 50 kDa - 500 kDa) — by subsequently PEGylating each of the 4 PHSA brackets. The MW, hydrodynamic diameter, zeta potential, osmolality, viscosity and COP of the PPHSA products were evaluated to determine the in vitro potential of PPHSA as a PE compared to PHSA. Future work will assess the in vivo response of transfused PPHSA brackets in animal models with normal and impaired endothelial function.

[0269] Materials and Methods

[0270] Materials: Glutaraldehyde (CsHsCh) (70%), sodium chloride (NaCl), potassium chloride (KC1), sodium hydroxide (NaOH), calcium chloride (CaCh-2H2O), sodium lactate (NaCsHsCh), N-acetyl-L-cysteine (C5H9NO3S), sodium cyanoborohydride (NaCNBHi), sodium phosphate dibasic (Na2HPO4), and sodium phosphate monobasic (NaffcPCh) were purchased from Sigma Aldrich (St. Louis, MO). 2-iminothiolane hydrochloride (IT) was procured from Fisher Scientific (Pittsburgh, PA), and 5 kDa mPEG-maleimide was purchased from Laysan Bio (Arab, AL). Polyethersulfone (PES) hollow fiber tangential flow filtration (TFF) modules with a pore size of 0.2 pm and polysulfone (PS) TFF modules with pore sizes of 500 kDa and 50 kDa were purchased from Repligen (Rancho Dominguez, CA). HSA (AlbuRx® 25, CSL Behring, King of Prussia, PA) was purchased from the Ohio State Outpatient Pharmacy (Columbus, Ohio).

[0271] PHSA Synthesis: HSA was polymerized at two different molar ratios of glutaraldehyde to HSA (43 : 1 and 60: 1). Briefly, HSA was diluted to 25 mg / mL in phosphate buffered saline (PBS) at a final volume of 1.5 L. Glutaraldehyde was added drop wise to the HSA solution at a controlled rate of 2 mL / min to achieve glutaraldehyde to HSA molar ratios of 43 : 1 and 60: 1. The reaction was allowed to proceed for 3 hours at 37 °C with constant stirring, after which it was quenched for 30 mins with sodium cyanoborohydride at a 3: 1 molar ratio of sodium cyanoborohydride:glutaraldehyde. The reaction vessel was then placed in a 4 °C refrigerator overnight. The polymerized product was initially filtered through a 0.2 pm PES TFF module and underwent 10 diafiltrations against a 500 kDa PS TFF module with PBS. The 500 kDa TFF permeate was then subjected to 10 diafiltrations on a 50 kDa PS TFF module with PBS. The 4 PHSA fractions were stored at -80 °C. Bracket A consisted of PHSA molecules between 500 kDa and 0.2 pm, while bracket B consisted of PHSA molecules between 50 - 500 kDa.

[0272] PPHSA Synthesis: Each PHSA fraction was thawed (4 °C) and diluted to 20 mg / mL in PBS to achieve a final volume of 200 mL. PHSA was incubated with 10x molar excess of IT and 6* molar excess of 5 kDa mPEG-maleimide in accordance with the amine basis of surface accessible amines on HSA. The reaction mixture was incubated at 4 °C with constant stirring overnight for 16 hours. At the end of this period, the reaction mixture was filtered through a 0.2 pm PES TFF module and underwent 10 diafiltrations against a 500 kDa PS TFF module with a modified lactated Ringer’s solution to facilitate removal of any unreacted reagents. The 500 kDa TFF permeate was then subjected to 10 diafiltrations on a 50 kDa PS TFF module with the modified lactated Ringer’s solution to facilitate additional reagent removal and buffer exchange. The final product was concentrated to above 60 mg / mL and stored at -80 °C for future characterization. Figure 10 shows the PEGylation of PHSA followed by TFF separation of the final product into two size brackets.

[0273] PHSA / PPHSA Concentration: The PHSA / PPHSA mass concentration was quantified from a known volume of solution via its’ dry mass using a Labconco Freeze dryer (Kansas City, MO). Samples were frozen prior to being placed under vacuum to facilitate sublimation over 24 hours, and dry mass determination of PHSA / PPHSA.

