METHOD FOR PREPARING ALBUMIN SOLUTIONS AND RELATED USES OF THE SAME AND OF THE SAME

Abstract

The present invention relates to human albumin solutions and in particular to methods and uses of preparing them. The invention further relates to human albumin solutions, which have an increased mercaptalbumin content and / or an increased transport capacity for reactive oxygen species, reactive nitrogen species as well as endogenous and exogenous ligands. Embodiments of the invention have been particularly developed for use in the treatment of disease and will be described hereinafter with reference to this application. However, it will be appreciated that the invention is not limited to this particular field of use.

Description

1 / 34 METHOD FOR PREPARING ALBUMIN SOLUTIONS AND RELATED USES THEREOF AND OF THE SAME FIELD OF THE INVENTION

[0001] The present invention relates to solutions of human albumin and, in particular, to methods and uses for preparing them. The invention also relates to solutions of human albumin having an increased content of human mercaptalbumin (HMA) and / or an increased transport capacity for reactive oxygen species, reactive nitrogen species, as well as endogenous and exogenous ligands. The embodiments of the invention have been particularly developed for use in the treatment of diseases and will be described herein with reference to this application. However, it will be appreciated that the invention is not limited to this particular field of use. BACKGROUND OF THE INVENTION

[0002] Any discussion of the prior art technique throughout the descriptive report should in no way be considered as an admission that such technique 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 is the main carrier of endogenous and exogenous ligands, particularly fatty acids (FAs), as well as nucleic acids, hormones, metals, toxins, and drugs. It is responsible for most of the pro- and antioxidant capacity of plasma and has (pseudo)enzymatic properties. Fatty acids are the physiological ligands of human albumin, which is characterized by nine fatty acid binding sites, FA1-FA9. Fatty acids bind to FA1 to FA9 sites with varying degrees of affinity: FA2, FA4, and FA5 are high-affinity sites, FA1 and FA3 have medium affinity, and FA6 and FA7 have low affinity. HSA plays an important role as a drug carrier (e.g., for antibiotics, anticoagulants, antineoplastics, antivirals, anesthetics, anxiolytics, and nonsteroidal anti-inflammatory drugs), influencing their Petition 870250083253, dated 09 / 16 / 2025, page 51 / 204 2 / 34 Pharmacokinetics and pharmacodynamics. The FA1, FA3-FA4, and FA7 pockets are the main drug-binding sites of HSA. FA1 recognizes antibiotics, antineoplastics, antiretrovirals, and nonsteroidal anti-inflammatory drugs. Ibuprofen is the prototypical ligand of the FA3-FA4 pocket, which also recognizes anesthetics, anxiolytics, and the orphan drug 4-phenylbutazone. The FA7 site is considered the main HSA binding site. The prototypical ligand is warfarin, a bulky heterocyclic anticoagulant. Several drugs bind to the FA7 site in the presence of myristate. This suggests that myristate induces one or more conformational changes at the binding site, resulting in the formation of three secondary overlapping sites capable of recognizing anesthetics, anticoagulants, antineoplastics, antiretrovirals, and nonsteroidal anti-inflammatory drugs (NSAIDs).

[0004] Furthermore, human albumin is a valuable biomarker for many diseases (e.g., cancer, rheumatoid arthritis, ischemia, obesity, and diabetes) and has clinical applications in the treatment of various pathologies, including shock, trauma, hemorrhage, acute respiratory distress syndrome, hemodialysis, acute liver failure, chronic liver disease, and hypoalbuminemia.

[0005] In recent years, there has been significant interest in the redox status of thiols in the context of disease. In human plasma, the largest cluster of thiols is again provided by human albumin. Human albumin comprises 585 amino acids in total (giving the molecule a weight of approximately 66 kDa) and contains 35 Cys residues. However, it contains only a single cysteine ​​(Cys-34), which is not involved in intramolecular disulfide bonds. As the main plasma protein, albumin has been studied as a major target of oxidative modification, and its oxidized form or redox state has been frequently discussed. Although there are several ways to oxidize albumin, Cys-34 is a particularly redox-sensitive site within albumin and, as such, one can refer to the redox state of albumin in terms of the redox state of Cys-34. The redox status of Cys-34 Petition 870250083253, dated 09 / 16 / 2025, p. 52 / 204 3 / 34 defines three fractions that generally allow the determination of the overall redox state of albumin. Namely, human mercaptalbumin (HMA), which contains a free thiol group, human non-mercaptalbumin-1 (HNA1), which contains a disulfide, and human non-mercaptalbumin-2 (HNA2), which contains a sulfinic or sulfonic acid group.

[0006] In plasma, mercaptalbumin provides several benefits, including: • Antioxidant protection: Mercaptalbumin can act as an antioxidant, helping to protect cells and tissues against damage caused by reactive oxygen species (such as H2O2, O2-, HOCl, etc.) and reactive nitrogen species (such as ONOO-, ONOOCO2-, etc.); • Detoxification: Mercaptalbumin can bind to and help remove toxic substances from the body, such as heavy metals and drugs; and • Immune function: Mercaptalbumin can help stimulate the immune system and protect against infections.

[0007] Consequently, a high percentage of non-mercaptalbumin may lead to a decreased capacity for protection against oxidative damage, detoxification of harmful substances, and / or support of immune function. Additionally, a low level of mercaptalbumin may be indicative of certain health conditions or diseases, such as liver disease or malnutrition.

[0008] For example, non-mercaptalbumin-1 is a reversible derivative, while non-mercaptalbumin-2 is irreversible. In healthy young people, about 70 to 80% of all albumin is mercaptalbumin, with non-mercaptalbumin-1 representing about 20 to 30%, and non-mercaptalbumin-2 representing only about 2 to 5%. Increased levels of non-mercaptalbumin-1 in plasma are associated with decreased antioxidant activity of human albumin. Non-mercaptalbumin-1 and non-mercaptalbumin-2 relate to the progression of inflammatory processes, while the concentration of mercaptalbumin in blood plasma is inversely correlated with atherosclerotic damage in the carotid artery. Petition 870250083253, dated 09 / 16 / 2025, page 53 / 204 4 / 34 Japanese residents.

[0009] Document WO 2013 / 024100 A2 describes methods for diagnosing and treating medical conditions associated with oxidative stress, particularly liver diseases. The methods are based on the determination and quantification of the fraction of albumin that is irreversibly oxidized to Cys34. The suggested therapeutic concept incorporates contacting the patient's plasma with an adsorbent capable of binding to non-mercaptalbumin-2, followed by infusion of fresh albumin.

[0010] 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 mercaptalbumin and non-mercaptalbumin in the plasma of patients suffering from various liver diseases. The plasma signals from one patient were compared with the signals from plasma incubated with glutathione at 25 °C from the same patient. Thus, the most prominent signal from the incubated plasma was identified as mercaptalbumin prolongation.

[0011] A similar analytical approach is known from Oettl and Marsche, 2010 (“Redox state of human serum albumin in terms of Cysteine-34 in health and disease”; Methods in Enzymology, Volume 474, 181-195), determining different albumin oxidation steps, namely, mercaptalbumin, non-mercaptalbumin 1 and non-mercaptalbumin 2, in the plasma of patients suffering from diabetes, liver damage or kidney damage.

[0012] Several approaches to modify albumin have been described, whether isolated from plasma or produced recombinantly, namely, producing albumin conjugates using the thiol reactivity of Cys-34. An exemplary process for producing albumin conjugates is known in document WO 2007 / 071068 A1. The process comprises the production and purification of recombinant albumin and the covalent linking of a compound to the Cys-34 thiol of albumin. An intermediate enrichment of Cys-34 thiol is suggested by contacting albumin with thioglycolic acid (TGA) or dithiothreitol. Petition 870250083253, dated 09 / 16 / 2025, p. 54 / 204 5 / 34 (DTT).

