Process for isolating glu-plasminogen from a solution

Abstract

The present invention relates to a method for isolating Glu-Plasminogen from a solution containing Glu-Plasminogen and one or more other polypeptides, wherein the method comprises the step of eluting the Glu-Plasminogen from the cation exchange solid phase with an elution buffer of a pH in the range of 6.2 to 6.5. Furthermore, the present invention relates to a composition comprising Glu-Plasminogen obtainable from the method of the invention.

Description

The present invention relates to a method for isolating Glu-Plasminogen from a solution containing Glu-Plasminogen and one or more other polypeptides, wherein the method comprises the step of eluting the Glu-Plasminogen from the cation exchange solid phase with an elution buffer of a pH in the range of 6.2 to 6.5. Furthermore, the present invention relates to a composition comprising Glu-Plasminogen obtainable from the method of the invention. Essentially pure Glu-Plasminogen solutions are of significant interest. Glu-Plasminogen is naturally occurring in blood. Glu-plasminogen is the native circulating precursor in the human plasma. Glu-plasminogen can be cleaved into Lys-plasminogen due to different isolation and purification processes. Glu-Plasminogen is the zymogen of the active serine protease plasmin. Its main role is the lysis of fibrin clots, which are formed because of blood coagulation processes. Insufficient cloth lysis can result in thrombotic events such as coronary infarcts, stroke, and pulmonary embolisms. Indeed, thrombotic events can cause serious health problems. For instance, coronary infarcts, stroke and pulmonary embolisms are some of the main causes of death in the developed world. There is a strong need to treat and prevent thrombotic events. As taught in WO 2018 / 162754 and WO 2020 / 152322, it has been experimentally found that Glu-Plasminogen can be effectively used for preventing or treating thrombotic events, in particular microthrombotic events in patients such as, e.g., in patients suffering from inherent or acquired plasminogen deficiency. It was found that treatment with Lys-Plasminogen can result in undesired side effects such as bleeding. Glu-plasminogen has a significantly longer half-life and a well-predictable activity. Therefore, the purification of Glu-plasminogen is preferred. Therefore, it is of interest to obtain rather pure Glu-Plasminogen and comparably high Glu-Plasminogen : Lys-Plasminogen ratios. It is of significant interest to obtain Glu-plasminogen in good yields and purity. Accordingly, there is the need for methods for isolating Glu-plasminogen from solutions and separating it from other polypeptides such as Lys-Plasm inogen and Plasmin, such as, e.g., from blood fractions. It was taught that mixtures of Glu- and Lys-Plasminogen can be isolated from plasma or plasma fractions (US 8,268,782, WO 2018 / 162754, WO 2022 / 258590). These methods typically result in mixtures of Glu- and Lys-Plasminogen, and optionally additionally Plasmin. There is an unmet need for a method for isolating Glu-Plasminogen, in particular separating it from Lys-Plasminogen and / or Plasmin. Surprisingly, it has been found that purifying Glu-Plasminogen via cation exchange chromatography including a step of eluting the Glu-Plasminogen from the cation exchange solid phase with an elution buffer of a pH in the range of 6.2 to 6.5 results in particularly pure Glu-Plasminogen. In particular, it was effectively separated from Lys-Plasminogen and Plasmin. A first aspect of the present invention relates to a method for isolating Glu-Plasminogen from a solution containing Glu-Plasminogen and one or more other polypeptides, wherein the method comprises the following steps: (i) contacting the solution with a cation exchange solid phase at a pH below 5.5 and thereby immobilizing the Glu-Plasminogen on the cation exchange solid phase; (ii) washing the cation exchange solid phase on which Glu-Plasminogen is immobilized obtained from step (i) with a washing buffer of a pH below 5.5; and (iii) eluting the Glu-Plasminogen from the cation exchange solid phase with an elution buffer of a pH in the range of 6.2 to 6.5. It was surprisingly found that the claimed method provides high yields of Glu-Plasminogen of high purity. The method could be conducted with comparatively low work efforts. Thus, the present invention is technically beneficial. In a preferred embodiment, contacting the solution with a cation exchange solid phase in step (i) is conducted at a pH value in a range of from 4.8 to 5.2, particularly at pH value of 5.0. In a preferred embodiment, washing the cation exchange solid phase in step (ii) is conducted with a washing buffer of a pH value in a range of from 5.0 to 5.4, particularly at pH value of 5.2. In a preferred embodiment, eluting the Glu-Plasminogen from the cation exchange in step (iii) is conducted with an elution buffer of a pH value in a range of from 6.2 to 6.4, particularly at pH value of 6.3. The structure of such proteins and the function of the Plasminogen / Plasmin system are generally known well. Glu-Plasminogen in the sense of the present invention may be Glu-Plasminogen of any species of interest. Preferably, Glu-Plasminogen is of human or mammalian origin. The terms “polypeptide”, “protein” and “peptide” may be understood interchangeably throughout the invention in the broadest sense as any chemical entity mainly composed of amino acid residues and comprising at least twenty amino acid residues consecutively linked with another via amide bonds. It will be understood that a polypeptide in the sense of the present invention may or may not be subjected to one or more posttranslational modification(s) and / or be conjugated with one or more non-amino acid moiety / moieties. The termini of the polypeptide may, optionally, be capped by any means known in the art, such as, e.g., amidation, acetylation, methylation, acylation. Posttranslational modifications are well-known in the art and may be, but may not be limited to, lipidation, phosphorylation, sulfatation, glycosylation, truncation, oxidation, reduction, decarboxylation, acetylation, amidation, deamidation, disulfide bond formation, amino acid addition, cofactor addition (e.g., biotinylation, heme addition, eicosanoid addition, steroid addition) and complexation of metal ions, nonmetal ions, peptides or small molecules and addition of iron-sulphide clusters. Moreover, optionally, co-factors, in particular cyclic guanidinium monophosphate (cGMP), but optionally also such as, e.g., ATP, ADP, NAD+, NADH+H+, NADP+, NADPH+H+, metal ions, anions, lipids, etc. may be bound to the protein, irrespective on the biological influence of these co-factors. In the context of Glu-Plasminogen, in particular, glycosylation and disulfide bonds may occur. An advantage of the method of the present invention is that two glycosylation patterns of Glu-Plasminogen may be observable in the isolated product. This may reflect the natural Glu-Plasminogen. Glu-Plasminogen is a plasma-derived proenzyme. It is known that Glu-Plasminogen has (essentially) no proteolytic activity. Preferably, the enzymatic activity of Glu-Plasminogen and / or the Glu-Plasminogen composition that may be obtained from the method of the present invention is below 70 units (U, i.e., pmol substrate / min) per 1.0 g / L of total polypeptide content, or below 50, below 25, below 10, below 9, below 8, below 