[0274] Size Exclusion Chromatography Elution Profile, MW Distribution and Hydrodynamic Diameter: The size exclusion elution profile of HSA, PHSA, and PPHSA brackets were measured via size exclusion high-performance liquid chromatography (SEC- HPLC) at a 280 nm absorbance. SEC-HPLC was performed on a SEC-1000 column (ThermoFisher Scientific, Waithan, MA) attached to an Ultimate 3000 HPLC / UHPLC system (ThermoFisher Scientific, Waithan, MA). Additionally, secondary confirmation of size was performed via SDS-PAGE of the low MW PHSA / PPHSA species. Each 2 pg sample was mixed at a 1 : 1 volume ratio with tris-glycine SDS buffer and incubated at 85 °C (15 mins). The sample mixture was then run on a polyacrylamide gel containing a 12% resolving gel and 4% stacking gel at 225 V (40 mins). The gel was stained with Coomassie blue (1 hour) and was destained (3x; 1 hour) with 30% methanol and 10% acetic acid. Lastly, the hydrodynamic diameter of PHSA / PPHSA was measured via dynamic light scattering (DLS). DLS measurements were performed on a Zetasizer Nano DLS spectrometer (Malvern Instruments, Worcestershire, UK).

[0275] Zeta Potential: The zeta potential ( ) of HSA, PHSA, and PPHSA fractions was measured at 1 mg / mL using a Zeta Potential Analyzer (ZetaPALS, Brookhaven, Holtsville, NY).

[0276] Osmolality: The osmolality of HSA, PHSA, and PPHSA solutions at a total protein concentration of 50 mg / mL was measured using an osmometer based on freezing point depression (OSMOMAT 030, Berlin, Germany).

[0277] Viscosity: The viscosity of HSA, PHSA, and PPHSA solutions was measured at 50 mg / mL using a viscometer DV-II plus with a cone spindle CPE-40 (Brookfield Engineering Laboratories, Middleboro, MA) at a shear rate of 316 s'1.

[0278] Colloid Oncotic Pressure (COP): The COP of HSA, PHSA, and PPHSA solutions was measured at 50 mg / mL using a Wescor 4420 Colloid Osmometer (Wescor, Logan, UT).

[0279] Circular Dichroism: The secondary structure of HSA, PHSA and PPHSA was assessed via circular dichroism (CD) on a JASCO J- 1500 CD spectrometer (JASCO, Easton, MD). Samples were prepared at 3 pM and measured between 200-250 nm for changes in secondary structure at 25 °C. Protein secondary structure, i.e., a-helical content, was assessed at the 222 nm minima.

[0280] Amine Quantification: A stock solution of 2,4,6-trinitrobenzenesulfonic acid (TNBSA; 0.01% w / v) was prepared from a stock reaction buffer, sodium bicarbonate (0.1 M; pH 8.5). Standard and protein samples (0.02-0.20 mg / mL) were diluted in the reaction buffer. Triplicates of TNBSA solution (50 pL) along with standard and protein samples (100 pL) were mixed in a microwell plate and incubated (37 °C; 2 h). SDS (10% w / v; 50 pL) and HC1 (1 N, 25 pL) were added to quench the reaction and the absorbance was measured (340 nm). The standard solution absorbance was subtracted from the protein samples allowing for amine quantification via the Beer Lambert Law with an extinction coefficient of 15,000 M^cm’1.

[0281] Data Analysis: In this Example, all statistical analysis was performed using the Tukey HSD test. Statistical significance was obtained if p < 0.05. Additionally, all results are presented as the mean ± standard deviation, and all statistical analyses were performed using JMP Pro v 16 (Cary, NC). Statistical comparisons were made between 1) unmodified HSA and all PPHSA brackets; 2) the same PPHSA bracket synthesized at the two different glutaraldehyde to HSA molar ratios; 3) the same PPHSA glutaraldehyde to HSA molar ratio fractionated into the two brackets; and 4) PPHSA and the respective PHSA precursor.

[0282] Results and Discussion

[0283] HSA, PHSA, and PPHSA biophysical properties are summarized in Table 2.