[0013] US patent 2013 / 329127 A1 describes the modification of thiol groups of various proteins, including human serum albumin, with PEG maleimide. A dilute albumin solution is incubated with Tris[2-carboxyethyl]phosphine hydrochloride (TCEP) for 10 minutes before the addition of a branched PEG reagent containing a terminal maleimide group. After the dark reaction, the resulting PEG-albumin conjugate is further processed and purified.

[0014] Document WO 2007 / 049941 A1 deals with bioactive albumin conjugates, in which the bioactive compound is covalently linked to albumin via a disulfide bridge. During subsequent analysis, the disulfide bridge is broken again by the reaction of the albumin conjugate with DTT at 37 °C.

[0015] Regarding the disease, document WO 2020 / 104458 A1 describes methods and kits for detecting liver dysfunction in an individual. In document WO 2020 / 104458 A1, the relative abundance of mercaptalbumin (HMA), non-mercaptalbumin-1 (HNA1), and non-mercaptalbumin-2 (HNA2) in a healthy person is revealed to be in the range of 70-80% mercaptalbumin, 20-30% non-mercaptalbumin-1, and <5% non-mercaptalbumin-2, which are ranges also revealed in Oettl and Marsche, 2010, cited above.

[0016] Because human albumin is an extremely robust protein with regard to pH stability (stable in the pH range of 4-9), temperature stability (can be heated to 60 °C for up to 10 h in the presence of stabilizers) and even exposure to organic solvents, there are several human albumin products. Typical clinical uses of human albumin products, whether purified from pooled donor plasma on an industrial scale or recombinantly produced, are the treatment of severe hypoalbuminemia or as a plasma expander.

[0017] However, commercial human albumin products have been described as comprising smaller amounts of mercaptalbumin and larger amounts of non-mercaptalbumin-1 and non-mercaptalbumin-2 compared to the respective amounts found in fresh plasma. Furthermore, the Petition 870250083253, dated 09 / 16 / 2025, page 55 / 204 6 / 34 commercially available albumins are formulated with substantial amounts (i.e., an excess) of fatty compounds, such as tryptophan and / or caprylic acid. As a result, the albumin:fatty compound ratio in commercially available albumin solutions is routinely between 1:5 and 1:8. For example, commercially available 5% octalbin albumin solution comprises 5% albumin (i.e., 50 g / l; 0.75 mmol), as well as tryptophan and caprylic acid, each at concentrations up to 4.2 mmol. 25% octalbin albumin solution comprises 25% albumin (i.e., 250 g / l; 3.8 mmol), as well as tryptophan and caprylic acid, each at concentrations up to 21 mmol.

[0018] Consequently, there is a need in the field for improved commercial products of human albumin and methods for their production.

[0019] The objective of the present invention is to overcome or improve at least one of the disadvantages of the prior art or to provide a useful alternative. In particular, it is an objective of the present invention to provide improved methods for the preparation of a human albumin solution from pooled donor plasma.

[0020] SUMMARY OF THE INVENTION

[0021] The problem stated above is solved by the invention disclosed in this document. Specifically, the disclosed methods for preparing a pooled donor plasma human albumin solution having an increased mercaptalbumin content and / or an increased carrying capacity for reactive oxygen species, reactive nitrogen species, as well as endogenous and exogenous ligands, as well as the related uses and prepared albumin solutions solve the above problem.

[0022] Consequently, 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, as well as endogenous and exogenous ligands, wherein said albumin solution Petition 870250083253, dated 09 / 16 / 2025, page 56 / 204 7 / 34 human is obtained in the course of a cold ethanol precipitation process from human blood plasma, wherein said method comprises the steps of (a) providing a solution of human albumin comprising mercaptalbumin and non-mercaptalbumin, wherein said non-mercaptalbumin comprises a disulfide fraction at the Cys-34 position; (b) combining glutathione, at least once, with said human albumin solution in a molecular ratio between albumin and glutathione (albumin:glutathione) in the range of 1:0.5 to 1:10; and (c) converting at least part of said non-mercaptalbumin into mercaptalbumin at a temperature of less than 15 °C, wherein said mercaptalbumin content in said human albumin solution and / or said transport capacity for reactive oxygen species, reactive nitrogen species, as well as endogenous and exogenous ligands of said human albumin solution is increased relative to the mercaptalbumin content of a human albumin solution prepared in a corresponding cold ethanol precipitation process from human blood plasma without steps (b) and (c).

[0023] In a second aspect, the present invention relates to a human albumin solution comprising an increased mercaptalbumin content when prepared by the method according to the first aspect, characterized in comprising a mercaptalbumin content of at least 85%, in particular at least 88%, preferably between 90 and 99% or between 90 and 95% or between 90 and 93%, relative to the total albumin content of said solution. In such embodiments, the non-mercaptalbumin-1 content is significantly reduced compared to an untreated sample. Namely, in such embodiments, the non-mercaptalbumin-1 content is less than 5%, preferably less than 4%. In particular embodiments, non-mercaptalbumin-1 is no longer detectable, i.e., the non-mercaptalbumin-1 content has been reduced to 0%. Specifically, in the modalities of this second aspect, when the mercaptalbumin content is increased to more than 90%, the non-mercaptalbumin-1 content is reduced to Petition 870250083253, dated 09 / 16 / 2025, page 57 / 204 8 / 34 less than 1%. More specifically, when the mercaptalbumin content is increased to more than 91%, the non-mercaptalbumin-1 content is reduced to 0%.

[0024] In a third aspect, the present invention relates to a human albumin solution of the second aspect for use in the treatment of a patient in need thereof.

[0025] Consequently, this third aspect of the present invention also encompasses treatment methods, wherein the method comprises administering the human albumin solution of the second aspect to a patient in need thereof and / or using a human albumin solution of the second aspect in the manufacture of a medicament for the treatment of a patient in need thereof.

[0026] Typically, treatment involves administering a human albumin solution with an increased mercaptalbumin content to a patient: - suffering from liver failure; and / or - suffering from chronic hepatitis, acute hepatitis, liver cirrhosis, fulminant hepatic failure, or hepatocellular carcinoma; and / or - suffering from kidney failure; and / or - who suffers from blood volume deficiency and for the restoration and maintenance of circulating blood volume in said patient.

[0027] In a fourth aspect, the present invention relates to the use of glutathione to increase the mercaptalbumin content, transport capacity for reactive oxygen species, reactive nitrogen species, as well as endogenous and exogenous ligands of a human albumin solution, wherein said human albumin solution is obtained in the course of a cold ethanol precipitation process of human blood plasma, during which glutathione is added at least once, wherein said use comprises: (a) supply of a human albumin solution comprising Petition 870250083253, dated 09 / 16 / 2025, page 58 / 204 9 / 34 mercaptalbumin and non-mercaptalbumin, wherein the said non-mercaptalbumin comprises a disulfide fraction in the Cys-34 position; (b) combining glutathione, at least once, with said human albumin solution in a molecular ratio between albumin and glutathione (albumin:glutathione) in the range of 1:0.5 to 1:10; and (c) converting at least part of said non-mercaptalbumin into mercaptalbumin at a temperature of less than 15 °C, wherein said mercaptalbumin content in said human albumin solution and / or said transport capacity for reactive oxygen species, reactive nitrogen species, as well as endogenous and exogenous ligands of said human albumin solution is increased relative to the mercaptalbumin content of a human albumin solution prepared in a corresponding cold ethanol precipitation process from human blood plasma without steps (b) and (c).

[0028] In a fifth aspect, the present invention relates to a human albumin solution comprising an increased mercaptalbumin content when obtained through the use of glutathione, according to the fourth aspect, characterized by comprising a mercaptalbumin content of at least 85%, in particular at least 88%, preferably between 90 and 99% or between 90 and 95% or between 90 and 93%, relative to the total albumin content of said solution. In such embodiments, the non-mercaptalbumin-1 content is significantly reduced compared to an untreated sample. Namely, in such embodiments, the non-mercaptalbumin-1 content is less than 5%, preferably less than 4%. In particular embodiments, non-mercaptalbumin-1 is no longer detectable, i.e., the non-mercaptalbumin-1 content has been reduced to 0%.Specifically, in the embodiments of this fifth aspect, when the mercaptalbumin content is increased to more than 90%, the non-mercaptalbumin-1 content is reduced to less than 1%. More specifically, when the mercaptalbumin content is increased to more than 91%, the non-mercaptalbumin-1 content is reduced to 0%. Petition 870250083253, dated 09 / 16 / 2025, page 59 / 204 10 / 34

[0029] In a sixth aspect, the present invention relates to a human albumin solution of the fifth aspect for use in the treatment of a patient in need thereof.