7, below 6, below 5, below 2, below 1, below 0.5, below 0.1, or below 0.01 U per 1.0 g / L of total polypeptide content. Such (essential) absence in proteolytic activity may be understood in the broadest sense as generally understood by a person skilled in the art. In this context, proteolytic activity may be determined by any means. For instance, it may be the activity determined by an S-2288 (Chromogenix) proteolytic activity assay. Alternatively, it may also be determined as the degradation of fibrin resulting in the generation of D-dimers. The specific enzyme activity of plasmin (that may be obtained from the Glu-Plasminogen after its activation) may be determined by measuring the generation of D-dimers from the degradation of fibrin. Preferably, proteolytic activity of Glu-plasminogen composition (preferably containing Glu-Plasminogen) that may be obtained from the method of the present invention is below the detection limit of the assay. Preferably, the Glu-Plasminogen that may be obtained from the method of the present invention has (essentially) no proteolytic activity. As used herein, the term “isolating Glu-Plasminogen” may be understood in the broadest sense as separating Glu-Plasminogen from other components, in particular other polypeptides, and increasing the content of Glu-Plasminogen, related to other polypeptides. The terms “isolating”, “purifying”, “increasing the content of”, etc. may be used interchangeably. It will be understood that “isolating” does not necessarily mean entire purity. In a preferred embodiment, the Glu-Plasminogen composition that may be obtained from the method of the present invention contains Glu-plasminogen in a purity of at least 75% (w / w), at least 80% (w / w), at least 85% (w / w), at least 90% (w / w), at least 95% (w / w), at least 96% (w / w), at least 97% (w / w), at least 98% (w / w), or at least 99% (w / w), based on the total polypeptide content. As used herein the term “purity” in the context of Glu-Plasminogen preferably refers to content related to the total polypeptide content, in particular Lys-Plasminogen and Plasmin (e.g., the Glu-Plasminogen : Lys-Plasminogen ratio). In a preferred embodiment, the Glu-Plasminogen composition that may be obtained from the method of the present invention has a mass ratio of Glu-Plasminogen : Lys-Plasminogen of at least 2 : 1, of at least 5 : 1, of at least 10 : 1, of at least 20 : 1, of at least 50 : 1, or of at least 100 : 1. In a preferred embodiment, the obtained Glu-Plasminogen contains no or only a low endotoxin content of below 1 Ell / mL, below 0.5 Ell / mL, below 0.1 Ell / mL, below 0.05 Ell / mL, or below 0.01 Ell / mL (as determined in a Limulus Amebocyte Lysate (LAL) endosafe endochrome assay according to European Pharmacopeia (version 5.0) chapter 2.6.14). In a preferred embodiment, the Glu-Plasminogen composition that may be obtained from the method of the present invention may contain no or only a low immunoglobulin content of below 5 g / L, below 2 g / L, below 1 g / L, below 0.5 g / L, or below 0.1 g / L of immunoglobulins (determined in a nephelometric assay). As used herein, other impurities, which are soluble in the basic aqueous buffer, may be any impurities such as one or more small molecular impurities such as, e.g., precipitation or dissolution agents (e.g., octanoic acid and / or one or more excipients), and / or may be one or more high-molecular weight impurities such as, e.g., one or more other polypeptides. As used herein, the term “aqueous buffer” may be understood in the broadest sense as any buffer solution that mainly (i.e., of more than 50% by weight) comprises water. A cation exchange solid phase may be any cation exchange material that is available in solid form. Preferably, it does not have size-exclusion properties. In a preferred embodiment, the cation exchange solid phase is based on a sulfoisobutyl moiety coupled to a solid support. Alternatively, also solid phases based on one or more sulfo-groups and / or on one or more carboxy groups may be used as a cation exchange solid phase. In a preferred embodiment, the cation exchange solid phase is Eshmuno CPX resin obtainable from Merck, Germany, which is a strong ion exchanger based on a sulfoisobutyl moiety coupled to a hydrophilic polyvinyl ether matrix. The cation exchange solid phase may be a cation exchange resin that may optionally be present in (essentially) spherical form. A cation exchange resin may be immobilized on a solid carrier. Such solid carrier may have any structure such as, e.g., a polysaccharide (e.g., Sepharose), silica, a synthetic polymer such as, e.g., polystyrene, or a combination of two or more thereof. The cation exchange solid phase may be used in a dispersion. The cation exchange solid phase may be used in a flow-through column or may be mixed manually with the solution containing Glu-Plasminogen of step (i). In a preferred embodiment, the cation exchange solid phase is used in a flowthrough column. Then, it may also be designated as stationary phase. Such stationary phase may be based on a cation exchange resin or may be a monolithic stationary phase. The solutions of loading of step (i), washing of step (ii) and eluting of step (iii) may sequentially flow through the column containing the cation exchange solid phase as stationary phase. The flow through may be set to allow sufficient time to contact between Glu-Plasminogen and the cation exchange solid phase. For example, such contact time may be in the range of at least 30 seconds, at least 1 min, at least 2 min, at least 5 min, 1 to 30 min, 2 to 15 min, or 3 to 10 min. When the cation exchange solid phase is mixed manually with the solution containing Glu-Plasminogen of step (i), the cation exchange solid phase may be mixed with the solution containing Glu-Plasminogen. Then, it may optionally be incubated to achieve an interaction with the Glu-Plasminogen. Subsequently, the cation exchange solid phase to which the Glu-Plasminogen is attached may be separated from the fluid by any means such as, e.g., by centrifugation, sedimentation or filtration. Optionally, it may be re-suspended once, twice, or more often and separated from the fluid again, which may be designated as a washing step. Finally, the Glu-Plasminogen may be eluted by mixing the cation exchange solid phase with the elution buffer of step (iii). Optionally, a washing step may be conducted each once or may be repeated twice or more often. The optional incubation to achieve an interaction with the Glu-Plasminogen may be at any range. For example, it may be conducted for a time range of from 1 min to 48 hours, 5 min to 24 hours, 10 min to 12 hours, 15 min to 6 hours, 20 min to 2 hours, 25 min to 1 hour, 25 min to 45 min, or approximately 30 min. The solution used in step (i) may be used directly when obtained from a source such as, e.g., blood or a blood fraction, e.g., from a plasma fractionation process or may be stored. Optionally, the method of the present invention may be coupled to a plasma fractionation process and conducted as part of the procedural flow (also designable as on-line). Alternatively, a stored solution used in step (i) may be used for the method of the present invention. Such stored solution used in step (i) may optionally be cooled or may be frozen and thawed when conducting the method of the present invention. As noted above, the solution used in step (i) has a pH below 5.5. In a preferred embodiment, the solution used in