Methods for preparing albumin solutions and uses and related uses thereof

Abstract

The present invention relates to human albumin solutions and in particular to methods of preparation and uses thereof. The invention also relates to a human albumin solution having an increased mercaptoalbumin content and / or an increased transport capacity for reactive oxygen species, reactive nitrogen species and endogenous and exogenous ligands. Some embodiments of the invention have been particularly developed for the treatment of disease and will be described hereinafter with reference to the present application. However, it is understood that the present invention is not limited to this particular field of application.

Description

Technical Field

[0001] The present invention relates to human albumin solutions and, in particular, to methods for their preparation and use. The present invention also relates to human albumin solutions having increased human mercaptalbumin (HMA) content and / or increased transport capacity for reactive oxygen species, reactive nitrogen species, and endogenous and exogenous ligands. Certain embodiments of the present invention have been specifically developed for the treatment of diseases and will be described below with reference to the present application. However, it should be understood that the present invention is not limited to this particular field of application. Background Art

[0002] Any discussion of the background art throughout the specification should in no way be considered as an admission that such art is widely known or forms part of the common general knowledge in the field.

[0003] Human albumin is the most abundant protein in plasma and serves as a primary carrier of endogenous and exogenous ligands, particularly fatty acids (FA), as well as nucleic acids, hormones, metals, toxins, and drugs. It is responsible for much of the pro- and antioxidant capacity of plasma and exhibits (pseudo-)enzymatic properties. Fatty acids are physiological ligands for human albumin, characterized by nine fatty acid binding sites, FA1 to FA9. Fatty acids bind to sites FA1 to FA9 with varying degrees of affinity: FA2, FA4, and FA5 are high-affinity sites, FA1 and FA3 have intermediate affinity, and FA6 and FA7 have low affinity. HSA plays an important role as a drug carrier, influencing the pharmacokinetics and pharmacodynamics of drugs such as antibiotics, anticoagulants, antitumor agents, antivirals, anesthetics, anxiolytics, and nonsteroidal anti-inflammatory drugs. FA1, FA3 to FA4, and the FA7 pocket are the primary drug-binding sites on HSA. FA1 recognizes antibiotics, antitumor agents, antiretrovirals, and nonsteroidal anti-inflammatory drugs. Ibuprofen is a typical ligand for the FA3 to FA4 pocket, which also recognizes anesthetics, anxiolytics, and the orphan drug 4-phenylbutazone. The FA7 site is considered the primary binding site for HSA. The typical ligand is warfarin, a bulky heterocyclic anticoagulant. Several drugs co-bind to the FA7 site in the presence of myristate. This suggests that myristate induces one or more conformational changes in the binding site, leading to the formation of three non-overlapping secondary sites capable of recognizing anesthetics, anticoagulants, antineoplastic agents, antiretrovirals, and non-steroidal anti-inflammatory drugs (NSAIDs).

[0004] Furthermore, human albumin is a valuable biomarker for many diseases, such as cancer, rheumatoid arthritis, ischemia, obesity, and diabetes, and has clinical applications in the treatment of several conditions, including shock, trauma, bleeding, acute respiratory distress syndrome, hemodialysis, acute liver failure, chronic liver disease, and hypoalbuminemia.

[0005] In recent years, the redox state of thiols has garnered significant interest in the context of disease. In human plasma, the largest thiol pool is also provided by human albumin. Human albumin comprises a total of 585 amino acids (giving it a molecular weight of approximately 66 kDa) and contains 35 Cys residues. However, it contains only one cysteine ​​(Cys-34), which does not participate in an intramolecular disulfide bond. As a major plasma protein, albumin has been studied as a primary target for oxidative modification, and its oxidized forms, or redox state, have been frequently discussed. While there are several ways to oxidize albumin, Cys-34 is a particularly redox-sensitive site within albumin, and therefore, its redox state can be described based on its redox state. The redox state of Cys-34 defines three fractions that generally allow for the determination of the overall redox state of albumin. These proteins include human mercaptalbumin (HMA) containing free thiol groups, human nonmercaptalbumin-1 (HNA1) containing disulfide bonds, and human nonmercaptalbumin-2 (HNA2) containing sulfinic acid or sulfonic acid groups.

[0006] In plasma, mercaptoalbumin provides several benefits, including:

[0007] • Antioxidant protection: Mercaptoalbumin acts as an antioxidant, helping to protect cells and tissues from oxidation by reactive oxygen species (e.g., H2O2, O2 - , HOCl, etc.) and reactive nitrogen species (such as ONOO - 、ONOOCO2 - damage caused by

[0008] • Detoxification: Mercaptoalbumin binds to toxic substances (such as heavy metals and drugs) and helps remove them from the body; and

[0009] • Immune function: Mercaptoalbumin may help stimulate the immune system and fight infection.

[0010] Therefore, a high percentage of non-mercaptoalbumin can lead to a reduced ability to protect against oxidative damage, detoxify harmful substances, and / or support immune function. Additionally, low levels of mercaptoalbumin can indicate certain health conditions or diseases, such as liver disease or malnutrition.

[0011] For example, non-mercaptoalbumin-1 is a reversible derivative, whereas non-mercaptoalbumin-2 is irreversible. In healthy young adults, approximately 70% to 80% of all albumin is mercaptoalbumin, of which non-mercaptoalbumin-1 accounts for approximately 20% to 30% and non-mercaptoalbumin-2 accounts for only approximately 2% to 5%. Elevated plasma levels of non-mercaptoalbumin-1 are associated with reduced antioxidant activity of human albumin. Both non-mercaptoalbumin-1 and non-mercaptoalbumin-2 are associated with the progression of inflammatory processes, and plasma mercaptoalbumin concentrations in Japanese residents are inversely correlated with atherosclerotic lesions in the carotid artery.

[0012] WO 2013 / 024100 A2 describes a method for diagnosing and treating medical conditions associated with oxidative stress, particularly liver disease. The method is based on the identification and quantification of the fraction of albumin that is irreversibly oxidized at Cys-34. The proposed treatment concept involves contacting the patient's plasma with an adsorbent capable of binding non-mercaptoalbumin-2, followed by an infusion of fresh albumin.

[0013] Using HPLC, Sogami et al., 1985 ("High-performance liquid chromatographic studies on non-mercapt to mercapt conversion of human serum albumin"; Journal of Chromatography, 332, 19-27) determined the relative occurrence of mercaptoalbumin and non-mercaptoalbumin in the plasma of patients with various liver diseases. The signal from the patient's plasma was compared with the signal from the same patient's plasma incubated with glutathione at 25°C. The more prominent signal from the incubated plasma was therefore attributed to mercaptoalbumin.

[0014] A similar analytical method is known from Oettl and Marsche, 2010 ("Redox state of humanserum albumin in terms of Cysteine-34 in health and disease"; Methods in Enzymology, Vol. 474, 181-195), which determined different oxidation phases of albumin, namely mercaptalbumin, non-mercaptoalbumin 1 and non-mercaptoalbumin 2, in the plasma of patients with diabetes, liver damage or renal damage.

[0015] Various methods have been described for modifying albumin, whether isolated from plasma or recombinantly produced, by exploiting the thiol reactivity of Cys-34 to produce conjugates of albumin. An exemplary method for producing albumin conjugates is known from WO 2007 / 071068 A1. This method involves producing and purifying recombinant albumin and covalently conjugating a compound to the Cys-34 thiol of albumin. Intermediate enrichment of the Cys-34 thiol group is proposed by contacting albumin with thioglycolic acid (TGA) or dithiothreitol (DTT).