[0030] Consequently, this sixth aspect of the present invention also encompasses treatment methods, wherein the method comprises administering the human albumin solution to a patient in need thereof and / or using a human albumin solution in the manufacture of a medicament for the treatment of a patient in need thereof.

[0031] Typically, treatment involves administering the fifth aspect human albumin solution to a patient: - suffering from liver failure; and / or - suffering from chronic hepatitis, acute hepatitis, liver cirrhosis, fulminant hepatic failure, or hepatocellular carcinoma; and / or - who suffers from kidney failure; and / or who suffers from blood volume deficiency, and for the restoration and maintenance of circulating blood volume in said patient. DETAILED DESCRIPTION OF THE INVENTION

[0032] In order to provide a clear and consistent understanding of the descriptive report and claims, and the scope to be given to such terms, the following definitions are provided.

[0033] In the context of the present disclosure, the terms albumin, human albumin or human serum albumin refer to unmodified human serum albumin (UniProtKB P02768; ALBUJHUMAN; (https: / / www.uniprot.org / uniprotkb / P02768 / entry) exclusively. Consequently, the terms do not encompass any modifications of albumin, such as fusion proteins (comprising whole human serum albumin or fragments thereof) or any conjugates of human serum albumin with other molecules (e.g., PEG or bioactive molecules). Petition 870250083253, dated 09 / 16 / 2025, pp. 60 / 204 11 / 34

[0034] The term Cohn process refers to the well-known series of purification steps for the purpose of extracting albumin from blood plasma developed by Edwin J. Cohn. The process is based on the differential solubility of albumin and other plasma proteins based on 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 Fluids1a,b,c,d. Journal of the American Chemical Society. 68 (3): 459-475.

[0035] The term Kistler-Nitschmann Process refers to the also well-known large-scale production process of human plasma fractions, including a series of alcohol precipitations, as published in 1962 (Kistler & Nitschmann 1962 Large scale production of human plasma fractions. Eight years experience with the alcohol fractionation procedure of Nitschmann, Kistler and Lergier”. Vox Sang.

[0036] Unless the context clearly requires otherwise, throughout the description and claims, the words “understand,” “that includes,” and similar words should be interpreted in an inclusive sense, as opposed to an exclusive or exhaustive sense; that is, in the sense of including, but not limited to.

[0037] Furthermore, throughout this descriptive report, reference to one (1) embodiment, some embodiments or one embodiment means that a particular feature, structure or characteristic described in conjunction with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the phrases in one (1) embodiment, in some embodiments or in one embodiment in various places throughout this descriptive report do not necessarily refer to the same embodiment, but may refer to it. Moreover, the particular features, structures or characteristics may be combined in any suitable manner, as would be evident to one skilled in the art of this disclosure, in one or more Petition 870250083253, dated 09 / 16 / 2025, p. 61 / 204 12 / 34 modalities.

[0038] As used in this document, unless otherwise specified, the use of the ordinal adjectives first, second, third, etc., to describe a common object, only indicates that different instances of similar objects are referring to them, and is not intended to imply that the objects thus described must be in a particular sequence, whether temporally, spatially, in classification or otherwise.

[0039] As used in this document, the term exemplificative is used in the sense of providing examples, rather than indicating quality. That is, an exemplificative modality is a modality provided as an example, rather than necessarily being a modality of exemplary quality.

[0040] As mentioned above, in plasma, mercaptalbumin provides several benefits, including: • Antioxidant protection: Mercaptalbumin can act as an antioxidant, helping to protect cells and tissues against damage caused by free radicals and reactive oxygen species; • Detoxification: Mercaptalbumin can bind to and help remove toxic substances from the body, such as heavy metals and drugs; and • Immune function: Mercaptalbumin can help stimulate the immune system and protect against infections.

[0041] Consequently, a high percentage of non-mercaptalbumin may lead to a decreased capacity for protection against oxidative damage, detoxification of harmful substances, and / or support of immune function. Additionally, a low level of mercaptalbumin may be indicative of certain health conditions or diseases, such as liver disease or malnutrition.

[0042] However, as also mentioned, it is known that commercially produced human albumin products, manufactured on an industrial scale, contain smaller amounts of beneficial mercaptalbumin and larger amounts of undesirable mercaptalbumins than plasma. Furthermore, albumins Petition 870250083253, dated 09 / 16 / 2025, page 62 / 204 13 / 34 commercially available solutions are formulated with substantial amounts (i.e., an excess) of fatty compounds, such as tryptophan and / or caprylic acid. As a result, the ratio of albumin to such fatty compounds in commercially available albumin solutions is routinely between 1:5 and 1:8. For example, commercially available 5% octalbin albumin solution comprises 5% albumin (i.e., 50 g / l; 0.75 mmol), as well as tryptophan and caprylic acid, each at concentrations up to 4.2 mmol. 25% octalbin albumin solution comprises 25% albumin (i.e., 250 g / l; 3.8 mmol), as well as tryptophan and caprylic acid, each at concentrations up to 21 mmol.Therefore, and without being bound by theory, an excess of fatty compounds likely causes most of the fatty acid binding sites FA1 to FA7 of albumin to be occupied, further limiting the albumin's ability to transport reactive oxygen species, reactive nitrogen species, as well as endogenous and exogenous ligands.

[0043] Since the conversion of mercaptalbumin to non-mercaptalbumin-1 is the result of the oxidation of the thiol group in Cys-34 of albumin, it is not entirely surprising that, during the industrial-scale purification process of albumin from pooled donor plasma batches, typically based on the Cohn process or the Kistler-Nitschmann process, and which comprises serial precipitation and reconstitution steps, a greater degree of Cys-34 oxidation is observed in the final albumin product of the purification process compared to fresh plasma.

[0044] However, since mercaptalbumin has the beneficial properties mentioned above, methods and procedures are needed to increase the mercaptalbumin content of industrially purified albumin products.

[0045] The methods of preparation and use of albumin according to the present invention achieve a higher mercaptalbumin content compared to the mercaptalbumin content of a human albumin solution prepared using known methods. However, the methods of Petition 870250083253, dated 09 / 16 / 2025, page 63 / 204 14 / 34 albumin preparation and uses according to the present invention can even achieve an increased mercaptalbumin content relative to the plasma mercaptalbumin content, i.e., a mercaptalbumin content of more than 85%. Methods of the invention and related albumin products

[0046] In the first aspect, the present invention relates to a method for preparing a human albumin solution having an increased mercaptalbumin content and / or an increased carrying capacity for reactive oxygen species, reactive nitrogen species, as well as endogenous and exogenous ligands, wherein said human albumin solution is obtained in the course of a cold ethanol precipitation process from human blood plasma, wherein said method comprises the steps of (a) providing a human albumin solution comprising mercaptalbumin and non-mercaptalbumin, wherein said non-mercaptalbumin comprises a disulfide fraction at the Cys-34 position; (b) combining glutathione, at least once, with said human albumin solution in a molecular ratio between albumin and glutathione (albumin:glutathione) in the range of 1:0.5 to 1:10; and (c) converting at least part of said non-mercaptalbumin into mercaptalbumin at a temperature of less than 15 °C, wherein said mercaptalbumin content in said human albumin solution and / or said transport capacity for reactive oxygen species, reactive nitrogen species, as well as endogenous and exogenous ligands of said human albumin solution is increased relative to the mercaptalbumin content of a human albumin solution prepared in a corresponding cold ethanol precipitation process from human blood plasma without steps (b) and (c).