[0284] Table 2. Biophysical properties of HSA and PHSA, and PPHSA solutions. Viscosity, osmolality, and COP were all measured at a total protein concentration of 50 mg / mL

[0285] . Colloid

[0286] Hydrodynamic Zeta _ , _

[0287] ,, . 'JViscosity _ . , Osmolality Osmotic

[0288] Fraction Diameter .JPotential „

[0289] , , (cP) , (Osm / kg) Pressure

[0290] ( vnm)7( vmV)67. „ . ' (mm Hg)

[0291] HSA 5.03± 0.15 1.23± 0.02 -17.77± 0.15 0.298± 0.001 20.60± 0.00

[0292] PHSA 43: 1 A 21.20± 0.20 3.56± 0.03 -26.08± 0.53 0.289± 0.007 17.55± 0.49

[0293] PHSA 43: I B 9.87± 1.11 2.42± 0.06 -21.27± 1.62 0.295± 0.005 24.00± 0.98

[0294] PHSA 60: 1 A 29.07± 4.21 2.42± 0.16 -28.97± 2.48 0.287± 0.006 22.20± 0.70

[0295] PHSA6O: 1 B 12.00± 2.05 2.68± 0.41 -19.68± 2.60 0.289± 0.001 23.20±0.70

[0296] PPHSA 43: 1 A 33.67± 4.56 4.01± 0.09 -12.95± 2.16 0.329± 0.005 45.60± 2.54

[0297] PPHSA 43: 1 B 12.60± 0.89 1.91± 0.00 -14.04± 0.42 0.335± 0.001 26.25± 3.04

[0298] PPHSA 60: 1 A 35.70± 2.50 3.59± 0.04 -12.51± 0.68 0.335± 0.003 33.40± 1.83

[0299] PPHSA 60: 1 B 14.43± 3.03 1.97± 0.05 -14.24± 1.26 0.338± 0.003 21.45± 1.90 SEC-HPLC: The elution profile of HSA, PHSA, and PPHSA was assessed via SEC- HPLC as shown in Figures 11 A-l IB. The control consisted of unmodified HSA that eluted at 9.48 ± 4.63 x 10'4min, while both PHSA and PPHSA fractions eluted at earlier times indicating that the PHSA / PPHSA species are larger in size compared to HSA. The PPHSA A and B fractions eluted earlier from the SEC-HPLC column than their respective precursor PHSA A and B fractions. This is indicative of an increase in PHSA size after PEGylation. Hence, the protein elution order for materials cross-linked at the same glutaraldehyde to HSA molar ratio from lowest to highest elution time was: PPHSA A, PHSA A, PPHSA B, PHSA B, and HSA.

[0300] PHSA 43: 1 B eluted at 9.05 ± 5.54 x 10'5minutes, while PPHSA 43: 1 B eluted at 8.39 ± 3.38 x 10'4minutes. The decrease in the SEC-HPLC elution time of PPHSA after PEGylation of PHSA indicated an increase in PPHSA size compared to PHSA. Similarly, PHSA 43 : 1 A eluted at 8.17 ± 1.18 x 10'3minutes, while PPHSA 43: 1 A eluted earlier at 7.76 ± 2.87 x 10'3minutes. A similar trend was observed for the 43 : 1 B bracket, where there was a decrease in the PHSA protein elution time after PEGylation; which corresponds to an increase in PPHSA size compared to its’ precursor PHSA. In addition, at the same glutaraldehyde to HSA molar ratio, the A fractions (500 kDa- 0.2 pm) eluted at earlier times compared to the B fractions (50 kDa- 500 kDa).

[0301] PHSA 60: 1 B eluted at 8.63 ± 1.29 x 10'3minutes, while PPHSA 60: 1 B eluted at 8.26 ± 1.18 x 10'3minutes. Furthermore, PHSA 60: 1 A eluted at 7.92 ± 2.45 x 10'3minutes, while PPHSA 60: 1 A eluted at 7.65 ± 8.18 x 10'5minutes. Therefore, the SEC-HPLC elution time decreased after the PHSA samples were PEGylated indicating the increased size of the PEGylated product compared to its’ PHSA precursor.