[0016] US 2013 / 329127 A1 describes the use of PEG-maleimide to modify the thiol groups of various proteins, including human serum albumin. A diluted albumin solution is incubated with tris[2-carboxyethyl]phosphine hydrochloride (TCEP) for 10 minutes, followed by the addition of a branched PEG reagent containing a terminal maleimide group. After the reaction is allowed to proceed in the dark, the resulting PEG-albumin conjugate is further processed and purified.

[0017] WO 2007 / 049941 A1 relates to bioactive albumin conjugates, wherein the bioactive compound is covalently bound to albumin via a disulfide bridge. In the course of further analysis, the disulfide bridge is cleaved again by reacting the albumin conjugate with DTT at 37°C.

[0018] Regarding diseases, WO 2020 / 104458 A1 describes methods and kits for detecting liver dysfunction in a subject. WO 2020 / 104458 A1 discloses that the relative abundance of mercaptoalbumin (HMA), non-mercaptoalbumin-1 (HNA1), and non-mercaptoalbumin-2 (HNA2) in healthy humans is 70% to 80% mercaptoalbumin, 20% to 30% non-mercaptoalbumin-1, and <5% non-mercaptoalbumin-2, which are also the ranges disclosed in Oettl and Marsche, 2010, cited above.

[0019] Because human albumin is an extremely robust protein with respect to pH stability (stable in the pH range of 4 to 9), temperature stability (heating at 60°C for up to 10 hours in the presence of stabilizers), and even exposure to organic solvents, a number of human albumin products exist. Whether purified from pooled donor plasma on an industrial scale or recombinantly produced, their typical clinical applications are in the treatment of severe hypoalbuminemia or as a plasma expander.

[0020] However, commercial human albumin products have been described as containing lower amounts of mercaptalbumin and higher amounts of both non-mercaptoalbumin-1 and non-mercaptoalbumin-2 than the corresponding amounts present in fresh plasma. Furthermore, commercial albumin is formulated with large (i.e., excess) amounts of fatty compounds, such as tryptophan and / or octanoic acid. Consequently, the albumin:fatty compound ratio in commercial albumin solutions is typically between 1:5 and 1:8. For example, a commercial 5% Octalbin albumin solution contains 5% albumin (i.e., 50 g / l; 0.75 mmol) and tryptophan and octanoic acid at concentrations of up to 4.2 mmol each. A 25% Octalbin albumin solution contains 25% albumin (i.e., 250 g / l; 3.8 mmol) and tryptophan and octanoic acid at concentrations of up to 21 mmol each.

[0021] Therefore, there is a need in the art for improved commercial human albumin products and methods for their production.

[0022] It is an object of the present invention to overcome or ameliorate at least one disadvantage of the prior art, or to provide a useful alternative.In particular, it is an object of the present invention to provide an improved method for preparing human albumin solutions from pooled donor plasma. Summary of the Invention

[0023] The above-mentioned problems are solved by the invention disclosed herein. Specifically, the disclosed methods for preparing human albumin solutions from pooled donor plasma, as well as related uses and the prepared albumin solutions, solve the above-mentioned problems. The human albumin solutions have an increased mercaptoalbumin content and / or an increased transport capacity for reactive oxygen species, reactive nitrogen species, and endogenous and exogenous ligands.

[0024] Therefore, in a first aspect, the present invention relates to a method for preparing a human albumin solution having an increased mercaptalbumin content and / or an increased transport capacity for reactive oxygen species, reactive nitrogen species and endogenous and exogenous ligands, wherein the human albumin solution is obtained in the course of a cold ethanol precipitation process of human plasma, wherein the method comprises the following steps:

[0025] (a) providing a human albumin solution comprising mercaptalbumin and non-mercaptoalbumin, wherein the non-mercaptoalbumin comprises a disulfide bond moiety at position Cys-34;

[0026] (b) combining glutathione with the human albumin solution at least once at a molecular ratio of albumin to glutathione (albumin:glutathione) of 1:0.5 to 1:10; and

[0027] (c) converting at least a portion of the non-mercaptoalbumin into mercaptalbumin at a temperature below 15°C,

[0028] wherein the mercaptoalbumin content in the human albumin solution and / or the transport capacity of the human albumin solution for reactive oxygen species, reactive nitrogen species and endogenous and exogenous ligands is increased relative to the mercaptoalbumin content of a human albumin solution prepared in a corresponding human plasma cold ethanol precipitation process lacking steps (b) and (c).

[0029] In a second aspect, the present invention relates to a human albumin solution comprising an increased mercaptalbumin content prepared by the method according to the first aspect, characterized in that it comprises a mercaptalbumin content of at least 85%, particularly at least 88%, preferably 90% to 99%, or 90% to 95%, or 90% to 93%, relative to the total albumin content of the solution. In such embodiments, the non-mercaptoalbumin-1 content is significantly reduced compared to the untreated sample. That is, in such embodiments, the non-mercaptoalbumin-1 content is less than 5%, preferably less than 4%. In some specific embodiments, the non-mercaptoalbumin-1 content is no longer detectable, i.e., the non-mercaptoalbumin-1 content has been reduced to 0%. In particular, in some embodiments of the second aspect, when the mercaptalbumin content is increased to greater than 90%, the non-mercaptoalbumin-1 content has been reduced to less than 1%. More particularly, when the mercaptalbumin content is increased to greater than 91%, the non-mercaptoalbumin-1 content has been reduced to 0%.

[0030] In a third aspect, the invention relates to the human albumin solution of the second aspect for use in treating a patient in need thereof.

[0031] Therefore, the third aspect of the present invention also covers a method of treatment and / or use of the human albumin solution of the second aspect in the preparation of a medicament for treating a patient in need thereof, wherein the method comprises administering the human albumin solution of the second aspect to a patient in need thereof.

[0032] Generally speaking, the treatment comprises administering the human albumin solution with increased mercaptalbumin content of the second aspect to a patient:

[0033] - Patients with liver failure; and / or

[0034] - Patients with chronic hepatitis, acute hepatitis, cirrhosis, fulminant hepatic failure or hepatocellular carcinoma; and / or

[0035] - Patients with renal failure; and / or

[0036] - Patients suffering from hypovolemia, and for the restoration and maintenance of circulating blood volume in said patients.

[0037] In a fourth aspect, the present invention relates to the use of glutathione for increasing the mercaptalbumin content of a human albumin solution and its transport capacity for reactive oxygen species, reactive nitrogen species, and endogenous and exogenous ligands, wherein the human albumin solution is obtained during a cold ethanol precipitation process of human plasma, during which glutathione is added at least once, wherein the use comprises:

[0038] (a) providing a human albumin solution comprising mercaptalbumin and non-mercaptoalbumin, wherein the non-mercaptoalbumin comprises a disulfide bond moiety at position Cys-34;

[0039] (b) combining glutathione with the human albumin solution at least once at a molecular ratio of albumin to glutathione (albumin:glutathione) of 1:0.5 to 1:10; and

[0040] (c) converting at least a portion of the non-mercaptoalbumin into mercaptalbumin at a temperature below 15°C,

[0041] wherein the mercaptoalbumin content in the human albumin solution and / or the transport capacity of the human albumin solution for reactive oxygen species, reactive nitrogen species and endogenous and exogenous ligands is increased relative to the mercaptoalbumin content of a human albumin solution prepared in a corresponding human plasma cold ethanol precipitation process lacking steps (b) and (c).