[0047] The endogenous and exogenous ligands of albumin are typically selected from among fatty acids (FAs), nucleic acids, hormones, metals, toxins, and drugs, where such drugs specifically include ibuprofen, Petition 870250083253, dated 09 / 16 / 2025, page 64 / 204 15 / 34 diazepam, warfarin, lidocaine, and indomethacin.

[0048] As indicated, the conversion step (c) of the above method is advantageously carried out at relatively low temperatures of less than 15 °C. Although conversion would be expected to be less efficient at lower temperatures, it has been found that lower temperatures achieve the desired equilibrium between a slower desirable conversion of non-mercaptalbumin to mercaptalbumin and a slower undesirable oxidation of Cys-34, so that the mercaptalbumin content can be increased as described.The temperature at which step (c) is performed is, in some cases, below 14 °C, below 13 °C, below 12 °C, below 11 °C, below 10 °C, below 9 °C, below 8 °C, below 7 °C, below 6 °C, below 5 °C, below 4 °C, below 3 °C, below 2 °C, below 1 °C, below 0 °C, below -1 °C, below -2 °C, below -3 °C, below -4 °C, below -5 °C, below -6 °C, below -7 °C, below -8 °C, below -9 °C, below -10 °C.

[0049] The temperature at which step (c) is performed, in some cases, ranges between -25 °C and -22 °C, between -22 °C and -19 °C, between -19 °C and -16 °C, between -16 °C and -13 °C, between -13 °C and -10 °C, between -10 °C and -7 °C, between -7 °C and -4 °C, between -4 °C and -1 °C, between -1 °C and 2 °C, between 2 °C and 5 °C, between 5 °C and 8 °C, between 8 °C and 11 °C, between 11 °C and 14 °C or between -25 °C and -23 °C, between -23 °C and -21 °C, between -21 °C and -19 °C, between -19 °C and -17 °C, between -17 °C and -15 °C, between -15 °C and -13 °C, between -13 °C and -11 °C, between -11 °C and -9 °C, between -9 °C and -7 °C, between -7 °C and -5 °C, between -5 °C and -3 °C, between -3 °C and -1 °C, between -1 °C and 1 °C, between 1 °C and 3 °C, between 3 °C and 5 °C, between 5 °C and 7 °C, between 7 °C and 9 °C, between 9 °C and 11 °C, between 11 °C and 13 °C, or between 13 °C and 15 °C. Preferably, between -10 °C and +10 °C.The temperature at which step (c) is performed is, in some cases, 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 particular modalities, the temperature at which step (c) is performed varies between 0 and 8 °C. Petition 870250083253, dated 09 / 16 / 2025, page 65 / 204 16 / 34

[0050] Preferably, the molecular ratio between albumin and glutathione (albumin:glutathione) is in the range of 1:0.5 to 1:5, such as 1:1, 1:2, 1:3, 1:4 or 1:5. In particular embodiments, the molecular ratio between albumin and glutathione (albumin:glutathione) is in the range of 1:0.5 to 1:2.5. In yet another embodiment, the ratio may be 1:0.75 to 1:1.5. Specifically, the ratio may be 1:1.

[0051] In some embodiments of the first aspect method, step (b), preferably also step (c), precedes at least one more 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.

[0052] In the context of the present disclosure, it is worth noting that, for example, the simple process of thawing a 2000-liter batch of a frozen donor plasma solution typically requires at least 3 hours, sometimes at least 5 hours.

[0053] Performing step (b) and, optionally, also step (c), before one (or several or all) of the time-consuming steps, which typically require at least 3 hours, sometimes at least 5 hours of time, in conventional plasma fractionation processes (i.e., the Cohn process or the Kistler-Nitschmann process and variations thereof), neutralizes the undesirable oxidation of desirable mercaptalbumin to undesirable non-mercaptalbumin during the course of each of the respective steps (or during the course of each of these respective steps) of the respective process. This has the advantage that the final albumin product has a higher mercaptalbumin content compared to an albumin product simply prepared by conventional processes.

[0054] Preferably, the method further comprises supplying said glutathione to be combined with the solution comprising Petition 870250083253, dated 09 / 16 / 2025, page 66 / 204 17 / 34 human albumin in step (b) in a container before the addition of human blood plasma or human whole blood to the container and in which, optionally, said human blood plasma or human whole blood is stored in the container. As such, glutathione can already be combined with the starting material of the Cohn or Kistler Nitschmann processes at the time of donation or shortly thereafter, when the whole blood or plasma (i.e., the human albumin solution) is placed in a plasma bag or bottle.

[0055] In some or all of the above embodiments of the first aspect method, step (b) may precede a cryo-poor plasma harvesting step. If so, the method further comprises supplying the glutathione to be combined with the solution comprising human albumin in step (b) into a container before: - to group donations of frozen human plasma in the container; - Thaw donations of frozen human plasma at a temperature of 0 °C to 5 °C; - to separate the still-solid cryoprecipitate from the cryo-poor liquid plasma; and - optionally, to further process the cryo-poor liquid plasma by capturing coagulation factors to generate a coagulation factor-poor plasma.

[0056] The thawing step of multiple batches of cryo-poor plasma to be pooled regularly takes between 3 and 10 hours, such as between 4 and 10 hours, 5 and 10 hours, 6 and 10 hours, 7 and 10 hours, 8 and 10 hours or 9 and 10 hours.

[0057] After the solid cryoprecipitate is separated from the cryo-poor liquid plasma, the cryo-poor liquid plasma can be processed onto synthetic resins, such as Sephadex or heparin-coupled resins, to capture coagulation factor IX, PPSB or FXI. PPSB stands for Prothrombin Complex Concentrate (PCC) or Prothrombin Complex Concentrate with Factor VIII Bypass-Inhibiting Activity and typically comprises the Petition 870250083253, dated 09 / 16 / 2025, page 67 / 204 18 / 34 Coagulation factors II, VII, IX and X.

[0058] In some or all of the above embodiments of the method in the first aspect, step (b) precedes a Fraction I+II+III formation step of the Cohn process or Precipitate A formation of the Kistler-Nitschmann (KN) process, and wherein the method further comprises the addition of glutathione to be combined with the human albumin solution to cryo-poor plasma or coagulation factor-poor plasma prior to the addition of ethanol. The Fraction I+II+III formation of the Cohn process or the Precipitate A formation of the Kistler-Nitschmann (KN) process typically involves the use of filter aids as well as a reduction in temperature to less than -1 °C, such as to between -1 °C and -6 °C. The entire step comprises several substeps which make it time-consuming, often requiring up to 32 hours.

[0059] In some or all of the above embodiments of the method in the first aspect, step (b) precedes a Fraction IV formation step of the Cohn process or Precipitate A formation of the KN process, wherein the method further comprises adding glutathione to be combined with the human albumin solution to the supernatant of Fraction I+II+III or to the supernatant of Precipitate A before the addition of ethanol. The formation of Fraction IV of the Cohn process or the formation of Precipitate A of the KN process typically involves the use of filter aids as well as reducing the temperature to less than -4 °C, such as to between -4 °C and -10 °C. The entire step comprises several substeps, which makes it time-consuming, often requiring up to 11 hours of time, such as between 8 and 11 hours of time (not including possible precipitation steps to capture Factor IV).

[0060] In some or all of the above embodiments of the method in the first aspect, step (b) precedes a Fraction V formation step of the Cohn process or Precipitate A formation step of the KN process, wherein the method further comprises adding glutathione to be combined with the human albumin solution to the Fraction IV supernatant before the addition of ethanol. The formation of Fraction V of the Cohn process or the formation of Petition 870250083253, dated 09 / 16 / 2025, pp. 68 / 204 19 / 34 Precipitate A of the KN process typically involves not the use of filter aids, but also a reduction in temperature to less than -8 °C, such as between -8 °C and -10 °C. The entire step comprises several sub-steps, which makes it time-consuming, often requiring up to 62 hours of time, such as between 18 and 62 hours, including certain waiting times.