step (i) has a pH in the range of pH 1 to 5.5, of pH 2 to 5.5, of pH 3 to 5.5, of pH 4 to 5.5, of pH 4.5 to 5.5, of pH 4.6 to 5.4, of pH 4.7 to 5.3, of pH 4.8 to 5.2, of pH 4.9 to 5.1, of approximately pH 5.0 or of exactly pH 5.0. Plasminogen and Plasmin have different isoelectric points (pl) due to their amino acid composition and glycosylation states. According to knowledge in the art as well as theoretical calculations based on amino acid composition, a lower pl is indicated for Glu-Plasminogen compared to Lys-Plasminogen and Plasmin. In steps (i) and (ii), the pH was chosen to result in positively charged Glu-Plasminogen, which does (essentially) not bind to the cation exchange (CEX) chromatography, while Lys-Plasminogen and Plasmin remain negatively charged and stay bound to the CEX chromatography. A noted above, the step (iii) is conducted with an elution buffer of a pH in the range of 6.2 to 6.5. It was surprisingly found that, in the step (iii), using pH ranges below pH 6.2 results in a loss of Glu-Plasminogen, which does not or purely elute at pH below 6.2. Furthermore, it was surprisingly found that, in the step (iii), at pH ranges above pH 6.5 (i.e., above pH 6.54), the content of Lys-Plasminogen and / or Plasmin undesirably increased and the purity of the Glu-Plasminogen composition decreased. Using pH ranges pf pH 6.2 to 6.5 results is reasonably pure Glu-Plasminogen and acceptable yields. In a preferred embodiment, the step (iii) is conducted with an elution buffer of a pH in the range of 6.15 to 6.54, of 6.2 to 6.4, of 6.3 to 6.5, of 6.3 to 6.4. of 6.4 to 6.5, of 6.45 to 6.54, of approximately pH 6.3 or of exactly pH 6.3. In a preferred embodiment, the step (iii) is conducted with an elution buffer of a pH of 6.5. In a preferred embodiment, the step (iii) is conducted with an elution buffer of a pH in a range of pH 6.45 to 6.54, of pH 6.46 to 6.53, of pH 6.47 to 6.52, of pH 6.48 to 6.51, of pH 6.49 to 6.51, or of pH 6.50. In another preferred embodiment, the step (iii) is conducted with an elution buffer of a pH in a range of pH 6.25 to 6.35, or of pH 6.3. In a preferred embodiment, the elution buffer has a pH in a range at or slightly below the isoelectric point of Glu-Plasminogen. The elution buffer may contain any buffer agent. In a preferred embodiment, the elution buffer of step (iii) contains citric acid, a citrate salt or both. In a preferred embodiment, the elution buffer of step (iii) is a citrate buffer, in particular a citrate / glycine buffer. In a preferred embodiment, the elution buffer of step (iii) is a citrate buffer containing 25 to 100 mM citric acid. In a preferred embodiment, the elution buffer of step (iii) is a citrate buffer containing 25 to 75 mM citric acid, 30 to 70 mM citric acid, 40 to 60 mM citric acid, or (approximately) 50 mM citric acid. In a preferred embodiment, the elution buffer of step (iii) is a citrate buffer containing 25 to 100 mM glycine. In a preferred embodiment, the elution buffer of step (iii) is a citrate buffer containing 25 to 75 mM glycine, 30 to 70 mM glycine, 40 to 60 mM glycine, or (approximately) 50 mM glycine. In a preferred embodiment, the elution buffer of step (iii) comprises 10 to 200 mM sodium chloride. In a preferred embodiment, the elution buffer of step (iii) comprises 20 to 150 mM sodium chloride, 50 to 125 mM sodium chloride, or (approximately) 100 mM sodium chloride. In a preferred embodiment, the elution buffer of step (iii) contains: (A)   25 to 100 mM citric acid, 25 to 75 mM citric acid, 30 to 70 mM citric acid, 40 to 60 mM citric acid, or (approximately) 50 mM citric acid; (B)   25 to 100 mM glycine, 25 to 75 mM glycine, 30 to 70 mM glycine, 40 to 60 mM glycine, or (approximately) 50 mM glycine; and (C)   10 to 200 mM sodium chloride, 20 to 150 mM sodium chloride, 50 to 125 mM sodium chloride, or (approximately) 100 mM sodium chloride. In a preferred embodiment, the elution buffer of step (iii) comprises citric acid and glycine in a molar ration of 2 : 1 to 1 : 2, of 1.5 : 1 to 1 : 1.5, or 1 :1. As noted above, the washing buffer of step (ii) has a pH below 5.5. In a preferred embodiment, the washing buffer of step (ii) has a pH in the range of pH 1 to 5.5, of pH 2 to 5.5, of pH 3 to 5.5, of pH 4 to 5.5, of pH 4.5 to 5.5, of pH 4.6 to 5.4, of pH 4.7 to 5.3, of pH 4.8 to 5.2, of pH 4.9 to 5.1, of pH 4.9 to 5.3, of pH 5.0 to 5.4, of pH 5.1 to 5.3, of pH 5.0 to 5.2, of pH 5.1 to 5.2, of pH 5.0 to 5.1, of approximately pH 5.0, or of approximately pH 5.2. The washing buffer may contain any buffer agent. In a preferred embodiment, the washing buffer of step (ii) contains citric acid, a citrate salt or both. In a preferred embodiment, the washing buffer of step (ii) is a citrate buffer, in particular a citrate / glycine buffer. In a preferred embodiment, the washing buffer of step (ii) is a citrate buffer containing 25 to 100 mM citric acid. In a preferred embodiment, the washing buffer of step (ii) is a citrate buffer containing 25 to 75 mM citric acid, 30 to 70 mM citric acid, 40 to 60 mM citric acid, or (approximately) 50 mM citric acid. In a preferred embodiment, the washing buffer of step (ii) is a citrate buffer containing 25 to 100 mM glycine. In a preferred embodiment, the washing buffer of step (ii) is a citrate buffer containing 25 to 75 mM glycine, 30 to 70 mM glycine, 40 to 60 mM glycine, or (approximately) 50 mM glycine. In a preferred embodiment, the washing buffer of step (ii) comprises 10 to 200 mM sodium chloride. In a preferred embodiment, the washing buffer of step (ii) comprises 20 to 150 mM sodium chloride, 50 to 125 mM sodium chloride, or (approximately) 100 mM sodium chloride. In a preferred embodiment, the washing buffer of step (ii) contains: (A)   25 to 100 mM citric acid, 25 to 75 mM citric acid, 30 to 70 mM citric acid, 40 to 60 mM citric acid, or (approximately) 50 mM citric acid; (B)   25 to 100 mM glycine, 25 to 75 mM glycine, 30 to 70 mM glycine, 40 to 60 mM glycine, or (approximately) 50 mM glycine; and (C)   10 to 200 mM sodium chloride, 20 to 150 mM sodium chloride, 50 to 125 mM sodium chloride, or (approximately) 100 mM sodium chloride. In a preferred embodiment, the washing buffer of step (ii) comprises citric acid and glycine in a molar ration of from 2 : 1 to 1 : 2, of from 1.5 : 1 to 1 : 1.5, or of (approximately) 1 : 1. Any of steps (i), (ii) and (iii) may be carried out at any conditions suitable for the respective purpose. Preferably, each step is carried out at a temperature in the range of between 0 °C and 30 °C, preferably in the range of between 4 °C and 25 °C, between 4 °C and 10 °C, or between 17 °C and 22 °C. The one or more other polypeptides that may be contained in the solution of step (i) from which Glu-Plasminogen is isolated may be any polypeptides, in particular any polypeptides that are present in blood, in particular blood plasma. In a preferred embodiment, the one or more other polypeptides comprise at least one polypeptide selected from the group consisting of Lys-Plasminogen and Plasmin. The solution of step (i) from which Glu-Plasminogen is isolated may be any solution containing Glu-Plasminogen and one or more other polypeptides such as, e.g., Lys-Plasminogen and / or Plasmin. In a preferred embodiment, the solution of step (i) originates from blood or a fraction thereof, preferably from a blood plasma fraction. As used herein, blood plasma (also designated as “plasma”, “plasm” or “blood plasm” etc.) may be obtained from