[0042] In a fifth aspect, the present invention relates to a human albumin solution comprising an increased mercaptalbumin content obtained by the use of glutathione according to the fourth aspect, characterized in that it comprises a mercaptalbumin content of at least 85%, in particular at least 88%, preferably 90% to 99%, or 90% to 95%, or 90% to 93%, relative to the total albumin content of the solution. In such embodiments, the non-mercaptoalbumin-1 content is significantly reduced compared to the untreated sample. That is, in such embodiments, the non-mercaptoalbumin-1 content is less than 5%, preferably less than 4%. In some specific embodiments, non-mercaptoalbumin-1 is no longer detectable, i.e., the non-mercaptoalbumin-1 content has been reduced to 0%. In particular, in some embodiments of the fifth aspect, when the mercaptalbumin content is increased to greater than 90%, the non-mercaptoalbumin-1 content has been reduced to less than 1%. More particularly, when the mercaptalbumin content is increased to greater than 91%, the non-mercaptoalbumin-1 content has been reduced to 0%.

[0043] In a sixth aspect, the present invention relates to the human albumin solution of the fifth aspect for use in treating a patient in need thereof.

[0044] Therefore, the sixth aspect of the present invention also covers a method of treatment and / or the use of a human albumin solution in the preparation of a medicament for treating a patient in need thereof, wherein the method comprises administering the human albumin solution to a patient in need thereof.

[0045] Generally speaking, the treatment comprises administering the human albumin solution of the fifth aspect to a patient:

[0046] - Patients with liver failure; and / or

[0047] - Patients with chronic hepatitis, acute hepatitis, cirrhosis, fulminant hepatic failure or hepatocellular carcinoma; and / or

[0048] - Patients with renal failure; and / or

[0049] Patients suffering from hypovolemia, and for restoring and maintaining circulating blood volume in said patients. DETAILED DESCRIPTION

[0050] In order to provide a clear and consistent understanding of the specification and claims, and the scope to be given to such terms, the following definitions are provided.

[0051] In the context of this disclosure, the term "albumin", "human albumin" or "human serum albumin" refers only to unmodified human serum albumin (UniProtKB P02768; ALBU_HUMAN; The term therefore does not encompass any modifications of albumin, such as fusion proteins (whether comprising full-length human serum albumin or fragments thereof) or any conjugates of human serum albumin with other molecules, such as PEG or biologically active molecules.

[0052] The term "Cohn process" refers to a well-known series of purification steps developed by Edwin J. Cohn for the extraction of albumin from plasma. The process is based on the differential solubility of albumin and other plasma proteins with respect to pH, ethanol concentration, temperature, ionic strength, and protein concentration, as published in 1946 (Cohn, EJ et al. 1946 "Preparation and Properties of Serum and Plasma Proteins. IV. A System for the Separation into Fractions of the Protein and Lipoprotein Components of Biological Tissues and Fluids 1a,b,c,d". Journal of the American Chemical Society. 68 (3): 459-475).

[0053] The term “Kistler-Nitschmann process” refers to the also well-known process for large-scale production of human plasma fractions, which includes a series of alcohol precipitations, as published in 1962 (Kistler & Nitschmann 1962 “Large scale production of human plasma fractions. Eightyears experience with the alcohol fractionation procedure of Nitschmann, Kistler and Lergier”. Vox Sang. 1962 Jul-Aug;7:414-24).

[0054] Unless the context clearly requires otherwise, throughout this specification and claims, the word "comprises" and variations thereof should be interpreted in an inclusive sense rather than an exclusive or exhaustive sense; that is, in the sense of "including but not limited to."

[0055] Furthermore, references throughout this specification to "one embodiment," "some embodiments," or "an embodiment" mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in one embodiment," "in some embodiments," or "in an embodiment" in various places throughout this specification are not necessarily all referring to (but may refer to) the same embodiment. Furthermore, as will be apparent to one of ordinary skill in the art from this disclosure, the particular features, structures, or characteristics of one or more embodiments may be combined in any suitable manner.

[0056] As used herein, unless otherwise indicated, ordinal adjectives "first," "second," "third," etc., used to describe common objects indicate merely that different instances of similar objects are being referenced and are not intended to imply that the objects so described are necessarily in a given order temporally, spatially, hierarchically, or in any other manner.

[0057] As used herein, the term "exemplary" is used to provide an example, rather than an indicative quality. That is, an "exemplary embodiment" is an embodiment provided as an example, but not necessarily an embodiment of exemplary quality.

[0058] As already mentioned above, in plasma, mercaptoalbumin provides several benefits, including:

[0059] • Antioxidant protection: Mercaptoalbumin acts as an antioxidant, helping to protect cells and tissues from damage caused by free radicals and reactive oxygen species;

[0060] • Detoxification: Mercaptoalbumin binds to toxic substances (such as heavy metals and drugs) and helps remove them from the body; and

[0061] • Immune function: Mercaptoalbumin may help stimulate the immune system and fight infection.

[0062] Therefore, a high percentage of non-mercaptoalbumin can lead to a reduced ability to protect against oxidative damage, detoxify harmful substances, and / or support immune function. Additionally, low levels of mercaptoalbumin can indicate certain health conditions or diseases, such as liver disease or malnutrition.

[0063] However, as already mentioned, commercial human albumin products produced on an industrial scale are known to contain lower amounts of the beneficial mercaptoalbumin and higher amounts of two undesirable non-mercaptoalbumins compared to plasma. Furthermore, commercial albumin is formulated with large (i.e., excessive) amounts of fatty compounds, such as tryptophan and / or octanoic acid. Consequently, the ratio of albumin to such fatty compounds in commercial albumin solutions is typically between 1:5 and 1:8. For example, a commercially available 5% Octalbin albumin solution contains 5% albumin (i.e., 50 g / l; 0.75 mmol) and tryptophan and octanoic acid at concentrations of up to 4.2 mmol each. A 25% Octalbin albumin solution contains 25% albumin (i.e., 250 g / l; 3.8 mmol) and tryptophan and octanoic acid at concentrations of up to 21 mmol each. Thus, and without being bound by theory, excess fatty compounds may result in the occupation of most of the fatty acid binding sites FA1 to FA7 of albumin, further limiting the transport capacity of albumin for reactive oxygen species, reactive nitrogen species, and endogenous and exogenous ligands.

[0064] Since the conversion from mercaptalbumin to non-mercaptoalbumin-1 is the result of oxidation of the thiol group at Cys-34 of albumin, it is not entirely surprising that during industrial-scale purification of albumin from pooled donor plasma batches (typically based on the Cohn process or the Kistler-Nitschmann process and involving sequential precipitation and reconstitution steps), a higher degree of Cys-34 oxidation is observed in the albumin end product of the purification process compared with fresh plasma.

[0065] However, because of the beneficial properties of mercaptalbumin described above, there is a need for methods and procedures for increasing the mercaptalbumin content of industrially purified albumin products.

[0066] The albumin preparation method and use according to the present invention achieves an increased mercaptoalbumin content relative to the mercaptoalbumin content of human albumin solutions prepared using known methods. Furthermore, the albumin preparation method and use according to the present invention can even achieve an increased mercaptoalbumin content relative to the mercaptoalbumin content of plasma, i.e. a mercaptoalbumin content of greater than 85%.

[0067] Methods of the present invention and related albumin products

[0068] In a first aspect, the present invention relates to a method for preparing a human albumin solution having an increased mercaptalbumin content and / or an increased transport capacity for reactive oxygen species, reactive nitrogen species and endogenous and exogenous ligands, wherein the human albumin solution is obtained in the course of a cold ethanol precipitation process of human plasma, wherein the method comprises the following steps:

[0069] (a) providing a human albumin solution comprising mercaptalbumin and non-mercaptoalbumin, wherein the non-mercaptoalbumin comprises a disulfide bond moiety at position Cys-34;

[0070] (b) combining glutathione with the human albumin solution at least once at a molecular ratio of albumin to glutathione (albumin:glutathione) of 1:0.5 to 1:10; and

[0071] (c) converting at least a portion of the non-mercaptoalbumin into mercaptoalbumin at a temperature below 15°C,

[0072] wherein the mercaptoalbumin content in the human albumin solution and / or the transport capacity of the human albumin solution for reactive oxygen species, reactive nitrogen species and endogenous and exogenous ligands is increased relative to the mercaptoalbumin content of a human albumin solution prepared in a corresponding human plasma cold ethanol precipitation process lacking steps (b) and (c).