[0061] In some or all of the above embodiments of the method in the first aspect, step (b) precedes a resuspension step of Fraction V of the Cohn process or resuspension of Precipitate C of the KN process, wherein the method further comprises the addition of glutathione to be combined with the human albumin solution to Fraction V prior to resuspension in water for injection (WFI). Resuspension of Fraction V of the Cohn process or resuspension of Precipitate C of the KN process typically comprises an increase in temperature to less than +1 °C, such as to between +1 °C and -4 °C. The entire step comprises several substeps, including homogenization, but excluding diafiltration, which makes it time-consuming, often requiring up to 22 hours of time, such as between 7.5 and 22 hours of time.

[0062] In this context, it is particularly useful that the conversion step (c) can be carried out at low temperatures, since the processes for preparing a human albumin solution from pooled donor plasma, as described above, routinely include the resuspension of frozen intermediates, such as the resuspension of the frozen paste of Fraction V of the Cohn process. In certain embodiments, advantageously, the human albumin solution of step (a) is provided as the respective thawing / resuspension solution containing the frozen human albumin concentrate or the frozen human albumin paste. In such cases, glutathione is combined with said solution in a molecular ratio between albumin and glutathione (albumin:glutathione) in the range of 1:0.5 to 1:10 based on the albumin content of the fully resuspended frozen intermediate, and said conversion of step (c) is carried out throughout the resuspension time, as Petition 870250083253, dated 09 / 16 / 2025, page 69 / 204 20 / 34 between 7:30 and 22:00.

[0063] 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 comprising: amino acids; sugars; and sugar alcohols are optionally added. Preferably, one or more stabilizers are selected from the group consisting of: glycine; glutamate; arginine; lysine; maltose; and sorbitol.

[0064] Preferably, the method further comprises carrying out the inactivation or removal of pathogens, in particular the inactivation or removal of viruses, in the human albumin solution, preferably by means of nanofiltration, solvent / detergent treatment (S / D treatment), low pH treatment or pasteurization of the human albumin solution, or a combination thereof.

[0065] In general, the pasteurization of human albumin solution is carried out as a one-step process or as a two-step process, optionally under an inert atmosphere. The one-step pasteurization process is carried out in the final container which includes the stabilizers N-acetyltryptophan and caprylic acid, while the two-step pasteurization process comprises additional pasteurization with the same stabilizers immediately before filling the albumin into the final containers.

[0066] If an S / D treatment is employed, preferably an environmentally friendly detergent will be used, i.e., a detergent other than Triton X-100. For example, polyethylene glycol ether, sec-alcohol ethoxylate, sec-alkoxy polyethylene glycol (Tergitol 15-S-9), Nereid, or a polysorbate, such as polysorbate 20 or polysorbate 80 (TWEEN 20 or TWEEN 80). However, in embodiments of the present invention where the removal of any S / D reagents is carried out without an oil extraction, i.e., in some embodiments of the present invention, an S / D treatment does not include an oil extraction step. Instead, the undesirable S / D reagents are removed using other means of Petition 870250083253, dated 09 / 16 / 2025, pp. 70 / 204 21 / 34 purification methods are available to those skilled in the art, such as, for example, (a) chromatographic separation, such as with the use of chromatographic gels or resins, (b) fractionation methods, such as precipitation methods, or (c) filtration, such as by diafiltration or with the use of adsorbents, such as activated carbon.

[0067] If an S / D treatment is employed, the S / D treatment may replace pasteurization of the human albumin solution as a virus inactivation step. Consequently, the S / D treatment performed on the human albumin solution for virus inactivation, which is suitable to completely replace pasteurization, is carried out without the addition of an indole stabilizer (such as N-acetyltryptophan) or a fatty acid stabilizer (such as caprylate). Instead, alternative stabilizers may be used, for example, stabilizers selected from the group of: sugars, amino acids and sugar alcohols. Preferably, one or more stabilizers are selected from the group consisting of: glycine; glutamate; arginine; lysine; maltose; and sorbitol.

[0068] In some embodiments, virus inactivation is achieved by employing a low pH treatment. Many viruses are irreversibly denatured and effectively destroyed at pH 5.0-5.5. However, several enveloped viruses are only effectively inactivated in the pH range of 3.5-4. Consequently, the low pH treatment in the context of the present invention is treatment at a pH of 5.5 or less, preferably at a pH between 3.0 and 5.5, such as a pH of about 3.0, 3.5, 4.0, 4.5, 5.0 or 5.5.

[0069] In some embodiments, low pH treatment in the pH range of 3.05.5 serves as a defatting step for albumin. Thus, simultaneous virus inactivation and albumin defatting are possible without the need to remove the virus-inactivating compounds. A simple pH adjustment after low pH treatment to values ​​compatible with albumin is sufficient.

[0070] Preferably, the method for preparing the human albumin solution comprising an increased mercaptalbumin content. Petition 870250083253, dated 09 / 16 / 2025, pp. 71 / 204 22 / 34 and / or an increased transport capacity for reactive oxygen species, reactive nitrogen species, as well as endogenous and exogenous ligands comprises filling the solution into a final container, which is subsequently washed with an inert gas. Uses of the invention

[0071] In a fourth aspect, the present invention relates to the use of glutathione to increase the mercaptalbumin content and / or an increased transport capacity for reactive oxygen species, reactive nitrogen species, as well as endogenous and exogenous ligands of a human albumin solution, wherein said human albumin solution is obtained in the course of a cold ethanol precipitation process of human blood plasma, during which glutathione is added at least once, wherein said use comprises: (a) supply of a human albumin solution comprising mercaptalbumin and non-mercaptalbumin, wherein the said non-mercaptalbumin comprises a disulfide fraction at the Cys-34 position; (b) combining glutathione, at least once, with said human albumin solution in a molecular ratio between albumin and glutathione (albumin:glutathione) in the range of 1:0.5 to 1:10, wherein: and (c) converting at least part of said non-mercaptalbumin into mercaptalbumin at a temperature of less than 15 °C, wherein said mercaptalbumin content in said human albumin solution and / or said transport capacity for reactive oxygen species, reactive nitrogen species, as well as endogenous and exogenous ligands of said human albumin solution is increased relative to the mercaptalbumin content of a human albumin solution prepared in a corresponding cold ethanol precipitation process from human blood plasma without steps (b) and (c).

[0072] As indicated, the conversion of (c) is advantageously relatively low and below 15 °C. Although the conversion is expected to be less Petition 870250083253, dated 09 / 16 / 2025, page 72 / 204 23 / 34 efficient at lower temperatures, it was found that lower temperatures achieve the desired equilibrium between a slower desirable conversion of non-mercaptalbumin to mercaptalbumin and a slower undesirable oxidation of Cys-34, so that the mercaptalbumin content can be increased as described. The temperature of (c) is, in some cases, 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, less than -10 °C.

[0073] The temperature of (c), in some cases, ranges between -25 °C and -22 °C, between -22 °C and -19 °C, between -19 °C and -16 °C, between -16 °C and -13 °C, between -13 °C and -10 °C, between -10 °C and -7 °C, between -7 °C and -4 °C, between -4 °C and -1 °C, between -1 °C and 2 °C, between 2 °C and 5 °C, between 5 °C and 8 °C, between 8 °C and 11 °C, between 11 °C and 14 °C or between -25 °C and -23 °C, between -23 °C and -21 °C, between -21 °C and -19 °C, between 19 °C and -17 °C, between -17 °C and -15 °C, between -15 °C and -13 °C, between -13 °C and -11 °C, between -11 °C and -9 °C, between -9 °C and -7 °C, between -7 °C and -5 °C, between -5 °C and -3 °C, between -3 °C and -1 °C, between -1 °C and 1 °C, between 1 °C and 3 °C, between 3 °C and 5 °C, between 5 °C and 7 °C, between 7 °C and 9 °C, between 9 °C and 11 °C, between 11 °C and 13 °C, or between 13 °C and 15 °C. Preferably, between -10 °C and +10 °C.The temperature of (c) is, in some cases, 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 particular embodiments, the temperature at which step (c) is carried out varies between 0 and 8 °C.