any source. It may for instance be obtained from a blood preservation from which the cells have been removed. Blood plasma is also commercially available from various suppliers. In the context of the present invention, the term “plasma fraction” may be understood in the broadest sense as any part separated from blood plasma that comprises Glu-plasminogen. The person skilled in the art knows several routes for preparing plasma fractions from blood plasma. One commonly known example is the Cohn process (also designated as Cohn method) based on freeze-thaw cycles and gradually increasing the concentration of ethanol in the solution. The solution of step (i) may originate from a blood plasma fraction. The solution of step (i) may originate from a precipitate of a blood plasma fraction. A precipitate of a blood plasma fraction as used herein may be blood plasma or any fraction thereof, which comprises Glu-Plasminogen. Preferably, the blood plasma fraction is obtained from a well-established blood fractionation process such as, e.g., the Cohn or Kistler-Nitschmann process. In a preferred embodiment, the solution of step (i) originates from a blood plasma fraction selected from the group consisting of: (a) cryo-poor plasma supernatant or cryo-poor plasma precipitate; (b) a fraction of any of paste I, II or III of the Cohn process, or a combination of two or all thereof; (c) a fraction of any of paste I, II or III of the Kistler-Nitschmann process, or a combination of two or all thereof; and (d) a combination of two or all thereof. In a preferred embodiment, the blood plasma fraction is selected from the group consisting of a fraction of paste I of the Cohn process, a fraction of paste II of the Cohn process, a fraction of paste III of the Cohn process, a fraction of pastes I and II of the Cohn process, a fraction of pastes I and III of the Cohn process, a fraction of pastes II and III of the Cohn process, a fraction of pastes I, II and III of the Cohn process. In a preferred embodiment, the blood plasma fraction is selected from the group consisting of a side or waste fraction of paste I of the Cohn process, a side or waste fraction of paste II of the Cohn process, a side or waste fraction of paste III of the Cohn process, a side or waste fraction of pastes I and II of the Cohn process, a side or waste fraction of pastes I and III of the Cohn process, a side or waste fraction of pastes II and III of the Cohn process, a side or waste fraction of pastes I, II and III of the Cohn process. In a preferred embodiment, the blood plasma fraction is a fraction of pastes II and III of the Cohn process. In a preferred embodiment, the blood plasma fraction is a fraction of pastes I and II of the Cohn process. In a preferred embodiment, the blood plasma fraction is a side or waste fraction of pastes II and III of the Cohn process. In a preferred embodiment, the blood plasma fraction is a side or waste fraction of pastes I and II of the Cohn process. In another preferred embodiment, the blood plasma fraction is selected from the group consisting of a fraction of paste I of the Kistler-Nitschmann process, a fraction of paste II of the Kistler-Nitschmann process, a fraction of paste III of the Kistler-Nitschmann process, a fraction of pastes I and II of the Kistler-Nitschmann process, a fraction of pastes I and III of the Kistler-Nitschmann process, a fraction of pastes II and III of the Kistler-Nitschmann process, a fraction of pastes I, II and III of the Kistler-Nitschmann process. In another preferred embodiment, the blood plasma fraction is selected from the group consisting of a side or waste fraction of paste I of the Kistler-Nitschmann process, a side or waste fraction of paste II of the Kistler-Nitschmann process, a side or waste fraction of paste III of the Kistler-Nitschmann process, a side or waste fraction of pastes I and II of the Kistler-Nitschmann process, a side or waste fraction of pastes I and III of the Kistler-Nitschmann process, a side or waste fraction of pastes II and III of the Kistler-Nitschmann process, a side or waste fraction of pastes I, II and III of the Kistler-Nitschmann process. In a preferred embodiment, the blood plasma fraction is Kistler-Nitschmann precipitate A (PPT-NA). In a preferred embodiment, the blood plasma fraction is a fraction of pastes II and III of the Kistler-Nitschmann process. In a preferred embodiment, the blood plasma fraction is a fraction of pastes I and II of the Kistler-Nitschmann process. In a preferred embodiment, the blood plasma fraction is a side or waste fraction of pastes II and III of the Kistler-Nitschmann process. In a preferred embodiment, the blood plasma fraction is a side or waste fraction of pastes I and II of the Kistler-Nitschmann process. In another preferred embodiment, the blood plasma fraction is selected from the group consisting of fraction of paste I of the Cohn process, fraction of paste II of the Cohn process, fraction of paste III of the Cohn process, fraction of pastes I and II of the Cohn process, fraction of pastes I and III of the Cohn process, fraction of pastes II and III of the Cohn process, fraction of pastes I, II and III of the Cohn process. In a preferred embodiment, the blood plasma fraction is fraction of pastes II and III of the Cohn process. In a preferred embodiment, the blood plasma fraction is fraction of pastes I and II of the Cohn process. In another preferred embodiment, the blood plasma fraction is selected from the group consisting of fraction of paste I of the Kistler-Nitschmann process, fraction of paste II of the Kistler-Nitschmann process, fraction of paste III of the Kistler-Nitschmann process, fraction of pastes I and II of the Kistler-Nitschmann process, fraction of pastes I and III of the Kistler-Nitschmann process, fraction of pastes II and III of the Kistler-Nitschmann process, fraction of pastes I, II and III of the Kistler-Nitschmann process. In a preferred embodiment, the blood plasma fraction is fraction of pastes II and III of the Kistler-Nitschmann process. In a preferred embodiment, the blood plasma fraction is fraction of pastes I and II of the Kistler-Nitschmann process. The solution contacted with the cation exchange solid phase in step (i) may be any solution such as, e.g., blood or a blood plasma faction such as described above, that may be used directly or further processed. In a preferred embodiment, the solution contacted with the cation exchange solid phase in step (i) is obtained from a precipitate containing Glu-Plasminogen. Such precipitate may be obtainable (obtained) by any means. In a preferred embodiment, the precipitate is obtained from contacting the blood fraction comprising Glu-Plasminogen with an organic solvent (e.g., octanoic acid, methanol, ethanol, pentanol, butanol, pentanol, hexanol, heptanol, octanol, phenol, acetone, etc.). In another preferred embodiment, the precipitate is obtained from contacting the blood fraction comprising Glu-Plasminogen with a chaotropic compound (e.g., a chaotropic salt (e.g., a barium salt, a calcium salt, a magnesium salt, a chlorate), guanidinium hydrochloride, thiocyanate such as guanidinium thiocyanate, perchlorate, iodide, urea, thiourea, high concentrations of sodium chloride, etc.). Such organic solvent or chaotropic salt can also be designated as precipitating agent. In another preferred embodiment, the precipitate is obtained from cooling the blood fraction comprising Glu-Plasminogen or subjecting