[0073] Endogenous and exogenous ligands to albumin are typically selected from fatty acids (FA), nucleic acids, hormones, metals, toxins, and drugs, wherein such drugs specifically include ibuprofen, diazepam, warfarin, lidocaine, and indomethacine.

[0074] As shown, the conversion step (c) of the above method is advantageously carried out at a relatively low temperature of less than 15°C. Although one would expect that the conversion efficiency would be lower at lower temperatures, it has been found that lower temperatures strike a desirable balance between the slower desired conversion of non-mercaptoalbumin to mercaptalbumin and the slower undesirable oxidation of Cys-34, such that the mercaptalbumin content can be increased as described. In some cases, the temperature at which step (c) is carried out is less than 14°C, less than 13°C, less than 12°C, less than 11°C, less than 10°C, less than 9°C, less than 8°C, less than 7°C, less than 6°C, less than 5°C, less than 4°C, less than 3°C, less than 2°C, less than 1°C, less than 0°C, less than -1°C, less than -2°C, less than -3°C, less than -4°C, less than -5°C, less than -6°C, less than -7°C, less than -8°C, less than -9°C, or less than -10°C.

[0075] In some cases, step (c) is performed at a temperature of -25°C to -22°C, -22°C to -19°C, -19°C to -16°C, -16°C to -13°C, -13°C to -10°C, -10°C to -7°C, -7°C to -4°C, -4°C to -1°C, -1°C to 2°C, 2°C to 5°C, 5°C to 8°C, 8°C to 11°C, 11°C to 14°C, or -25°C to -23°C, -23°C to -21°C. ℃, -21 ℃ to -19 ℃, -19 ℃ to -17 ℃, -17 ℃ to -15 ℃, -15 ℃ to -13 ℃, -13 ℃ to -11 ℃, -11 ℃ to -9 ℃, -9 ℃ to -7 ℃, -7 ℃ to -5 ℃, -5 ℃ to -3 ℃, -3 ℃ to -1 ℃, -1 ℃ to 1 ℃, 1 ℃ to 3 ℃, 3 ℃ to 5 ℃, 5 ℃ to 7 ℃, 7 ℃ to 9 ℃, 9 ℃ to 11 ℃, 11 ℃ to 13 ℃ or 13 ℃ to 15 ℃. Preferably, it is -10 ℃ to +10 ℃. In some cases, the temperature at which step (c) is performed is 15° C., 14° C., 13° C., 12° C., 11° C., 10° C., 9° C., 8° C., 7° C., 6° C., 5° C., 4° C., 3° ​​C., 2° C., 1° C., 0° C., -1° C., -2° C., -3° C., -4° C., -5° C., -6° C., -7° C., -8° C., -9° C., -10° C., -11° C., -12° C., -13° C., -14° C., -15° C., -16° C., -17° C., -18° C., -19° C., -20° C., -21° C., -22° C., -23° C., -24° C., or -25° C. In some specific embodiments, the temperature at which step (c) is performed is 0° C. to 8° C.

[0076] Preferably, the molecular ratio of albumin to glutathione (albumin:glutathione) is 1:0.5 to 1:5, for example 1:1, 1:2, 1:3, 1:4, or 1:5. In some specific embodiments, the molecular ratio of albumin to glutathione (albumin:glutathione) is 1:0.5 to 1:2.5. In another embodiment, the ratio may be 1:0.75 to 1:1.5. Specifically, the ratio may be 1:1.

[0077] In some embodiments of the method of the first aspect, step (b), and optionally step (c), are performed before at least one additional time-consuming step, wherein the at least one additional time-consuming step is selected from the following: a liquid-solid separation step, preferably a filtration, sedimentation, precipitation or centrifugation step; a reconstitution step; a resuspension step; a concentration step, preferably an ultrafiltration or diafiltration step; and a recovery step from storage.

[0078] In the context of the present disclosure, it is noteworthy that the simple process of thawing, for example, a 2000 liter batch of frozen pooled donor plasma solution typically requires a time period of at least 3 hours, and sometimes at least 5 hours.

[0079] Carrying out step (b), and optionally also step (c), before any (or several or all) of the time-consuming steps (generally requiring at least 3 hours, sometimes at least 5 hours) of a conventional plasma fractionation process (i.e., the Cohn process or the Kistler Nitschmann process and variations thereof) counteracts the undesirable oxidation of the desired mercaptalbumin to undesirable non-mercaptoalbumin during the course of each of the respective steps of the respective process (or during the course of each of these respective steps). This has the advantage that the final albumin product has an increased mercaptalbumin content compared to an albumin product simply prepared by a conventional process.

[0080] Preferably, the method further comprises providing the glutathione to be combined with the solution containing human albumin in step (b) in a container, and then adding human plasma or human whole blood to the container, and wherein optionally, the human plasma or human whole blood is stored in the container. Thus, glutathione can be combined with the starting material of the Cohn or Kistler Nitschmann process at the time of donation or shortly thereafter, when the whole blood or plasma (i.e., the human albumin solution) is filled into the plasma bag or bottle. In some or all of the above embodiments of the method of the first aspect, step (b) may be performed before the step of harvesting cryo-poor plasma. If so, the method further comprises providing the glutathione to be combined with the solution containing human albumin in step (b) in a container, and then:

[0081] - combining frozen human plasma donations in said container;

[0082] - Thaw frozen human plasma donations at 0°C to 5°C;

[0083] - separation of the still solid cryoprecipitate from the liquid cryo-poor plasma; and

[0084] - Optionally, the liquid cryo-poor plasma is further processed by capturing coagulation factors to produce coagulation factor-poor plasma.

[0085] The step of thawing several cryo-poor plasma batches for pooling typically takes a time of 3 to 10 hours, such as 4 to 10 hours, 5 to 10 hours, 6 to 10 hours, 7 to 10 hours, 8 to 10 hours or 9 to 10 hours.

[0086] Once the solid cryoprecipitate has been separated from the liquid cryo-poor plasma, the liquid cryo-poor plasma can be further processed on a synthetic resin (such as Sephadex or a heparin-coupled resin) to capture coagulation factor IX (PPSB or FXI). PPSB stands for prothrombin complex concentrate (PCC) or prothrombin complex with factor VIII inhibitor-bypassing activity and typically contains coagulation factors II, VII, IX, and X.

[0087] In some or all of the above embodiments of the method of the first aspect, step (b) is performed before the step of forming Fractions I+II+III by the Cohn process or the step of forming Precipitate A by the Kistler-Nitschmann (KN) process, and the method further comprises adding glutathione to be combined with the human albumin solution to the cryoprecipitated plasma or coagulation factor-poor plasma, followed by the addition of ethanol. Forming Fractions I+II+III by the Cohn process or forming Precipitate A by the Kistler-Nitschmann (KN) process typically involves the use of a filter aid and lowering the temperature to below -1°C, for example, -1°C to -6°C. The entire process involves several substeps, making it time-consuming, typically requiring up to 32 hours.

[0088] In some or all of the above embodiments of the method of the first aspect, step (b) is performed before the step of forming Fraction IV by the Cohn process or the step of forming Precipitate A by the KN process, wherein the method further comprises adding glutathione to be combined with the human albumin solution to the supernatant of Fractions I+II+III or the supernatant of Precipitate A, followed by the addition of ethanol. Forming Fraction IV by the Cohn process or forming Precipitate A by the KN process typically involves the use of a filter aid and lowering the temperature to below -4°C, for example, -4°C to -10°C. The entire process includes several sub-steps that make it time-consuming, typically requiring up to 11 hours, for example, 8 to 11 hours (excluding the potential precipitation step for capturing Factor IV).