[0074] Preferably, the molecular ratio between albumin and glutathione (albumin:glutathione) is in the range of 1:0.5 to 1:5, such as 1:1, 1:2, 1:3, 1:4 or 1:5. In particular embodiments, the molecular ratio between albumin and glutathione (albumin:glutathione) is in the range of 1:0.5 to 1:2.5. In yet another embodiment, the ratio may be 1:0.75 to 1:1.5. Specifically, the ratio may be 1:1. Petition 870250083253, dated 09 / 16 / 2025, pp. 73-204 24 / 34

[0075] In some embodiments, (b), optionally also (c), precedes at least one more 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.

[0076] Performing (b) and optionally also (c), before one (or several or all) of the time-consuming steps, which typically require at least 3 hours, sometimes at least 5 hours of time, in conventional plasma fractionation processes (i.e., the Cohn process or the Kistler-Nitschmann process and variations thereof), neutralizes the undesirable oxidation of desirable mercaptalbumin to undesirable non-mercaptalbumin during the course of each of the respective steps (or during the course of each of these respective steps) of the respective process. This has the advantage that an increased mercaptalbumin content can be achieved through the use of glutathione in a human albumin solution compared to a human albumin solution that has not been treated with glutathione as described, but which has simply been subjected to conventional processing.

[0077] Preferably, the glutathione to be combined with the solution comprising human albumin in (b) may be used in a container before the addition of human blood plasma or human whole blood to the container, so that, additionally, said human blood plasma or human whole blood may be stored in the container.

[0078] In some or all of the above embodiments of the fourth aspect, (b) may precede a cryo-poor plasma harvesting step. If so, the method further comprises providing the glutathione to be combined with the solution comprising human albumin in (b) into a container before: - to group donations of frozen human plasma in the container; Petition 870250083253, dated 09 / 16 / 2025, pp. 74 / 204 25 / 34 - Thaw donations of frozen human plasma at a temperature of 0 °C to 5 °C; - to separate the still-solid cryoprecipitate from the cryo-poor liquid plasma; and - optionally, to further process the cryo-poor liquid plasma by capturing coagulation factors to generate a coagulation factor-poor plasma.

[0079] In some or all embodiments above the fourth aspect, (b) precedes a Fraction I+II+III formation step of the Cohn process or Precipitate A formation of the Kistler-Nitschmann (KN) process and wherein the method further comprises adding glutathione to be combined with the human albumin solution to cryo-poor plasma or coagulation factor-poor plasma before the addition of ethanol.

[0080] In some or all of the above embodiments of the fourth aspect, (b) precedes a Fraction IV formation step of the Cohn process or Precipitate A formation 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 to the Precipitate A supernatant before the addition of ethanol.

[0081] In some or all of the above embodiments of the fourth aspect, (b) precedes a Fraction V formation step of the Cohn process or Precipitate A formation of the KN process, wherein the method further comprises adding glutathione to be combined with the human albumin solution to the Fraction IV supernatant before the addition of ethanol.

[0082] In some or all of the above embodiments of the fourth aspect, (b) precedes a resuspension step of Fraction V of the Cohn process or resuspension of Precipitate 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 resuspension in water for injection (WFI). Petition 870250083253, dated 09 / 16 / 2025, pp. 75 / 204 26 / 34

[0083] 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 comprising: amino acids; sugars; and sugar alcohols are optionally added. Preferably, one or more stabilizers are selected from the group consisting of: glycine; glutamate; arginine; lysine; maltose; and sorbitol.

[0084] Preferably, the use further comprises performing pathogen inactivation or removal, in particular virus inactivation or removal, in the human albumin solution, preferably by means of performing nanofiltration, solvent / detergent treatment (S / D treatment), low pH treatment or pasteurization of the human albumin solution, or a combination thereof.

[0085] In general, pasteurization of human albumin solution is carried out as a one-step process or as a two-step process, optionally under an inert atmosphere.

[0086] If an S / D treatment is employed, preferably an environmentally friendly detergent will be used, i.e., a detergent other than Triton X-100. For example, polyethylene glycol ether, sec-alcohol ethoxylate, sec-alkoxy polyethylene glycol (Tergitol 15-S-9), Nereid, or a polysorbate, such as polysorbate 20 or polysorbate 80 (TWEEN 20 or TWEEN 80). However, in embodiments of the present invention where the removal of any S / D reagents is carried out without an oil extraction, i.e., in some embodiments of the present invention, an S / D treatment does not include an oil extraction step. Instead, undesirable S / D reagents are removed using other purification methods available to those skilled in the art, such as, for example, (a) chromatographic separation, such as with the use of chromatography gels or resins, (b) fractionation methods, such as precipitation methods, or (c) filtration, such as by diafiltration or with the use of adsorbents, such as activated carbon.

[0087] If an S / D treatment is employed, the S / D treatment may Petition 870250083253, dated 09 / 16 / 2025, pp. 76 / 204 27 / 34 replace pasteurization of the human albumin solution as a virus inactivation step. Consequently, the S / D treatment performed on the human albumin solution for virus inactivation, which is suitable to completely replace pasteurization, is carried out without the addition of an indole stabilizer (such as N-acetyltryptophan) or a fatty acid stabilizer (such as caprylate). Instead, alternative stabilizers can be used, for example, stabilizers selected from the group of: sugars, amino acids and sugar alcohols. Preferably, one or more stabilizers are selected from the group consisting of: glycine; glutamate; arginine; lysine; maltose; and sorbitol.

[0088] In some embodiments, virus inactivation and degreasing are performed using a low pH treatment. Many viruses are irreversibly denatured and effectively destroyed at pH 5.0-5.5. However, several enveloped viruses are only effectively inactivated in the pH range of 3.5-4. Consequently, the low pH treatment in the context of the present invention is treatment at a pH of 5.5 or less, preferably at a pH between 3.0 and 5.5, such as a pH of about 3.0, 3.5, 4.0, 4.5, 5.0 or 5.5.

[0089] Preferably, the use of glutathione to increase the mercaptalbumin content and / or to increase the transport capacity for reactive oxygen species, reactive nitrogen species, as well as endogenous and exogenous ligands of the human albumin solution comprising an increased mercaptalbumin content and / or an increased transport capacity for reactive oxygen species, reactive nitrogen species, as well as endogenous and exogenous ligands comprises filling the solution into a final container, which is subsequently washed with an inert gas.

[0090] In a fifth aspect, the present invention relates to a human albumin solution comprising an increased mercaptalbumin content when obtained through use according to the fourth aspect, as described. Petition 870250083253, dated 09 / 16 / 2025, page 77 / 204 28 / 34 above, characterized in that the human albumin solution comprises a mercaptalbumin content of at least 80%, in particular at least 85%, relative to the total albumin content of the solution.

[0091] The human albumin solutions of the present invention can be used in the treatment of diseases. Specifically, it is to be used in the treatment of a patient in need thereof. In particular, for use in the treatment of a patient: - suffering from liver failure; and / or - suffering from chronic hepatitis, acute hepatitis, liver cirrhosis, fulminant hepatic failure, or hepatocellular carcinoma; and / or - suffering from kidney failure; and / or - who suffers from blood volume deficiency and for the restoration and maintenance of circulating blood volume in said patient. Albumin products of the invention

[0092] In the second and third aspects, as well as in the fifth and sixth aspects, the present invention relates to human albumin solutions comprising an increased mercaptalbumin content and / or an increased transport capacity for reactive oxygen species, reactive nitrogen species, as well as endogenous and exogenous ligands of said human albumin solution when prepared by the method according to the first aspect or by use according to the fourth aspect, respectively.

[0093] The respective human albumin solutions can be used as a pharmaceutical product.