[0302] SDS-PAGE: As a secondary form of PEGylation confirmation, HSA, PHSA, and PPHSA were analyzed via SDS-PAGE in Figure 12. The HSA control has a prominent band above 55 kDa which is near the actual molecular weight (MW) of HSA. The smearing in the PHSA lanes compared to unmodified HSA is indicative of the presence of larger molecular species compared to HSA. The B fraction at both PHSA molar ratios has unmodified HSA present and is justified by the TFF modules used to fractionate the material (50 - 500 kDa). All PPHSA samples did not travel through the gel as well as their respective PHSA precursors indicating that these materials have a higher MW compared to their unPEGylated precursors. All chemically modified species were well above 250 kDa

[0303] Amine Quantification: HSA has only one accessible cysteine residue in its amino acid sequence where maleimide PEG conjugation is possible. Therefore, to increase the number of thiol binding sites to enable maleimide PEG conjugation, primary amines on the surface of PHSA must be converted to thiol groups. Therefore, lysine residues on the surface of PHSA were converted to thiol groups via reaction with iminothiolane (IT). Once the PHSA surface thiolation was complete, maleimide PEG was conjugated to PHSA. To quantify maleimide PEG attachment, amine quantification was performed on PHSA and PPHSA samples, prior to and after PEGylation. The fractionated PHSA at both cross-link densities did not have all possible lysine residues accessible for thiolation due to steric hinderance in the PHSA product. Of the accessible amines, half were PEGylated for all batches. The following are the accessible amines that were successfully converted to thiol groups and subsequently conjugated to maleimide PEG per PHSA molecule: 6, 9, 4, and 5 for PPHSA 43 : 1 A, PPHSA 43 : 1 B, PPHSA 60: 1 A, and PPHSA 60: 1 B, respectively. The B brackets are likely more PEGylated than the A brackets due to the presence of free HSA molecules in solution.

[0304] DLS: Previous SEC-HPLC and SDS-PAGE data indicate an increase in MW when PHSA species are PEGylated. To further support these observations, DLS measurements of the hydrodynamic diameter of these species are shown in Figure 13. All PPHSA species were larger in size compared to unmodified HSA (p < 0.05). Additionally at the same glutaraldehyde to HSA molar ratio, the PPHSA A bracket was significantly larger in size compared to the PPHSA B bracket (p < 0.05). Interestingly, PPHSA 43 : 1 A is significantly larger than PHSA 43 : 1 A (p < 0.05).

[0305] Zeta Potential: To analyze the potential colloidal stability of PPHSA, zeta potential measurements were performed on the HSA control, PHSA, and PPHSA and shown in Figure 14. The zeta potential of all PPHSA molecules are statistically higher (i.e. more positive charge) than their precursor PHSA molecules (p < 0.05). In addition, all PHSA molecules possess statistically larger negative values compared to unmodified HSA (p < 0.05).

[0306] Viscosity: All sample measurements were performed at 50 mg / mL due to the difficulty in concentrating the PEGylated material above 50 mg / mL.

[0307] HSA is significantly less viscous compared to each PHSA and PPHSA at all crosslinking densities (p < 0.05), as shown in Figure 15. The A fraction for PPHSA synthesized at the same cross-linking density was significantly more viscous than the B fraction. Additionally, while not observed for the 43 : 1 cross-linking density, the PPHSA 60: 1 A and B fractions were significantly different from their parent PHSA (p < 0.05). The PHSA A brackets increase in viscosity when PEGylated. In contrast, the PHSA B brackets decrease in viscosity when PEGylated. This is likely due to the presence of free HSA in the B fractions.

[0308] Osmolality: Figure 16 shows the osmolality of HSA, PHSA and PPHSA solutions. It is evident that all PPHSA solutions, regardless of glutaraldehyde to HSA molar ratio and bracket, possess statistically higher osmolality compared to unmodified HSA (p < 0.05). In addition, all PPHSA solutions have statistically higher osmolality than their precursor PHSA.

[0309] Colloid Oncotic Pressure (COP): In general, PHSA maintained the COP near that of unpolymerized HSA. However, in general, a substantial increase in COP was observed when PHSA was PEGylated — regardless of the cross-linking density and size bracket as shown in Figure 17. The COP of the HSA control is statistically lower compared to the PPHSA 43: 1 A (p < 0.05), PPHSA 43: 1 B (p < 0.05), and PPHSA 60: 1 A (p < 0.05) fractions. Additionally, the COP of the parent PHSA A bracket is significantly less that the PEGylated material at both crosslinking densities (p < 0.05), however, the same trend is not true for the B brackets. The lack of significance between the parent PHSA B bracket and its’ PEGylated derivative (p > 0.05) can be attributed to the presence of free HSA molecules in solution, which lowers the observed COP for the PPHSA B brackets. Furthermore, there is a significance between the two sized brackets A and B between the two cross-linking densities, i.e., PPHSA 43 : 1 A and PPHSA 43 : 1 B, and PPHSA 60: 1 A and PPHSA 60: 1 B.