[0089] In some or all of the above embodiments of the method of the first aspect, step (b) is performed before the step of forming Fraction V by the Cohn process or the step of forming Precipitate A by the KN process, wherein the method further comprises adding glutathione to be combined with the human albumin solution to the supernatant of Fraction IV, followed by the addition of ethanol. Forming Fraction V by the Cohn process or forming Precipitate A by the KN process typically does not include the use of a filter aid, but does include lowering the temperature to below -8°C, for example, -8°C to -10°C. The entire step includes several substeps that make it time-consuming, typically requiring up to 62 hours, for example, 18 to 62 hours, including some hold time.

[0090] In some or all of the above embodiments of the method of the first aspect, step (b) is performed before the step of resuspending Fraction V using the Cohn process or the step of resuspending Precipitate C using the KN process, wherein the method further comprises adding glutathione to be combined with the human albumin solution to Fraction V before resuspending in water for injection (WFI). Resuspending Fraction V using the Cohn process or resuspending Precipitate C using the KN process typically involves raising the temperature to below +1°C, for example, from +1°C to -4°C. This entire step, including homogenization but not diafiltration, is time-consuming and typically requires up to 22 hours, for example, from 7.5 to 22 hours.

[0091] In this context, it is particularly useful that the conversion step (c) can be performed at low temperatures, since the process for preparing a human albumin solution from pooled donor plasma, as described above, conventionally includes resuspension of a frozen intermediate, such as a Cohn process frozen Fraction V paste. In certain embodiments, it is advantageous that the human albumin solution of step (a) is provided as a corresponding thawed / resuspended solution containing a frozen human albumin concentrate or a frozen human albumin paste. In such cases, glutathione is combined with the solution at a molecular ratio of albumin to glutathione (albumin:glutathione) of 1:0.5 to 1:10, based on the albumin content of the fully resuspended frozen intermediate, and the conversion of step (c) is performed throughout the resuspension time (e.g., within a period of 7.5 to 22 hours).

[0092] Typically, the method further comprises a final adjustment of the protein content of the human albumin solution, during which one or more stabilizers selected from the group consisting of amino acids, sugars, and sugar alcohols are optionally added. Preferably, the one or more stabilizers are selected from the group consisting of glycine, glutamic acid, arginine, lysine, maltose, and sorbitol.

[0093] Preferably, the method further comprises pathogen inactivation or pathogen removal, in particular virus inactivation or virus removal, of the human albumin solution, preferably by nanofiltration, solvent / detergent treatment (S / D treatment), low pH treatment or pasteurization of the human albumin solution, or a combination thereof.

[0094] Typically, pasteurization of human albumin solutions is performed in a one-step or two-step process, optionally under an inert atmosphere. The one-step pasteurization process is performed in the final container containing the stabilizers N-acetyl-tryptophan and octanoic acid, while the two-step pasteurization process involves an additional pasteurization using the same stabilizers just before filling the albumin into the final container.

[0095] If an S / D treatment is employed, it is preferred to use an environmentally friendly detergent, i.e., a detergent other than Triton X-100. For example, polyethylene glycol ethers, secondary alcohol ethoxylates, secondary alkoxy (sec-alcoxy) polyethylene glycol (Tergitol 15-S-9), Nereid, or a polysorbate (e.g., polysorbate 20 or polysorbate 80 (Tween 20 or Tween 80)). However, in some embodiments of the present invention, removal of any S / D agent is performed without oil extraction, i.e., in some embodiments of the present invention, the S / D treatment does not include an oil extraction step. Instead, the undesired S / D agent is removed using other purification means available to the skilled person, such as, for example, by (a) chromatography, e.g., by using a chromatographic gel or resin, (b) fractionation methods, e.g., precipitation methods, or (c) filtration, e.g., by diafiltration or by using an adsorbent (e.g., activated carbon).

[0096] If S / D treatment is employed, it can replace pasteurization of the human albumin solution as a viral inactivation step. Therefore, S / D treatment of the human albumin solution for viral inactivation (which is suitable for completely replacing pasteurization) is performed without the addition of indole stabilizers (e.g., N-acetyl-tryptophan) or fatty acid stabilizers (e.g., caprylate). Instead, alternative stabilizers can be used, for example, stabilizers selected from the group consisting of sugars, amino acids, and sugar alcohols. Preferably, the one or more stabilizers are selected from the group consisting of glycine; glutamic acid; arginine; lysine; maltose; and sorbitol.

[0097] In some embodiments, viral inactivation is performed by using a low pH treatment. Many viruses are irreversibly denatured and effectively destroyed at a pH of 5.0 to 5.5. However, several enveloped viruses are only effectively inactivated at a pH range of 3.5 to 4. Therefore, a low pH treatment in the context of the present invention is a treatment at a pH of 5.5 or lower, preferably at a pH of 3.0 to 5.5, for example, at a pH of about 3.0, 3.5, 4.0, 4.5, 5.0, or 5.5.

[0098] In some embodiments, the low pH treatment at a pH range of 3.0 to 5.5 acts as an albumin defatting step. Thus, simultaneous viral inactivation and albumin defatting are possible without the need to remove viral inactivating compounds. Simply adjusting the pH to an albumin-compatible value after the low pH treatment is sufficient.

[0099] Preferably, the method for preparing a human albumin solution comprising an increased mercaptalbumin content and / or an increased transport capacity for reactive oxygen species, reactive nitrogen species and endogenous and exogenous ligands comprises filling the solution into a final container followed by final flushing of the final container with an inert gas.

[0100] Uses of the present invention

[0101] In a fourth aspect, the present invention relates to the use of glutathione for increasing the mercaptalbumin content and / or increasing the transport capacity of a human albumin solution for reactive oxygen species, reactive nitrogen species, and endogenous and exogenous ligands, wherein the human albumin solution is obtained during a cold ethanol precipitation process of human plasma, during which glutathione is added at least once, wherein the use comprises:

[0102] (a) providing a human albumin solution comprising mercaptalbumin and non-mercaptoalbumin, wherein the non-mercaptoalbumin comprises a disulfide bond moiety at position Cys-34;

[0103] (b) combining glutathione with the human albumin solution at least once at a molecular ratio of albumin to glutathione (albumin:glutathione) of 1:0.5 to 1:10; wherein; and

[0104] (c) converting at least a portion of the non-mercaptoalbumin into mercaptoalbumin at a temperature below 15°C,

[0105] wherein said mercaptoalbumin content in said human albumin solution and / or said transport capacity of said human albumin solution for reactive oxygen species, reactive nitrogen species and endogenous and exogenous ligands is increased relative to the mercaptoalbumin content of a human albumin solution prepared in a cold ethanol precipitation process of corresponding human plasma in the absence of steps (b) and (c).

[0106] As shown, the conversion of (c) is advantageously carried out at a relatively low temperature, less than 15°C. Although the inventors expected that the conversion efficiency would be lower at lower temperatures, it has been found that lower temperatures strike a desired balance between the slower desired conversion of non-mercaptoalbumin to mercaptoalbumin and the slower undesirable oxidation of Cys-34, so that the mercaptoalbumin content can be increased as described. In some cases, the temperature of (c) is less than 14°C, less than 13°C, less than 12°C, less than 11°C, less than 10°C, less than 9°C, less than 8°C, less than 7°C, less than 6°C, less than 5°C, less than 4°C, less than 3°C, less than 2°C, less than 1°C, less than 0°C, less than -1°C, less than -2°C, less than -3°C, less than -4°C, less than -5°C, less than -6°C, less than -7°C, less than -8°C, less than -9°C, or less than -10°C.