[0094] Specifically, the respective human albumin solutions are used in the treatment of a patient in need of them. In particular, for use in the treatment of a patient: - suffering from liver failure; and / or - suffering from chronic hepatitis, acute hepatitis, liver cirrhosis, fulminant hepatic failure, or hepatocellular carcinoma; and / or Petition 870250083253, dated 09 / 16 / 2025, pp. 78 / 204 29 / 34 - who suffers from kidney failure; and / or who suffers from blood volume deficiency, and for the restoration and maintenance of circulating blood volume in said patient. Examples Evaluation of free cysteine ​​34 in albumin by mass spectrometry.

[0095] A mass spectrometry (MS) method was established to detect the oxidation status of Cysteine ​​34 (Cys-34) in human albumin. A control sample to verify the ability of the established MS method to identify oxidized fractions of human non-mercaptablumine-1 (HNA1) and non-mercaptablumine-2 (HNA2) was prepared by oxidizing albumin with 3% H2O2. An untreated reference sample was also prepared.

[0096] For the conversion of HNA1 to human mercaptalbumin (HMA), Fraction V was resuspended in the presence of a reducing agent. For sample preparation for analysis, this was followed by depth filtration, pH adjustment, and a subsequent diafiltration step to remove residual glutathione. This process was carried out with a reference sample without glutathione and five samples with an increasing molar ratio between albumin and glutathione as a reducing agent (albumin:glutathione) of 1:0.5, 1:1.5, 1:2.5, 1:5, as well as 1:10.

[0097] As a control, a resuspension sample of Fraction V, untreated with a reducing agent, was taken after depth filtration, pH adjustment, and ultrafiltration / diafiltration. This sample represents the untreated albumin concentrate. After determining the protein concentration, several control samples were generated from smaller aliquots, each comprising 10% albumin. The untreated control samples comprised only albumin at a concentration of 10%. The oxidized control samples comprised 3% H2O2 in the 10% albumin solution. The reduction control sample comprised 5 mM DTT in the 10% albumin solution.

[0098] All samples, that is, control samples and samples from the inventive process, were measured to verify the method and show the impact. Petition 870250083253, dated 09 / 16 / 2025, pp. 79 / 204 30 / 34 of different albumin:glutathione in the oxidation status of the albumin molecule.

[0099] Merck LiChrosolv ultra-pure water was used for the preparation of all stock solutions, buffers, and dilutions to avoid interference with the mass spectrometry method. All materials used were single-use to avoid any interference with the mass spectrometry measurement. Table 1 shows the temperature and duration of each unit operation. Unit operation Temperature ranges [°C] Duration [h] Resuspension of Fraction V -1.5 ± 1 °C 20 Depth filtration 3.5 ± 1.5 °C 2.5 pH adjustment 3.5 ± 1.5 °C 1 Diafiltration < 10 °C 10

[0100] All reagents were pre-cooled to 4 °C and resuspension of Fraction V occurred in a cooling chamber at a temperature of -1.5 ± 1 °C.

[0101] 10 g of Fraction V were used in 50 ml tubes for each sample and 1.6 times the amount of water / glutathione mixture was added as described below. A protein solution of approximately 100 g / l was prepared. Resuspension was performed for 20 hours under agitation in a shaker. Table 2 Composition of process samples. Sample Albumin molar glutathione ratio Glutathione Concentration [mM] Glutathione Volume [ml] Water Volume [ml] Control 0 0 0 16 Petition 870250083253, dated 09 / 16 / 2025, pages 80 / 204 31 / 34 Glutathione 0.5x 0.5 0.753 0.2 15.8 Glutathione 1.5x 1.5 2.259 0.59 15.41 Glutathione 2.5x 2.5 3.765 0.98 15.02 Glutathione 5x 5 7.53 1.96 14.04 Glutathione 10x 10 15.06 3.92 12.08

[0102] After resuspension, the cooling chamber temperature was increased to 3.5 ± 1.5 °C and the solution was filtered using a 0.22 µm Millipore filter. After filtration, the solution was incubated for a further 2 hours. During the unit operation, the pH was adjusted by slowly adding a calculated amount (21.1 g) of 1.25 M NaOH at 4 °C to the solution. The sample was incubated for a total incubation time of approximately 1 hour. 8 ml of each sample were then dialyzed at 4 °C using Thermo Fisher Slide-A-Lyzer G3 dialysis cassettes (prepared as described in the manufacturer's protocol) with a molecular weight cutoff of 20 kD. The buffer solution was changed twice during sample dialysis for 10 hours in a 60 mM NaCl solution. Instead of dialysis, ultrafiltration / diafiltration can also be used, which will preferably be used on an industrial scale.

[0103] For LC-MS, the following solvents, gradients and flow rates were applied. Table 3 Solvents used during MS. SOLVENT C Water with 0.1% formic acid SOLVENT D Acetonitrile with 0.1% formic acid

[0104] A gradient of 5-85% D over 3.5 min was applied with a rate of Petition 870250083253, dated 09 / 16 / 2025, pp. 81 / 204 32 / 34 flow rate of 0.4 ml / min.

[0105] LC-MS with 4 modules: 1. Xevo G2-XS QTof mass spectrometer with ESI source (Waters; Cat. No.: e.g., 186010532) 2. Oil-free vacuum pump (Edwards; Cat. No.: e.g., XDS46) 3. ACQUITY UPLC H-Class Bio Systems (A) Sample manager (Waters; Cat. No.: e.g., 186015040) (B) Quaternary solvent manager (Waters; Cat. No.: e.g., 186015041) (C) Column manager (Waters; Cat. No.: e.g., 186015043) 4. Computer with Waters Connect software, including the UNIFI application with the Large Molecule option.

[0106] Column: ACQUITY UPLC® Protein BEH C4 2.1x50 mm Column (Waters; Catalog number: for example, 186004495) Results - control samples Table 4: Results of the control samples. Albumin Species Control Untreated DTT at 5mM H2O2 at 3% HMA 64.46 0.00 HNA1 29.84 34.79 HNA2 1.63 65.21 Unknown 4.07 0.00

[0107] Analysis of the reduction control sample comprising DTT did not produce interpretable results due to the high heterogeneity of the sample, which did not allow for the discernment of clear peaks of HMA, HNA1, or HNA2. Unintentionally Petition 870250083253, dated 09 / 16 / 2025, pp. 82 / 204 33 / 34 getting stuck on theory, this heterogeneity is probably the result of the reduction and breaking of other disulfide bonds within albumin.

[0108] Oxidation with 3% H2O2 showed complete oxidation of HMA to HNA1 and HNA2 Results - experimental samples Table 5 shows the results obtained from the process samples. Albumin Species Control and 0.5x Glutathione 1.5x Glutathione 2.5x Glutathione 5x Glutathione 10x Glutathione HMA 75.04 88.87 91.60 92.59 91.42 91.63 HNA1 17.97 3.50 0.00 0.00 0.00 0.00 HNA2 1.97 2.07 2.32 1.47 2.47 2.36 Unknown 5.02 5.56 6.08 5.94 6.11 6.01 Conclusion

[0109] As can be seen from the results above, the addition of glutathione to albumin at a ratio of 1:0.5 already leads to a favorable increase in HMA compared to an untreated control sample, in particular, to an HMA content of more than 85%. Increasing the ratio to more than 1:1.5 further increases the HMA content to more than 90%, reaching a plateau between 91 and 93%. The maximum HMA content can be achieved when the albumin:glutathione ratio is 1:2.5. At the same time, the HNA1 content of the experimental samples is significantly reduced, substantially to 0% in most experimental samples.

[0110] Although albumin oxidation worked very well, reduction of the protein with DTT resulted in a very diverse spectrum, in which it was impossible to assign individual peaks. This is probably due to the breaking of internal disulfide bonds.