[0310] Circular Dichroism: Circular dichroism was used to assess changes in HSA secondary structure prior to and after polymerization and PEG conjugation. Unmodified HSA mainly exhibits a-helices in its secondary structure, which is consistent with the two minima at 208 and 222 nm. Figure 18 shows the circular dichroism spectra of all materials. All PHSA fractions possess higher alpha helical content compared to HSA. Once PEGylated, all PHSA fractions exhibited a loss in alpha helical content when compared to PHSA and HSA. The loss in a helical content of PPHSA brackets compared to their precursor PHSA brackets was assessed by the 222 nm a helix peak ratio between PHSA and PPHSA brackets and is as follows: PPHSA 43 : 1 A loses 37% of its’ alpha helical content, PPHSA 43: 1 B loses 28% of its’ alpha helical content, PPHSA 60: 1 A loses 18% of its’ alpha helical content, and PPHSA 60: 1 B loses 30% of its alpha helical content.

[0311] Conclusion

[0312] Upon PEGylation, all PPHSA brackets increased in MW, hydrodynamic diameter, osmolality, and COP, while the zeta potential was decreased compared to their precursor PHSA brackets. Future work will assess the in vivo response of the PPHSA brackets in animal models of normal and impaired endothelial function.

[0313] References

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[0321] 8. J. Elmer, P. Cabrales, Q. Wang, N. Zhang, and A. F. Palmer, "Synthesis and biophysical properties of polymerized human serum albumin," (in eng), Biotechnol Prog, vol. 27, no. 1, pp. 290-6, Jan-Feb 2011, doi: 10.1002 / btpr.53 L

[0322] 9. D. A. Belcher, A. T. Williams, A. F. Palmer, and P. Cabrales, "Polymerized albumin restores impaired hemodynamics in endotoxemia and polymicrobial sepsis," Scientific Reports, vol. 11, no. 1, p. 10834, 2021 / 05 / 25 2021, doi: 10.1038 / s41598-021-90431-z.

[0323] 10. S. Chatpun, P. Nacharaju, and P. Cabrales, "Improving cardiac function with new- generation plasma volume expanders," (in eng), Am J Emerg Med, vol. 31, no. 1, pp. 54-63, Jan 2013, doi: 10.1016 / j.ajem.2012.05.031.

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[0325] 12. J. K. Dozier and M. D. Distefano, "Site-Specific PEGylation of Therapeutic Proteins," (in eng), Int J Mol Sci, vol. 16, no. 10, pp. 25831-64, Oct 28 2015, doi: 10.3390 / ijmsl61025831. 13. S. Lee and D. K. Lee, "What is the proper way to apply the multiple comparison test?," (in eng), Korean J Anesthesiol, vol. 71, no. 5, pp. 353-360, Oct 2018, doi:

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[0329] Other advantages which are obvious, and which are inherent to the invention will be evident to one skilled in the art. It will be understood that certain features and sub-combinations are of utility and may be employed without reference to other features and sub-combinations. This is contemplated by and is within the scope of the claims. Since many possible embodiments may be made of the invention without departing from the scope thereof, it is to be understood that all matter herein set forth or shown in the accompanying drawings is to be interpreted as illustrative and not in a limiting sense.

Claims

WHAT IS CLAIMED IS:

1. A composition comprising polymerized albumin, wherein the composition comprises less than 5% by weight low molecular weight albumin species having a molecular weight less than 100 kDa; and less than 5% by weight high molecular weight albumin species.

2. The composition of claim 1, wherein the polymerized albumin comprises polymerized human serum albumin (PolyHSA).

3. The composition of any of claims 1-2, wherein albumin comprises a recombinant albumin.

4. The composition of any of claims 1-3, wherein the high molecular weight albumin species comprise albumin species that are retained on a filtration membrane having a pore size of 0.2 pm.