the fraction to a freezethaw cycle. A precipitate may be obtained by any means known in the art. In a preferred embodiment, the precipitate of blood plasma fraction comprising Glu-Plasminogen is obtained from freezing and thawing the blood plasma or a fraction thereof (also: subjecting the blood plasma or a fraction thereof to a freeze-thaw cycle), change of temperature, change of pH, addition of at least one precipitating agent, or a combination of two or more thereof. In a preferred embodiment, the precipitating agent is selected from the group consisting of octanoic acid, ethanol, a salt, or polyethylene glycol, or a combination of two or more thereof to the blood plasma fraction. In a preferred embodiment, the precipitate of blood plasma fraction comprising Glu-Plasminogen is obtained from addition of octanoic acid to the blood plasma fraction. It will be understood that the term “octanoic acid” as used herein is understood in the broadest sense as any compound comprising an octanoate anion, i.e., including the free acid as well as a salt thereof. A precipitate may be provided in any form. In a preferred embodiment, the precipitate is obtained by filtration, preferably dead-end filtration. Then, it can be also designated as filter cake. In a preferred embodiment, the precipitate is an octanoic acid (OA) filter cake, i.e., a precipitate obtained by contacting a blood fraction comprising Glu-Plasminogen with octanoic acid and subsequent filtration. Alternatively, the precipitate of step (i) is obtained by centrifugation. The precipitate obtainable from a plasma fractionation process may be used directly when obtained from the plasma fractionation process or may be stored. Optionally, the method of the present invention may be coupled to a plasma fractionation process and conducted as part of the procedural flow (also designable as on-line). Alternatively, a stored precipitate may be used for the method of the present invention. Such stored precipitate may optionally be cooled or may be frozen and thawed when conducting the method of the present invention. In a preferred embodiment, the solution contacted with the cation exchange solid phase in step (i) is obtained from the following steps: (0-a) dispersing the octanoic acid precipitate, in particular an octanoic acid precipitate of blood or a fraction thereof as defined herein, in a basic aqueous buffer of pH 8 to 10 more preferably pH 8.5 to 9.5, and optionally incubating the obtained dispersion to allow the dissolution of at least parts of the polypeptides and other impurities which are soluble in the basic aqueous buffer; (0-b) separating the solid parts of the dispersion from the liquid parts of step (i-a), in particular by filtration, centrifugation dialysis, phase separation, sedimentation, centrifugation, or a combination thereof, and optionally washing the solid parts; (0-c) dissolving a fraction containing Glu-Plasminogen from the solid parts obtained from step (i-b), in particular a buffer of pH 2 to 6.6, preferably pH 4.5 to pH 5; and (0-d) obtaining the solution containing Glu-Plasminogen. The basic aqueous buffer of step (0-a) may have a pH in the range of pH 8 to 10. In a preferred embodiment, the basic aqueous buffer of step (I) is of pH 8.1 to 9.9, of pH 8.2 to 9.8, of pH 8.3 to 9.7, of pH 8.4 to 9.6, of pH 8.5 to 9.5, of pH 8.6 to 9.4, of pH 8.7 to 9.3, of pH 8.8 to 9.2, of pH 8.9 to 9.1, or of pH 9. The buffer used in step (0-c) may have a pH in the range of pH 2 to 6.6. In a preferred embodiment, the acidic aqueous buffer in step (0-c) is pH 2 to 6, of pH 2.5 to 5.9, of pH 3 to 5.7, of pH 3.5 to 5.6, of pH 4 to 6, of pH 4.5 to 5.5, of pH 4.6 to 5.4, of pH 4.7 to 5.3, of pH 4.8 to 5.2, of pH 4.9 to 5.1,or of pH 5. Preferably, the pH of the acidic aqueous buffer in step (0-c) is not above, more preferably below, pH 6.6, i.e. the isoelectric point of Glu-plasminogen. The acidic buffer and / or the basic buffer may contain any buffer agent or combination thereof suitable for such purpose. Preferably, the buffer agents are pharmaceutically acceptable buffer agents. In a preferred embodiment, the acidic aqueous buffer comprises formic acid, acetate, carbonate, hydrogen carbonate, and / or citrate, preferably sodium formate, sodium acetate, citric acid or sodium citrate, in particular sodium acetate. In a preferred embodiment, the acidic aqueous buffer is an acetate buffer, in particular a sodium acetate buffer. For instance, acidic aqueous buffers as used herein may comprise a sodium acetate I glycine buffering system or a citric acid I glycine buffering system. It will be understood that the acidic aqueous buffer may optionally also comprise one or more further ingredients. In a preferred embodiment, it comprises a water-soluble or emulsifiable polymer such as, e.g., polyethylene glycol (PEG), preferably having a mean molecular weight (determinable by size exclusion chromatography) of 200 to 35.000 Da, 1.000 to 100.000 Da, 200 to 1.000 Da, 500 to 5.000 Da, 1.000 to 10.000 Da, 5.000 to 50.000 Da, or 10.000 to 100.000 Da. Additionally or alternatively, the solution of step (i) may be supplemented with one or more agents that support dissolution of Glu-Plasminogen. Dispersing may be carried out by any means. For instance, dispersing may be performed by adding the basic aqueous buffer and shaking, mixing (manually or via a stirrer, mixer or blender), or a combination thereof. As used herein, dispersing may be preparing any (precipitate) : (basic aqueous buffer) ratio. In a preferred embodiment, the (precipitate): (basic aqueous buffer) weight ratio is in the range of from 20 : 1 to 1 : 20, of from 10 : 1 to 1 : 10, of from 2 : 1 to 1 : 20, of from 1 : 1 to 1 : 20, of from 1 : 2 to 1 : 10, of from 1 : 3 to 1 : 7, of from 1 : 4 to 1 : 6, or in the range of approximately 1 : 5. In a preferred embodiment, the solution of step (i) comprises dissolved lysine and / or at least one other compound of formula (I) or a salt thereof: (H2N)n-R-(A)m (I), wherein: n is an integer of 1 or 2; m is an integer of 0, 1 or 2; A is at each occurrence independently from each other a carboxyl group, or an amino group; R is a linear or branched C3-Ci2-alkylene, a linear or branched C3-C12-heteroalkylene, a C6-Ci2-arylene optionally substituted by one or more halogens or one or more Ci-C4-(hetero)alkyl residues, a C3-Ci2-heteroarylene optionally substituted by one or more halogens or one or more Ci-C4-(hetero)alkyl residues, a C3-Ci2-alkylene-C6-Ci2-arylene optionally substituted by one or more halogens or one or more Ci-C4-(hetero)alkyl residues, a C3-Ci2-alkylene-C3-Ci2- heteroarylene optionally substituted by one or more halogens or one or more Ci-C4-(hetero)alkyl residues, preferably wherein the compound of formula (I) is selected from the group consisting of aminohexanoic acid, aminopentanoic acid, aminoheptanoic acid, aminooctanoic acid, aminononanoic acid, 1,6-diaminohexane, aminodecanoic acid, ornithine, aminomethyl benzoic acid, oxalysine, and a salt thereof, in particular wherein the at least one compound of formula (I) is 6-aminohexanoic acid or a salt thereof. In a preferred embodiment, the at least one compound of formula (I) is selected from the group consisting of aminohexanoic acid, aminopentanoic acid, aminoheptanoic acid, aminooctanoic acid, aminononanoic acid, 6-diaminohexane, aminodecanoic acid, ornithine, aminomethyl benzoic acid, and oxalysine, in particular wherein the at least one compound of formula (I) is 6-aminohexanoic acid, in particular aminohexanoic acid. In a preferred embodiment, the solution of step (i) comprises dissolved lysine, in particular L-lysine. In a preferred embodiment, the solution of step (i) comprises dissolved 6-aminohexanoic acid. The solution containing Glu-Plasminogen obtained in step (iii) may be used directly or may be further treated. In a preferred embodiment, the solution containing isolated Glu-Plasminogen obtained in step (iii) is further subjected to a further step of increasing the purity and / or the concentration of the Glu-Plasminogen by means of performing chromatography based on a solid phase comprising immobilized lysine and / or at least one immobilized compound of formula (I) wherein preferably first a buffer allowing the interaction of the Glu-Plasminogen with the solid phase, preferably of a pH in the range of from 4.4 to 5.5, is used which is followed by eluting the Glu-Plasminogen by means of a buffer, preferably of a pH in the range of 2 to 4, that decreases the interaction of the Glu-Plasminogen with the Glu-Plasminogen. In a preferred embodiment, the buffer allowing the interaction of the Glu-Plasminogen has a pH in the range of from 4.4 to 5.5, of from 4.5 to 5.4, of from 4.6 to 5.3, of from 4.7 to 5.2, of from 4.8 to 5.1, or of approximately 5.0. In a preferred embodiment, the buffer for eluting the Glu-Plasminogen has a pH in the range of from 2 to 4, of from 2.1 to 3.9, of from 2.5 to 3.5, of from 2.7 to 3.2, of from 2.9 to 3.1, or of approximately 3.0. In a preferred embodiment, the method further comprises a step of filtration, including dead-end filtration, tangential flow filtration, or a combination thereof, dialysis, and / or phase separation, preferably by sedimentation, and / or centrifugation, optionally comprising addition of a filtration aid, preferably wherein the filtration aid is selected from the group consisting of polymers, preferably high-molecular weight polyethylene glycol (PEG), cellulose, or cellulose derivatives, diatomaceous earth, perlite, detergents, and combinations of two or more thereof. A filtration aid may be any filtration aid known in the art. In a preferred embodiment, the filtration aid is selected from the group consisting of polymers, preferably high-molecular weight polyethylene glycol (PEG), cellulose, or cellulose derivatives, diatomaceous earth, perlite, detergents (e.g., Tween-20), and combinations of two or more thereof. It will be understood that also a combination of two or more filtration aids may be used. Optionally, the solution containing isolated Glu-Plasminogen obtained may be subjected to at least one further step selected from the group consisting of: subjecting the solution to at least one precipitation-washing, cycle; chromatography based on a stationary phase comprising immobilized lysine and / or at least one immobilized compound of formula (I); affinity chromatography selective for the Glu-Plasminogen; molecular size chromatography; dialysis; ultrafiltration, including dead-end ultrafiltration, tangential flow ultrafiltration, or a combination thereof; and a combination of two or more thereof. In a preferred embodiment, the method comprises the following steps: (I) dispersing an octanoic acid precipitate containing Glu-Plasminogen, in particular an octanoic acid precipitate of blood or a fraction thereof as defined herein, in a basic aqueous buffer of pH 8 to 10, more preferably pH 8.5 to 9.5; (II) incubating the dispersion obtained from step (I) to allow the dissolution of at least parts of the polypeptides and other impurities which are soluble in the basic aqueous buffer; (III) separating solid parts and liquid parts of the incubated dispersion of step (II) from each other by means of filtration, including dead-end filtration, tangential flow filtration, or a combination thereof, dialysis, phase separation, sedimentation, and / or centrifugation, or a combination thereof; (IV) mixing the solid parts obtained from step (III) with an acidic aqueous buffer of pH 2 to 6.6, preferably pH 4.5 to pH 5, wherein the acidic aqueous buffer further comprises dissolved lysine, at least one compound of formula (I) or a salt or combination thereof, and optionally incubating the mixture to allow the Glu-Plasminogen to dissolve, and removing solid parts; and (V) conducting the method of the present invention, wherein the solution contacted with a cation exchange solid phase in step (i) is obtained in step (IV), and obtaining a solution containing isolated Glu-Plasminogen; (VI) optionally increasing the purity and / or the concentration of the Glu-Plasminogen obtained from step (V) by means of performing chromatography based on a solid phase comprising immobilized lysine and / or at least one immobilized compound of formula (I), wherein first a buffer allowing the interaction of the Glu-Plasminogen with the solid phase is used which is followed by eluting the Glu-Plasminogen by means of a buffer that decreases the interaction of the Glu-Plasminogen with the solid phase; and obtaining a solution containing isolated Glu-Plasminogen. The basic aqueous buffer of step (I) may have a pH in the range of pH 8 to 10. In a preferred embodiment, the basic aqueous buffer of step (I) is of pH 8.1 to 9.9, of pH 8.2 to 9.8, of pH 8.3 to 9.7, of pH 8.4 to 9.6, of pH 8.5 to 9.5, of pH 8.6 to 9.4, of pH 8.7 to 9.3, of pH 8.8 to 9.2, of pH 8.9 to 9.1, or of pH 9. The acidic aqueous buffer in step (IV) may have a pH in the range of pH 2 to 6.6. In a preferred embodiment, the acidic aqueous buffer in step (IV) is pH 2 to 6, of pH 2.5 to 5.9, of pH 3 to 5.7, of pH 3.5 to 5.6, of pH 4 to 6, of pH 4.5 to 5.5, of pH 4.6 to 5.4, of pH 4.7 to 5.3, of pH 4.8 to 5.2, of pH 4.9 to 5.1, or of pH 5. Preferably, the pH of the acidic aqueous buffer in step (iv) is not above, more preferably below, pH 6.6, i.e., the isoelectric point of Glu-plasminogen. In a preferred embodiment, the pH-difference in between the basic aqueous buffer of step (I) and the aqueous buffer in step (IV) is at least 0.5, at least 1, at least 1,25, at least 1.5, at least 1.75, at least 2, at least 2.25, at least 2.5, at least 2.75, at least 3, at least 3.25, at least 3.5, at least 3.75, or at least 4. In a preferred embodiment, the pH-difference in between the basic aqueous buffer of step (i) and the aqueous buffer in step (iv) is between 0.5 and 10, between 1 and 9, between 2 and 8, between 2.5 and 7, between 3 and 6, between 3.5 and 5, or between 3.5 and 4.5. In a preferred embodiment, the method further comprises: (a) a virus inactivation step of the obtained composition containing isolated Glu-Plasminogen; (b) adjusting the pH of the solution containing isolated Glu-Plasminogen to a desired range, in particular to a pH below 5.5, preferably approximately pH 5.0; (c) freeze drying or drying of the Glu-Plasminogen; or (d) a combination of two or more thereof. Optionally, the method of the present invention comprises contacting the obtained solution with at least one small pore anion exchange resin interacting with at least part of the precipitating agent, in particular octanoic acid. In a preferred embodiment, such at least one small pore anion exchange resin interacting with at least part of the precipitating agent is a resin having a microporous structure such as, e.g. pores in the range of from 0.1 to 10 nm, from 1 to 100 nm, from 0.1 to 10 pm, or from 1 to 100 pm. In a preferred embodiment, such at least one small