[0107] In some cases, the temperature of (c) is -25°C to -22°C, -22°C to -19°C, -19°C to -16°C, -16°C to -13°C, -13°C to -10°C, -10°C to -7°C, -7°C to -4°C, -4°C to -1°C, -1°C to 2°C, 2°C to 5°C, 5°C to 8°C, 8°C to 11°C, 11°C to 14°C, or -25°C to -23°C, -23°C to -21°C, -21°C to -19°C, -19°C to -17°C, -17°C to -15°C, -15°C to -13°C, -13°C to -11°C, -11°C to -9°C, -9°C to -7°C, -7°C to -5°C, -5°C to -3°C, -3°C to -1°C, -1°C to 1°C, 1°C to 3°C, 3°C to 5°C, 5°C to 7°C, 7°C to 9°C, 9°C to 11°C, 11°C to 13°C, or 13°C to 15°C. Preferably, -10°C to +10°C. In some cases, the temperature of (c) is 15° C., 14° C., 13° C., 12° C., 11° C., 10° C., 9° C., 8° C., 7° C., 6° C., 5° C., 4° C., 3° ​​C., 2° C., 1° C., 0° C., -1° C., -2° C., -3° C., -4° C., -5° C., -6° C., -7° C., -8° C., -9° C., -10° C., -11° C., -12° C., -13° C., -14° C., -15° C., -16° C., -17° C., -18° C., -19° C., -20° C., -21° C., -22° C., -23° C., -24° C., or -25° C. In some embodiments, step (c) is performed at a temperature between 0° C. and 8° C.

[0108] Preferably, the molecular ratio of albumin to glutathione (albumin:glutathione) is 1:0.5 to 1:5, for example 1:1, 1:2, 1:3, 1:4, or 1:5. In some specific embodiments, the molecular ratio of albumin to glutathione (albumin:glutathione) is 1:0.5 to 1:2.5. In another embodiment, the ratio may be 1:0.75 to 1:1.5. Specifically, the ratio may be 1:1.

[0109] In some embodiments, (b), and optionally (c), are performed before at least one further time-consuming step selected from the group consisting of: a liquid-solid separation step, preferably a filtration, sedimentation, precipitation or centrifugation step; a reconstitution step; a resuspension step; a concentration step, preferably an ultrafiltration or diafiltration step; and a recovery step from storage.

[0110] Carrying out (b), and optionally also (c), before any (or several or all) of the time-consuming steps (generally requiring at least 3 hours, sometimes at least 5 hours) of conventional plasma fractionation methods (i.e., the Cohn process or the Kistler-Nitschmann process and their variations) counteracts the undesired oxidation of the desired mercaptalbumin to undesired non-mercaptoalbumin during (or during) each of the respective steps of the respective method. This has the advantage that, by using glutathione, an increase in the mercaptalbumin content in the human albumin solution can be achieved compared to a human albumin solution that has not been treated with glutathione as described but has only undergone conventional treatment.

[0111] Preferably, the glutathione to be combined with the solution comprising human albumin in (b) is available in the container before adding human plasma or human whole blood to the container, so that the human plasma or human whole blood can optionally be stored in the container.

[0112] In some or all of the above embodiments of the fourth aspect, (b) may be performed before the step of harvesting cryo-depleted plasma. If so, the method further comprises providing the glutathione in (b) to be combined with the solution comprising human albumin in a container, and then:

[0113] - combining frozen human plasma donations in said container;

[0114] - Thaw frozen human plasma donations at 0°C to 5°C;

[0115] - separation of the still solid cryoprecipitate from the liquid cryo-poor plasma; and

[0116] - Optionally, the liquid cryo-poor plasma is further processed by capturing coagulation factors to produce coagulation factor-poor plasma.

[0117] In some or all of the above embodiments of the fourth aspect, (b) is performed before the step of forming fractions I+II+III of the Cohn process or the step of forming precipitate A of the Kistler-Nitschmann (KN) method, and wherein the method further comprises adding glutathione to be combined with the human albumin solution to the cryoprecipitated plasma or coagulation factor-poor plasma, and then adding ethanol.

[0118] In some or all of the above embodiments of the fourth aspect, (b) is performed before the step of forming Fraction IV of the Cohn process or the step of forming Precipitate A of the KN process, wherein the method further comprises adding glutathione to be combined with the human albumin solution to the Fraction I+II+III supernatant or the Precipitate A supernatant, and then adding ethanol.

[0119] In some or all of the above embodiments of the fourth aspect, (b) is performed before the step of forming Fraction V of the Cohn process or the step of forming Precipitate A of the KN process, wherein the method further comprises adding glutathione to be combined with the human albumin solution to the Fraction IV supernatant, followed by the addition of ethanol.

[0120] In some or all of the above embodiments of the fourth aspect, (b) is performed before the step of resuspending fraction V of the Cohn process or the step of resuspending pellet C of the KN process, wherein the method further comprises adding glutathione to be combined with the human albumin solution to fraction V prior to resuspending in water for injection (WFI).

[0121] Typically, the use further comprises a final adjustment of the protein content of the human albumin solution, during which one or more stabilizers selected from the group consisting of amino acids, sugars, and sugar alcohols are optionally added. Preferably, the one or more stabilizers are selected from the group consisting of glycine, glutamic acid, arginine, lysine, maltose, and sorbitol.

[0122] Preferably, the use further comprises pathogen inactivation or pathogen removal, in particular virus inactivation or virus removal, of the human albumin solution, preferably by nanofiltration, solvent / detergent treatment (S / D treatment), low pH treatment or pasteurization of the human albumin solution, or a combination thereof.

[0123] Typically, pasteurization of human albumin solutions is performed in a one-step process or a two-step process, optionally under an inert atmosphere.

[0124] If an S / D treatment is employed, it is preferred to use an environmentally friendly detergent, i.e., a detergent other than Triton X-100. For example, polyethylene glycol ethers, secondary alcohol ethoxylates, secondary alkoxy polyethylene glycols (Tergitol 15-S-9), Nereid, or polysorbates (e.g., polysorbate 20 or polysorbate 80 (Tween 20 or Tween 80)). However, in some embodiments of the present invention, removal of any S / D agent is performed without oil extraction, i.e., in some embodiments of the present invention, the S / D treatment does not include an oil extraction step. Instead, the undesired S / D agent is removed using other purification means available to the skilled person, such as, for example, by (a) chromatography, e.g., using a chromatographic gel or resin, (b) fractionation methods, e.g., precipitation methods, or (c) filtration, e.g., by diafiltration or by using an adsorbent (e.g., activated carbon).

[0125] If S / D treatment is employed, it can replace pasteurization of the human albumin solution as a viral inactivation step. Therefore, S / D treatment of the human albumin solution for viral inactivation (which is suitable for completely replacing pasteurization) is performed without the addition of indole stabilizers (e.g., N-acetyl-tryptophan) or fatty acid stabilizers (e.g., caprylate). Instead, alternative stabilizers can be used, for example, stabilizers selected from the group consisting of sugars, amino acids, and sugar alcohols. Preferably, the one or more stabilizers are selected from the group consisting of glycine; glutamic acid; arginine; lysine; maltose; and sorbitol.

[0126] In some embodiments, viral inactivation and defatting are performed by using a low pH treatment. Many viruses are irreversibly denatured and effectively destroyed at pH 5.0 to 5.5. However, several enveloped viruses are effectively inactivated only at a pH range of 3.5 to 4. Therefore, a low pH treatment in the context of the present invention is a treatment at a pH of 5.5 or lower, preferably at a pH of 3.0 to 5.5, for example, at a pH of about 3.0, 3.5, 4.0, 4.5, 5.0, or 5.5.