5. The composition of any of claims 1-4, wherein the high molecular weight albumin species comprise albumin species having a molecular weight greater than 2,000 kDa.

6. The composition of any of claims 1-5, wherein the high molecular weight albumin species comprise albumin species having a molecular weight greater than 500 kDa.

7. The composition of any of claims 1-6, wherein the composition comprises less than 5% by weight albumin species having a molecular weight less than 100 kDa; and less than 5% by weight albumin species having a molecular weight greater than 500 kDa.

8. The composition of any of claims 1-6, wherein the composition comprises less than 5% by weight albumin species having a molecular weight less than 500 kDa; and less than 5% by weight albumin species that are retained on a filtration membrane having a pore size of 0.2 pm.

9. The composition of any of claims 1-8, wherein the polymerized albumin is prepared by a process that comprises: polymerizing albumin;filtering the polymerized albumin by ultrafiltration against a first filtration membrane having a pore size that separates the low molecular weight albumin species from the polymerized albumin; and filtering the polymerized albumin by ultrafiltration against a second filtration membrane having a pore size that separates the high molecular weight albumin species from the polymerized albumin.

10. The composition of claim 9, wherein the ultrafiltration comprises tangential -flow filtration.

11. The composition of any of claims 9-10, wherein the first filtration membrane is rated for retaining solutes having a molecular weight greater than 100 kDa, thereby forming a retentate fraction comprising the polymerized albumin and a permeate fraction comprising low molecular weight albumin species having a molecular weight less than 100 kDa.

12. The composition of claim 11, wherein the second filtration membrane is rated for retaining solutes having a molecular weight greater than 500 kDa, thereby forming a retentate fraction comprising the high molecular weight albumin species and a permeate fraction comprising the polymerized albumin.

13. The composition of claim 11, wherein the second filtration membrane has a pore size of 0.2 pm, thereby forming a retentate fraction comprising the high molecular weight albumin species and a permeate fraction comprising the polymerized albumin.

14. The composition of any of claims 9-10, wherein the first filtration membrane is rated for retaining solutes having a molecular weight greater than 500 kDa, thereby forming a retentate fraction comprising the polymerized albumin and a permeate fraction comprising low molecular weight albumin species having a molecular weight less than 500 kDa.

15. The composition of claim 14, wherein the second filtration membrane has a pore size of 0.2 pm, thereby forming a retentate fraction comprising the high molecular weight albumin species and a permeate fraction comprising the polymerized albumin.

16. The composition of any of claims 1-15, wherein the low molecular weight albumin species comprises unreacted albumin, albumin oligomers, or any combination thereof.

17. The composition of any of claims 1-16, wherein the composition comprises less than 1% by weight low molecular weight albumin species having a molecular weight less than 100 kDa and less than 1% by weight high molecular weight albumin species, such as less than 0.1% by weight low molecular weight albumin species having a molecular weight less than 100 kDa and less than 0.1% by weight high molecular weight albumin species.

18. The composition of any of claims 1-17, wherein the polymerized albumin has a crosslinker to albumin molar ratio of at least about 10:1, such as a cross-linker to albumin molar ratio of from 20:1 to 100:1.

19. The composition of claim 18, wherein the cross-linker comprises glutaraldehyde, succindialdehyde, activated forms of polyoxyethylene and dextran, -hydroxy aldehydes, such as glycolaldehyde, N-maleimido-6-aminocaproyl-(2'-nitro,4'-sulfonic acid)-phenyl ester, m- maleimidobenzoic acid-N-hydroxysuccinimide ester, succinimidyl 4-(N- maleimidomethyl)cyclohexane-l -carboxylate, sulfosuccinimidyl 4-(N- maleimidomethyl)cyclohexane-l -carboxylate, m-maleimidobenzoyl-N-hydroxy succinimide ester, m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester, N-succinimidyl(4- iodoacetyl)aminobenzoate, sulfosuccinimidyl(4-iodoacetyl)aminobenzoate, succinimidyl 4-(p- maleimidophenyl)butyrate, sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate, l-ethyl-3-(3- dimethylarninopropyl)carbodiimide hydrochloride, N,N'-phenylene dimaleimide, or a combination thereof.