[0111] Many modifications and other embodiments of the invention presented Petition 870250083253, dated 09 / 16 / 2025, pp. 83 / 204 34 / 34 in this document can be viewed by one skilled in the art to which the invention relates, having the benefit of the teachings set forth in the preceding 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 should be included within the scope of the appended claims. Although specific terms are employed in this document, they are used only in a generic and descriptive sense and not for purposes of limitation. Petition 870250083253, dated 09 / 16 / 2025, pp. 84 / 204

Claims

1 / 6 CLAIMS 1. Method for the preparation of a human albumin solution having an increased mercaptalbumin content and / or an increased transport capacity for reactive oxygen species, reactive nitrogen species, as well as endogenous and exogenous ligands, wherein said human albumin solution is obtained in the course of a cold ethanol precipitation process from human blood plasma, wherein said method is characterized by comprising the steps of (a) providing a human albumin solution comprising mercaptalbumin and non-mercaptalbumin, wherein said non-mercaptalbumin comprises a disulfide fraction at the Cys-34 position; (b) combining glutathione, at least once, with said human albumin solution in a molecular ratio between albumin and glutathione (albumin:glutathione) in the range of 1:0.5 to 1:10;and (c) conversion of at least part of said non-mercaptalbumin into mercaptalbumin at a temperature of less than 15 °C, wherein said mercaptalbumin content in said human albumin solution and / or said transport capacity for reactive oxygen species, reactive nitrogen species, as well as endogenous and exogenous ligands of said human albumin solution is increased relative to the mercaptalbumin content of a human albumin solution prepared in a corresponding cold ethanol precipitation process from human blood plasma without steps (b) and (c).

2. Method according to claim 1, characterized in that the molecular ratio between albumin and glutathione (albumin:glutathione) is in the range of 1:0.5 to 1:5, preferably 1:0.5 to 1:2.5, more preferably 1:0.75 to 1:1.

5. Petition 870250083253, dated 09 / 16 / 2025, page 85 / 204 2 / 6 3. Method according to claim 1 or 2, characterized in that step (b) precedes at least one more 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, wherein, optionally, said time-consuming step requires at least 3 hours, preferably at least 5 hours, of time.

4. Method, according to any one of claims 1 to 3, wherein said method is further characterized by comprising providing said glutathione to be combined with said human albumin solution in step (b) in a container prior to the addition of human blood plasma or human whole blood to said container and wherein, optionally, said human blood plasma or human whole blood is stored in said container.

5. Method, according to any one of claims 1 to 3, characterized in that step (b) precedes a cryo-poor plasma harvesting step and in that said method further comprises supplying said glutathione to be combined with said solution comprising human albumin in step (b) in a container before: - pooling frozen human plasma donations in said container; - thawing said frozen human plasma donations at a temperature of 0 °C to 5 °C; - separating the still solid cryoprecipitate from said cryo-poor liquid plasma; and - optionally, further processing said cryo-poor liquid plasma by capturing coagulation factors to generate a coagulation factor-poor plasma.

6. Method according to any one of claims 1 to 5, characterized in that: step (b) precedes a step of forming Fraction I+II+III of the Cohn process or of forming Precipitate A of the Kistler-Nitschmann (KN) process and in which said method further comprises adding said glutathione to be combined with said human albumin solution to cryo-poor plasma or coagulation factor-poor plasma before the addition of ethanol; and / or in which step (b) precedes a step of forming Fraction IV of the Cohn process or of forming Precipitate A of the KN process, in which said method further comprises adding said glutathione to be combined with said human albumin solution to the supernatant of Fraction I+II+III or to the supernatant of Precipitate A before the addition of ethanol;and / or step (b) precedes a Fraction V formation step of the Cohn process or Precipitate A formation step of the KN process, wherein said method further comprises adding said glutathione to be combined with said human albumin solution to the supernatant of Fraction IV before the addition of ethanol; and / or step (b) precedes a Fraction V resuspension step of the Cohn process or Precipitate C resuspension step of the KN process, wherein said method further comprises adding said glutathione to be combined with said human albumin solution to Fraction V before resuspension in water for injection (WFI).

7. Method, according to any one of claims 1 to 6, wherein said method is characterized by further comprising a final adjustment of the protein content of said human albumin solution during which one or more stabilizers selected from the group consisting of: amino acids; sugars; and sugar alcohols are, Petition 870250083253, dated 16 / 09 / 2025, page 87 / 204 4 / 6 optionally added.

8. Method according to claim 7, characterized in that one or more stabilizers are selected from the group consisting of: glycine; glutamate; arginine; lysine; maltose; and sorbitol.

9. A method according to any one of claims 1 to 8, wherein said method is characterized by further comprising carrying out the inactivation or removal of pathogens, in particular the inactivation or removal of viruses, in said human albumin solution, preferably by means of carrying out nanofiltration, solvent / detergent treatment (S / D treatment), low pH treatment or pasteurization of said human albumin solution, or a combination thereof, and optionally, the pasteurization of said human albumin solution is carried out as a one-step process or as a two-step process, optionally under an inert atmosphere.

10. A method according to any one of claims 1 to 9, characterized by comprising filling said solution into a final container, which is subsequently rinsed with an inert gas.

11. Method according to any one of claims 1 to 10, characterized in that (a) said reactive oxygen species are selected from the group consisting of: H2O2, O2- and HOCl; (b) said reactive nitrogen species are selected from the group consisting of: ONOO- and ONOOCO2-; (c) the endogenous and exogenous ligands are selected from the group consisting of: fatty acids (FAs), nucleic acids, hormones, metals, toxins and drugs.

12. Human albumin solution comprising an increased mercaptalbumin content when prepared by the method as defined in any of claims 1 to 11, characterized by Petition 870250083253, dated 16 / 09 / 2025, page 88 / 204 5 / 6 comprising a mercaptalbumin content of at least 85%, in particular at least 88%, preferably between 90 and 99% or between 90 and 95% or between 90 and 93%, in relation to the total albumin content of said solution.

13. Use of human albumin solution comprising an increased mercaptalbumin content as defined in claim 12, characterized in being for the preparation of a medicament for the treatment of a patient in need thereof.

14. Use of human albumin solution comprising an increased mercaptalbumin content as defined in claim 12, characterized in being for the preparation of a medicament for the treatment of a patient suffering from hepatic insufficiency.

15. Use of human albumin solution comprising an increased mercaptalbumin content, according to claim 14, characterized in that said medicament is adapted for administering the human albumin solution to a patient suffering from chronic hepatitis, acute hepatitis, hepatic cirrhosis, fulminant hepatic failure or hepatocellular carcinoma.

16. Use of human albumin solution comprising an increased mercaptalbumin content as defined in claim 12, characterized in being for the preparation of a medicament for the treatment of a patient suffering from renal insufficiency.

17. Use of human albumin solution comprising an increased mercaptalbumin content as defined in claim 12, characterized in being for the preparation of a medicament for the restoration and maintenance of circulating blood volume in a patient where blood volume deficiency has been demonstrated in said patient.

18. Use of glutathione to increase the mercaptalbumin content of a human albumin solution, characterized by the fact that said Petition 870250083253, dated 09 / 16 / 2025, p.89 / 204 6 / 6 human albumin solution is obtained in the course of a cold ethanol precipitation process of human blood plasma, during which glutathione is added at least once, and wherein said use comprises: (a) providing a human albumin solution comprising mercaptalbumin and non-mercaptalbumin, wherein said non-mercaptalbumin comprises a disulfide fraction at the Cys-34 position; (b) combining glutathione, at least once, with said human albumin solution in a molecular ratio between albumin and glutathione (albumin:glutathione) in the range of 1:0.5 to 1:10; and (c) convert said non-mercaptalbumin into mercaptalbumin at a temperature of less than 15 °C, wherein said mercaptalbumin content in said human albumin solution is increased relative to the mercaptalbumin content of a human albumin solution prepared in a corresponding cold ethanol precipitation process from human blood plasma without steps (b) and (c).

19. Human albumin solution comprising an increased mercaptalbumin content when obtained through the use of glutathione as defined in claim 17, characterized by comprising a mercaptalbumin content of at least 85%, in particular at least 88%, preferably between 90 and 99% or between 90 and 95% or between 90 and 93%, relative to the total albumin content of said solution. Petition 870250083253, dated 09 / 16 / 2025, pp. 90 / 204