pore anion exchange resin interacting with at least part of the precipitating agent is a resin based on one or more styrene-based (co)polymers and may contain quaternary ammonium groups and / or sulfonate groups. In a preferred embodiment, such at least one small pore anion exchange resin interacting with at least part of the precipitating agent is a Dowex resin, in particular Dowex 1x8 having 50 to 100 mesh (0.15 to 0.3 mm average particle size), 100 to 200 mesh (0.08 to 0.15 mm average particle size), or 200 to 400 mesh (0.04 to 0.08 mm average particle size). The obtained Glu-plasminogen may optionally be formulated, stored or may be directly used for any purpose. For instance, it may be used for therapeutic uses. Then, the Glu-Plasminogen may be administered to an individual for use in a method for treating or preventing one or more thrombotic events. Then, preferably, the pH or a solution is adapted to the desired range before administration. Optionally, one or more further pharmaceutically acceptable ingredients may be added. Pharmaceutical and medicinal uses of Glu-plasminogen are described in detail in WO 2018 / 162754 and WO 2020 / 152322. When storage is desired, the obtained Glu-plasminogen may optionally be lyophilized (freeze-dried) or frozen. It will be understood that the method of the present invention does not only allow the preparation of Glu-Plasminogen, but also of one or more further components as desirable side products. As indicated above, the Glu-plasminogen obtainable (obtained) by the method of the present invention bears special structural characteristics as such, such as particularly high purity and optionally minor amounts of residuals of the agents used in the procedural steps. Accordingly, a further aspect of the present invention relates to a composition comprising Glu-Plasminogen obtainable from a method of the present invention, wherein the mass ratio of Glu-Plasminogen : Lys-Plasminogen is at least 10:1. It will be understood that the embodiments and definitions as laid out in the context of the method above mutatis mutandis apply to the composition comprising Glu-Plasminogen obtainable therefrom. In a preferred embodiment, the mass ratio of Glu-Plasminogen : Lys-Plasminogen is at least 2 : 1, at least 5 : 1, at least 10 : 1, at least 20 : 1, at least 50 : 1, or at least 100 : 1. In a preferred embodiment, the Glu-plasminogen makes up at least 70% (w / w) of the total polypeptide content of the composition. In a preferred embodiment, the Glu-plasminogen makes up at least 75% (w / w), at least 80% (w / w), at least 85% (w / w), at least 90% (w / w), at least 95% (w / w), at least 96% (w / w), at least 97% (w / w), at least 98% (w / w), or at least 99% (w / w), of the total polypeptide content of the composition. In one embodiment of the present invention, the Glu-Plasminogen obtainable (or obtained) from a method of the present invention is provided in a pharmaceutical composition. Accordingly, it may be admixed with one or more pharmaceutically acceptable carriers. Accordingly, a further aspect of the present invention refers to a pharmaceutical composition comprising Glu-Plasminogen obtainable (or obtained) from a method of the present invention and one or more pharmaceutically acceptable carriers. The terms “pharmaceutical composition” and “pharmaceutical formulation” may be understood interchangeably. As used herein, the terms “pharmaceutically acceptable carrier”, “pharmaceutically acceptable excipient”, “carrier” and “excipient” may be understood interchangeably in the broadest sense as any substance that may support the pharmacological acceptance of the Glu-Plasminogen and the at least one plasminogen activator, respectively. Such pharmaceutical composition may be ready to use and may preferably be a liquid formulation, in particular an injection portion. The storage form may also be liquid, but may also be a dried form (e.g. a powder such as a powder comprising dried or freeze-dried Glu-Plasminogen and the at least one plasminogen activator, respectively) or may be a paste or syrup or the like. Optionally, a dried form, paste or syrup may be dissolved or emulsified prior to being administered to the patient. A pharmaceutically acceptable carrier may exemplarily be selected from the list consisting of an aqueous buffer, saline, water, dimethyl sulfoxide (DMSO), ethanol, vegetable oil, paraffin oil or combinations of two or more thereof. Furthermore, the pharmaceutically acceptable carrier may optionally contain one or more detergent(s), one or more foaming agent(s) (e.g., sodium lauryl sulfate (SLS), sodium dodecyl sulfate (SDS)), one or more coloring agent(s) (e.g., food coloring), one or more vitamin(s), one or more salt(s) (e.g., sodium, potassium, calcium, zinc salts), one or more humectant(s) (e.g., sorbitol, glycerol, mannitol, propylene glycol, polydextrose), one or more enzyme(s), one or more preserving agent(s) (e.g., benzoic acid, methylparaben, one or more antioxidant(s), one or more herbal and plant extract(s), one or more stabilizing agent(s), one or more chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA), and / or one or more uptake mediator(s) (e.g., polyethylene imine (PEI), a cell-penetrating peptide (CPP), a protein transduction domain (PTD), an antimicrobial peptide, etc.). The present invention also relates to a dosage unit of the pharmaceutical composition usable in the context of the treatment or prevention of the present invention. Exemplarily, the present invention may refer to a single dose container or to a multiple dosage form. The present invention also relates to a composition comprising Glu-plasminogen of the present invention or a pharmaceutical composition of the present invention for use as a medicament. The present invention also relates to a composition comprising Glu-plasminogen of the present invention or a pharmaceutical composition of the present invention for use in a method for treating or preventing a thrombotic event. Such treatment with a composition comprising Glu-plasminogen is described in detail in WO 2018 / 162754 and WO 2020 / 152322. It will be understood that the numerical values taught herein may be understood in the broadest sense as generally understood as rounded values that embrace the subsequent decimal number. For instance, pH 5 includes values from pH 4.5 to 5.4 as generally understood. As used throughout the present invention, number ranges may be understood as generally understood in the art. Thus, the given numbers are to be understood as rounded ranges (rounded from the subsequent digit). For instance, a given number value of “6.5” should be understood as a range of from 6.46 to 6.54. However, in particular when it is an edge value, the given values should additionally also be considered as a disclosure of the exact value as given such as, in the above example, “below 6.54” as well as “below 6.50” or “above 6.45” as well as for “above 6.50”. The following examples and figures are intended to provide illustrative embodiments of the present invention described and claimed herein. These examples are not intended to provide any limitation on the scope of the invented subject-matter. The following figures, examples and claims further illustrate the invention. Brief description of the Figures Figure 1 shows the comparison of plasminogen products from different processes and fractions via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Herein from left to right, the lanes show Lys-Plasminogen (1), Plasmin standard (2), protein marker standard indicating the molecular mass (3), Glu- Plasminogen standard (4); plasminogen isolated by a method described in WO 2018 / 162754 (found to be rather pure Glu-Plasminogen) (5); plasminogen isolated from octanoic acid (OA) paste without CEX step yields only Lys-Plasminogen (6); Plasminogen isolated from paste l / lll (of a Cohn-like process) by a method described in WO 2022 / 258590 yields a mix of Glu- and Lys-Plasminogen (7); plasminogen isolated from octanoic acid (OA) paste by a method described in WO 2022 / 258590 yields a mix of Glu- and Lys-plasminogen (8); plasminogen isolated from isolated from octanoic acid (OA) paste according to the present invention yields pure Glu-Plasminogen (9). 