20. The composition of any of claims 18-19, wherein the cross-linker is from at least one of a dialdehyde class, bisimidate class, the acyl diazide class, or the aryl dihalide class.

21. The composition of any of claims 1-20, wherein the polymerized albumin is covalently modified with a polymer.

22. The composition of claim 21, wherein the polymer comprises a polyalkylene oxide, such as polyethylene glycol (PEG).

23. The composition of claim 21, wherein the polymer comprises a zwitterionic polymer.

24. The composition of any of claims 21-23, wherein the polymer has a molecular weight of from 200 Da to 1,000,000 Da, such as from 1,000 Da to 10,000 Da, or from 3,000 Da to 6,000 Da, or about 5,000 Da.

25. The composition of any of claims 21-24, wherein the polymerized albumin is covalently modified with the polymer in a process that comprises: conjugating one or more polymers to the polymerized albumin to form a polymer- modified polymerized albumin; filtering the polymer-modified polymerized albumin by ultrafiltration against a first filtration membrane having a pore size that separates low molecular weight contaminants from the polymer-modified polymerized albumin; and filtering the polymer-modified polymerized albumin by ultrafiltration against a second filtration membrane having a pore size that separates the high molecular weight contaminants from the polymer-modified polymerized albumin.

26. The composition of claim 25, wherein the ultrafiltration comprises tangential -flow filtration.

27. The composition of any of claims 25-26, wherein the first filtration membrane is rated for retaining solutes having a molecular weight greater than 100 kDa, thereby forming a retentate fraction comprising the polymer-modified polymerized albumin and a permeate fraction comprising low molecular weight contaminants having a molecular weight less than 100 kDa.

28. The composition of claim 27, wherein the second filtration membrane is rated for retaining solutes having a molecular weight greater than 500 kDa, thereby forming a retentate fraction comprising the high molecular weight contaminants and a permeate fraction comprising the polymer-modified polymerized albumin.

29. The composition of claim 27, wherein the second filtration membrane has a pore size of 0.2 pm, thereby forming a retentate fraction comprising the high molecular weight contaminants and a permeate fraction comprising the polymer-modified polymerized albumin.

30. The composition of any of claims 25-26, wherein the first filtration membrane is rated for retaining solutes having a molecular weight greater than 500 kDa, thereby forming a retentate fraction comprising the polymer-modified polymerized albumin and a permeate fraction comprising low molecular weight contaminants having a molecular weight less than 500 kDa.

31. The composition of claim 30, wherein the second filtration membrane has a pore size of 0.2 pm, thereby forming a retentate fraction comprising the high molecular weight albumin species and a permeate fraction comprising the polymerized albumin.

32. The use of the composition of any of claims 1-31 as a plasma expander or plasma substitute.

33. A method of replacing lost blood volume in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the composition of any of claims 1-31.

34. A method of treating hypercytokinemia in a subject in need thereof, the method comprising: administering to the subject a therapeutically effective amount of the composition of any of claims 1-31 to reduce circulating cytokine levels by at least 5%, such as from 5% to 70%.

35. The method of claim 34, wherein the hypercytokinemia is induced by an infectious agent such as influenza (e.g., H1N1 influenza or H5N1 influenza), coronavirus infection (e.g., avian coronavirus (IBV), porcine coronavirus HKU15 (PorCoV HKU15), Porcine epidemic diarrhea virus (PEDV), HCoV-229E, HCoV-OC43, HCoV-HKUl, HCoV-NL63, SARS-CoV, SARS- CoV-2, or MERS-CoV), Influenza B, Parainfluenza virus, Ebola, Epstein-Barr virus, cytomegalovirus, or group A streptococcus.

36. The method of claim 34, wherein the hypercytokinemia is associated with graft-versus- host disease.

37. A method of preventing hypercytokinemia in a subject, the method comprising:administering to the subject a therapeutically effective amount of the composition of any of claims 1-31 to reduce or prevent an increase in circulating cytokine levels.