[0127] Preferably, the use of glutathione for increasing the mercaptalbumin content and / or increasing the transport capacity of a human albumin solution for reactive oxygen species, reactive nitrogen species, and endogenous and exogenous ligands comprises filling the solution into a final container, followed by flushing the final container with an inert gas, the human albumin solution comprising an increased mercaptalbumin content and / or an increased transport capacity for reactive oxygen species, reactive nitrogen species, and endogenous and exogenous ligands.

[0128] In a fifth aspect, the present invention relates to a human albumin solution comprising an increased mercaptoalbumin content obtained by the use according to the fourth aspect as described above, characterised in that the human albumin solution comprises a mercaptoalbumin content of at least 80%, in particular at least 85%, relative to the total albumin content of the solution.

[0129] The human albumin solution of the present invention can be used to treat diseases. Specifically, it is used to treat patients in need thereof. In particular, it is used to treat the following patients:

[0130] - Patients with liver failure; and / or

[0131] - Patients with chronic hepatitis, acute hepatitis, cirrhosis, fulminant hepatic failure or hepatocellular carcinoma; and / or

[0132] - Patients with renal failure; and / or

[0133] - Patients suffering from hypovolemia, and for the restoration and maintenance of circulating blood volume in said patients.

[0134] Albumin product of the present invention

[0135] In the second and third aspects and in the fifth and sixth aspects, the present invention relates to a human albumin solution comprising an increased mercaptalbumin content and / or an increased transport capacity of the human albumin solution for reactive oxygen species, reactive nitrogen species and endogenous and exogenous ligands when prepared by the method according to the first aspect or the use according to the fourth aspect, respectively.

[0136] The corresponding human albumin solution can be used as a medicine.

[0137] Specifically, the corresponding human albumin solution is used to treat patients in need thereof. In particular, it is used to treat the following patients:

[0138] - Patients with liver failure; and / or

[0139] - Patients with chronic hepatitis, acute hepatitis, cirrhosis, fulminant hepatic failure or hepatocellular carcinoma; and / or

[0140] - Patients with renal failure; and / or

[0141] Patients suffering from hypovolemia, and for restoring and maintaining circulating blood volume in said patients.

[0142] Example

[0143] Evaluation of free cysteine ​​34 in albumin by mass spectrometry

[0144] A mass spectrometry (MS) method was developed to determine the oxidation state of cysteine ​​34 (Cys-34) in human albumin. A control sample was prepared by oxidizing albumin with 3% H₂O₂ to validate the ability of the established MS method to identify oxidized human non-mercaptoalbumin-1 (HNA1) and human non-mercaptoalbumin-2 (HNA2) moieties. An untreated reference sample was also prepared.

[0145] To convert HNA1 to human mercaptalbumin (HMA), fraction V was resuspended in the presence of a reducing agent. To prepare the sample for analysis, depth filtration, pH adjustment, and a subsequent diafiltration step were performed to remove residual glutathione. This method was performed using a reference sample without glutathione and five samples with increasing molar ratios of albumin to glutathione as the reducing agent (albumin:glutathione) of 1:0.5, 1:1.5, 1:2.5, 1:5, and 1:10.

[0146] As a control, a sample of the resuspended fraction V that was not treated with a reducing agent was removed after depth filtration, pH adjustment, and ultrafiltration / diafiltration. This sample represents the untreated albumin concentrate. After determining the protein concentration, several smaller aliquot control samples were generated, each containing 10% albumin. The untreated control sample contained only 10% albumin. The oxidized control sample contained 3% H2O2 in a 10% albumin solution. The reduced control sample contained 5 mM DTT in a 10% albumin solution.

[0147] All samples (ie control samples and samples according to the method of the invention) were measured to validate the method and to show the effect of different albumin:glutathione on the oxidation state of the albumin molecule.

[0148] All stock solutions, buffers, and dilutions were prepared using ultrapure water (LiChrosolv) from Merck to avoid interference with the mass spectrometry method. All materials used were single-use only to avoid any interference with the mass spectrometry measurements.

[0149] Table 1 shows the temperature and duration of each unit operation.

[0150]

[0151] All reagents were pre-cooled to 4°C and resuspension of fraction V took place in a cooling chamber at a temperature of -1.5 ± 1°C.

[0152] For each sample, 10 g of fraction V was added to a 50 ml tube and a 1.6-fold amount of the water / glutathione mixture was added as described below. This resulted in a protein solution of approximately 100 g / l. Resuspend the protein solution with stirring on a shaker for 20 hours.

[0153] Table 2 Composition of method samples.

[0154]

[0155] After resuspension, the temperature of the cooling chamber was raised to 3.5 ± 1.5°C, and the solution was filtered using a Millipore 0.22 µm filter. After filtration, the solution was incubated for an additional 2 hours. During the unit operation, the pH was adjusted by slowly adding a calculated amount (21.1 g) of 1.25 M NaOH to the solution at 4°C. The samples were incubated for a total incubation duration of approximately 1 hour. Then, 8 ml of each sample was dialyzed at 4°C using Slide-A-Lyzer G3 dialysis cassettes from Thermo Fisher (prepared as described in the manufacturer's protocol) with a molecular weight cutoff of 20 kD. The buffer solution was changed twice during the 10-hour dialysis of the sample against 60 mM NaCl solution. Ultrafiltration / diafiltration can also be used instead of dialysis and is preferred at an industrial scale.

[0156] For LC-MS, the following solvents, gradients, and flow rates were applied.

[0157] Table 3. Solvents used during MS.

[0158]

[0159] A gradient of 5% to 85% D in 3.5 min was applied, and the flow rate was 0.4 ml / min.

[0160] LC-MS with 4 modules:

[0161] 1. Mass spectrometer Xevo G2-XS QTof with ESI source (Waters; catalog number: e.g., 186010532)

[0162] 2. Oil-free vacuum pump (Edwards; catalog number: e.g. XDS46)

[0163] 3. ACQUITY UPLC H-Class Bio System

[0164] (A) Sample manager (Waters; catalog number: e.g., 186015040)

[0165] (B) Quaternary solvent manager (Waters; catalog number: e.g., 186015041)

[0166] (C) Column manager (Waters; catalog number: e.g., 186015043 )

[0167] 4. Computer with Waters Connectivity Software (including the UNIFI App with the Large Molecule option)

[0168] Column: ACQUITY UPLC® Protein BEH C4 column 2.1 × 50 mm (Waters; catalog number: e.g., 186004495)

[0169] Results - Control Sample

[0170] Table 4 Results for control samples.

[0171]

[0172] Analysis of the reduced control sample containing DTT did not yield interpretable results due to the high heterogeneity of the sample, which did not allow for the identification of clear HMA, HNA1, or HNA2 peaks. Without wishing to be bound by theory, this heterogeneity may be a result of additional disulfide bond reduction and breakage within the albumin.

[0173] Oxidation with 3% H2O2 showed complete oxidation of HMA to HNA1 and HNA2.

[0174] Results - Experimental Samples

[0175] Table 5 shows the obtained results for the process samples.

[0176]

[0177]

[0178] in conclusion

[0179] As can be seen from the above results, compared to the untreated control sample, the addition of glutathione to albumin at a ratio of 1:0.5 has led to a favorable increase in HMA, specifically resulting in an HMA content exceeding 85%. Increasing this ratio to greater than 1:1.5 further increased the HMA content to above 90%, stabilizing between 91% and 93%. The maximum HMA content was achieved when the albumin:glutathione ratio was 1:2.5. At the same time, the HNA1 content of the experimental samples was significantly reduced, essentially decreasing to 0% in most experimental samples.