38. The method of claim 37, wherein the subject is infected with or has been exposed to an infectious agent such as influenza (e.g., H1N1 influenza or H5N1 influenza), coronavirus infection (e.g., avian coronavirus (IBV), porcine coronavirus HKU15 (PorCoV HKU15), Porcine epidemic diarrhea virus (PEDV), HCoV-229E, HCoV-OC43, HCoV-HKUl, HCoV- NL63, SARS-CoV, SARS-CoV-2, or MERS-CoV), Influenza B, Parainfluenza virus, Ebola, Epstein-Barr virus, cytomegalovirus, or group A streptococcus.

39. The method of claim 37, wherein the subject has received or will receive transplanted cells, transplanted tissue, a transplanted organ, or any combination thereof.

40. The method of claim 39, wherein the transplanted cells, transplanted tissue, a transplanted organ, or any combination thereof comprise an allograft or a xenograft.

41. A method of treating endothelial dysfunction in a subject, the method comprising: administering to the subject a therapeutically effective amount of the composition of any of claims 1-31 to reduce circulating levels of a biomarker for endothelial dysfunction in the subject.

42. A method of preventing endothelial dysfunction in a subject, the method comprising: administering to the subject a therapeutically effective amount of the composition of any of claims 1-31 to reduce or prevent an increase in circulating levels of a biomarker for endothelial dysfunction in the subject.

43. The method of claim 42, wherein the composition of any of claims 1-31 is administered in an effective amount to prevent circulating levels of the biomarker for endothelial dysfunction rising above normal levels for subjects without endothelial dysfunction.

44. The method of any of claims 42-43, wherein the biomarker for endothelial dysfunction comprises syndecan-1.

45. A method of treating endothelial dysfunction in a subject in need thereof, the method comprising: administering to the subject a therapeutically effective amount of the composition of any of claims 1-31 to reduce endothelial barrier permeability.

46. A method of protecting endothelial tissue in a subject, the method comprising: administering to the subject the composition of any of claims 1-31 in a therapeutically effective amount to protect endothelial tissue from damage.

47. The method of any one of claims 33-46, wherein the subject has a normal blood pressure.

48. The method of any one of claims 33-47, wherein the composition of any of claims 1-31 is administered via infusion or exchange transfusion.

49. The method of any one of claims 33-47, wherein the composition of any of claims 1-31 is administered via infusion.

50. The method of claim 49, wherein the infusion comprises infusion of a volume of the composition of any of claims 1-31, and wherein the volume comprises from 10% to 30% of the subject’s total blood volume.

51. The method of any one of claims 33-47, wherein the composition of any of claims 1-31 is administered via exchange transfusion.

52. The method of claim 51, wherein the exchange transfusion comprises exchange transfusion of from 5% to 90% of the subject’s total blood volume with the composition of any of claims 1-31.

53. The method of any one of claims 33-52, wherein the composition of any of claims 1-31 is administered in an amount effective to reduce circulating cytokine levels by at least 5%, such as from 5% to 70%.

54. The method of any one of claims 33-53, wherein the composition of any of claims 1-31 is administered in a therapeutically effective amount to reduce an immune response.

55. The method of any one of claims 33-54, wherein the composition of any of claims 1-31 is administered in a therapeutically effective amount to reduce the number of leukocytes adhered to endothelial tissue in the subject.

56. The method of any one of claims 33-55, wherein the composition of any of claims 1-31 is administered in a therapeutically effective amount to improve vascular integrity.

57. The use of the composition of any of claims 1-31 for the ex vivo perfusion of an organ or tissue.

58. The use of the composition of any of claims 1-31 for ex vivo cell or tissue culture.

59. The use of the composition of any of claims 1-31 in extracorporeal device, such as an ECMO device.

60. A method of binding or sequestering a metabolite in vivo or ex vivo, the method comprising contacting the metabolite with the composition of any of claims 1-31.

61. The method of claim 60, wherein the metabolite comprises free heme.

62. A method of treating a subject comprising administering to the composition of any of claims 1-31.

63. The method of claim 62, wherein the subject is suffering from a loss of blood due to injury, hemolytic anemia, equine infectious anemia, feline infectious anemia, a bacterial infection, Factor IV fragmentation, hypersplenation, splenomegaly, hemorrhagic syndrome, hypoplastic anemia, aplastic anemia, idiopathic immune hemadytic conditions, iron deficiency, isoimmune hemdytic anemia, microangiopathic hemolytic anemia, parasitism, or any combination thereof.

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