[0180] While albumin oxidation was very effective, reduction of the protein with DTT produced a very diverse spectrum in which no single peak could be assigned. This was likely due to disruption of internal disulfide bonds.

[0181] Many modifications and other embodiments of the invention described herein will come to mind to one skilled in the art having the benefit of the teachings presented in the foregoing description and the associated drawings. Therefore, it should be understood that the invention is not limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

Claims

1. A method for preparing a human albumin solution having an increased mercaptalbumin content and / or an increased transport capacity for reactive oxygen species, reactive nitrogen species, and endogenous and exogenous ligands, wherein the human albumin solution is obtained in the course of a cold ethanol precipitation process of human plasma, wherein the method comprises the following steps: (a) providing a human albumin solution comprising mercaptalbumin and non-mercaptoalbumin, wherein the non-mercaptoalbumin comprises a disulfide bond moiety at position Cys-34; (b) combining glutathione with the human albumin solution at least once at a molecular ratio of albumin to glutathione (albumin:glutathione) of 1:0.5 to 1:10; and (c) converting at least a portion of the non-mercaptoalbumin into mercaptalbumin at a temperature below 15°C, wherein the mercaptoalbumin content in the human albumin solution and / or the transport capacity of the human albumin solution for reactive oxygen species, reactive nitrogen species, and endogenous and exogenous ligands is increased relative to the mercaptoalbumin content of a human albumin solution prepared in a corresponding human plasma cold ethanol precipitation process lacking steps (b) and (c).

2. The method according to claim 1, wherein the molecular ratio of albumin to glutathione (albumin:glutathione) is 1:0.5 to 1:5, preferably 1:0.5 to 1:2.5, more preferably 1:0.75 to 1:1.

5.

3. The method according to claim 1 or claim 2, wherein step (b) is performed before at least one further time-consuming step, the further time-consuming step being selected from the group consisting of: a liquid-solid separation step, preferably a filtration, sedimentation, precipitation or centrifugation step; a reconstitution step; a resuspension step; a concentration step, preferably an ultrafiltration or diafiltration step; and a recovery step from storage, and wherein optionally, the time-consuming step requires a time of at least 3 hours, preferably at least 5 hours.

4. The method according to any one of claims 1 to 3, wherein the method further comprises providing the glutathione to be combined with the human albumin solution in step (b) in a container, and then adding human plasma or human whole blood to the container, and wherein optionally, the human plasma or human whole blood is stored in the container.

5. The method according to any one of claims 1 to 3, wherein step (b) is performed before the step of harvesting cryo-poor plasma, and wherein the method further comprises providing the glutathione to be combined with the solution comprising human albumin in step (b) in a container, and then: - combining frozen human plasma donations in said container; - thawing the frozen human plasma donation at a temperature of 0°C to 5°C; - separating the still solid cryoprecipitate from the liquid cryo-depleted plasma; and - Optionally, further processing the liquid cryo-poor plasma by capturing coagulation factors to produce coagulation factor-poor plasma.

6. The method according to any one of claims 1 to 5, wherein: step (b) is performed before the step of forming fractions I+II+III by the Cohn process or the step of forming precipitate A by the Kistler-Nitschmann (KN) process, and wherein the method further comprises adding the glutathione to be combined with the human albumin solution to cryoprecipitated plasma or coagulation factor-poor plasma, and then adding ethanol; and / or wherein step (b) is performed before the step of forming fraction IV by the Cohn process or the step of forming precipitate A by the KN process, wherein the method further comprises adding the glutathione to be combined with the human albumin solution to the supernatant of fractions I+II+III or the supernatant of precipitate A, and then adding ethanol; and / or Step (b) is performed before the step of forming fraction V by the Cohn process or the step of forming precipitate A by the KN process, wherein the method further comprises adding the glutathione to be combined with the human albumin solution to the supernatant of fraction IV, and then adding ethanol; and / or Step (b) is performed before the step of resuspending fraction V in the Cohn process or the step of resuspending precipitate C in the KN process, wherein the method further comprises adding the glutathione to be combined with the human albumin solution to fraction V, and then resuspending it in water for injection (WFI).

7. The method according to any one of claims 1 to 6, wherein the method further comprises a final adjustment of the protein content of the human albumin solution, during which one or more stabilizers selected from the group consisting of: amino acids; sugars; and sugar alcohols are optionally added.

8. The method of claim 7, wherein the one or more stabilizers are selected from the group consisting of: glycine; glutamic acid; arginine; lysine; maltose; and sorbitol.

9. The method according to any one of claims 1 to 8, wherein the method further comprises subjecting the human albumin solution to pathogen inactivation or pathogen removal, in particular virus inactivation or virus removal, preferably by subjecting the human albumin solution to nanofiltration, solvent / detergent treatment (S / D treatment), low pH treatment or pasteurization, or a combination thereof, and Optionally, pasteurization of the human albumin solution is performed in a one-step process or a two-step process, optionally under an inert atmosphere.

10. The method according to any one of claims 1 to 9, comprising filling the solution into a final container and subsequently flushing the final container with an inert gas.

11. The method according to any one of claims 1 to 10, wherein: (a) The active oxygen species is selected from: H2O2, O2 - and HOCl; (b) The active nitrogen species is selected from: ONOO - and ONOOCO2 - ; (c) The endogenous and exogenous ligands are selected from the group consisting of fatty acids (FA), nucleic acids, hormones, metals, toxins, and drugs.

12. A human albumin solution comprising an increased mercaptalbumin content prepared by the process according to any one of claims 1 to 11, characterized in that it comprises a mercaptalbumin content of at least 85%, in particular at least 88%, preferably 90% to 99%, or 90% to 95%, or 90% to 93%, relative to the total albumin content of the solution.

13. The human albumin solution comprising an increased mercaptalbumin content according to claim 12, for use in treating a patient in need thereof.

14. The human albumin solution comprising an increased mercaptalbumin content according to claim 12, for use in treating patients suffering from liver failure.

15. The human albumin solution comprising an increased mercaptoalbumin content for use according to claim 14, wherein the use comprises administering the human albumin solution to a patient suffering from chronic hepatitis, acute hepatitis, cirrhosis, fulminant hepatic failure or hepatocellular carcinoma.

16. The human albumin solution comprising an increased mercaptalbumin content according to claim 12, for use in treating patients suffering from renal failure.

17. The human albumin solution comprising an increased mercaptalbumin content of claim 12 for use in restoring and maintaining circulating blood volume in a patient who has exhibited hemorrhage volume depletion.

18. Use of glutathione for increasing the mercaptalbumin content of a human albumin solution, wherein the human albumin solution is obtained during a cold ethanol precipitation process of human plasma, during which glutathione is added at least once, wherein the use comprises: (a) providing a human albumin solution comprising mercaptalbumin and non-mercaptoalbumin, wherein the non-mercaptoalbumin comprises a disulfide bond moiety at position Cys-34; (b) combining glutathione with the human albumin solution at least once at a molecular ratio of albumin to glutathione (albumin:glutathione) of 1:0.5 to 1:10; and (c) converting the non-mercaptoalbumin into mercaptoalbumin at a temperature below 15°C, wherein the mercaptoalbumin content in the human albumin solution is increased relative to the mercaptoalbumin content of a human albumin solution prepared in a corresponding human plasma cold ethanol precipitation process lacking steps (b) and (c).

19. A human albumin solution comprising an increased mercaptoalbumin content obtained by the use of glutathione according to claim 17, said human albumin solution being characterized in that it comprises a mercaptoalbumin content of at least 85%, in particular at least 88%, preferably from 90% to 99%, or from 90% to 95%, or from 90% to 93%, relative to the total albumin content of said solution.

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