0.6 pg total protein content is each loaded in lanes 1, 2 and 4. 1.5 pg total protein content is each loaded in lanes 5 to 9. The further lanes are empty. Examples Materials and methods a) Dispersing the caprylate precipitate in basic aqueous buffer A filter cake of an OA-precipitate was dispersed in a basic aqueous buffer (containing 0.1 M sodium acetate, 0.05 M glycine, 0.02 M NaCI and 3.5% (w / v) PEG 20000, the generated suspension was adjusted to a pH of 9.0 with NaOH) at a ratio of filter cake : buffer of 1 : 5) b) Incubation of the dispersion The dispersion was incubated and stirred for 1 hour under moderate cooled conditions of 4 to 10°C (tempering unit, Lauda, Germany). A filter aid (5 g / kg Harborlite 900, Imerys Filtration France S.A.S.) was added and further incubated and stirred for 30 min at moderate cooled conditions of 4 to 10°C. c) Separation of solid parts from liquid parts of the incubated dispersion The dispersion of containing the filter aid was filtered by means of a standard filter (Micro-Media XL Filter Pads, Grade M-503, Die 100L). The filter cake was washed with up to half of the buffer volume used for dispersing the precipitate. Filtration time was adjusted to the individual experiment. It was pursued until filtration was completed. Typically, it was in the range of approximately 22 min at 73 kg / m3. It was surprisingly found that the use of a basic aqueous buffer for dispersing the precipitate followed by filtration enabled efficient essential removal of impurities such as, e.g., numerous proteins other than (Glu-)Plasminogen, fatty acids, and residuals of octanoic acid. Impurities could be removed in the filtrate. The remaining filter cake contained (Glu-)Plasminogen. d)    Solubilization of (Glu-)Plasminogen The filter cake containing (Glu-)Plasminogen was mixed with an acidic aqueous buffer (containing 0.1 M sodium acetate and 0.2 M lysine analogue 6-aminohexanoic acid, adjusted to pH 5.0) at a mass ratio of filter cake : buffer in the preferable range of 1 : 3. 30 g Dowex 1x8 (DuPont Dow Chemicals, USA) per 180 g OA-precipitate were added. The mixture was incubated for 1.5 hours under moderate cooled conditions of 4 to 10°C (tempering unit, Lauda, Germany). Then, residuals were removed by filtration by means of a standard filter (Micro-Media XL Filter Pads, Grade M-503, Die 100L). Filtration time was adjusted to the individual experiment. It was pursued until filtration was completed. Typically, it was in the range of approximately 6 min, but was adapted to the filter load. The (Glu-)Plasminogen essentially remained in the solution. To improve yields, the filter residuals were washed with the same volume of the acidic aqueous buffer used for solubilization. The two solutions were subsequently combined prior to further processing. e) Cation exchange (CEX) chromatography The solutions containing Glu-Plasminogen obtained from the above were diluted in an acidic aqueous buffer (10 mM sodium acetate, 50 mM glycine, adjusted to pH 5.0) in a volume ratio 1 : 2. The strong cation exchange (CEX) chromatography resin Eshmuno CPX (Merck, Germany) was used as stationary phase. This was used on an NGC Chromatography System (BioRad, USA) equipped with a 12 mL chromatography column (126 x 11 mm) using a flow-through of 2.4 mL / min (5 min contact time). This column was equilibrated by an equilibrating buffer (50 mM sodium acetate, adjusted to pH 5.0). The diluted solutions containing Glu-Plasminogen were loaded. Then, the column was washed with a washing buffer (50 mM citric acid, 50 mM glycine, 100 mM NaCI, adjusted to pH in the range of 5.0 to 5.2). The fraction containing Glu-Plasminogen was eluted with different buffers as depicted in Table 1 below. The fraction containing Lys-Plasminogen, Plasmin and other highly active proteases remained in the adsorbed phase. It was found that the lysine analogue 6-aminohexanoic acid could effectively be removed from the solution. Furthermore, conductivity during cation exchange (CEX) chromatography was investigated. It was found that, in particular, a conductivity below 20 mS / cm is favorable for the CEX elution and the isolation of Glu-Plasminogen from Lys-Plasminogen and Plasmin. Particularly, conductivity in a range from 10 to 20 mS / cm is preferable in embodiments of the inventive method. (Data not shown.) f) Chromatography with lysine-conjugated Sepharose The solutions containing Glu-Plasminogen obtained from the above were diluted in an acidic aqueous buffer (50 mM sodium acetate, 50 mM glycine, adjusted to pH 5.0) in a volume ratio 1 : 2 (in further comparative experiments, broader ranges were used). ECH-Lysine Sepharose 4 Fast-Flow (based on crosslinked 4% agarose, GE Healthcare / Cytiva, USA) was used as stationary phase. This was used on an NGC Chromatography System (BioRad, USA) equipped with a 6 mL chromatography column (63 x 11 mm) using a flow-through of 0.6 mL / min (10 min contact time). The column was equilibrated by a 50 mM citric acid buffer of pH 5.0. The solutions containing Glu-plasminogen were loaded. The column was washed with a first washing buffer (50 mM citric acid, 50 mM glycine, 1 M NaCI, adjusted to pH 5.0). Then, the column was washed with a second washing buffer (10 mM citric acid, 50 mM glycine, adjusted to pH 5.0). Finally, the fraction containing Glu-plasminogen was eluted by a citrate-containing buffer of low pH (50 mM citrate, 50 mM glycine, 20 mM NaCI, adjusted to pH 3.0). g) Determination of elution efficacy and purity The Glu-Plasminogen and Lys-Plasminogen / Plasmin content in relation to the loaded content as loaded on the CEX was finally determined by non-reduced sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (in gels having an acrylamide content of 10%) and determination of the band intensity. The results were compared for different elution buffers as shown in Table 1. Herein, the Glu-Plasminogen and Lys-Plasminogen / Plasmin content applied to the column (as determined by omitting the CEX step) were defined as 100% each. This means that a yield of “>90%” means essentially complete elution of the respective protein from the CEX column. For example, “ca. 50%” means that approximately half of the respective protein was retarded in the CEX column and approximately the other half thereof was eluted from the column, “ca. 80%” means that approximately 1 / 5 of the respective protein was retarded in the column and approximately 4 / 5 thereof were eluted from the CEX column. “<10%” means that the respective protein was essentially completely retarded on the CEX column. The fraction of Lys-Plasminogen / Plasmin was determined as a common fraction, because these two proteins cannot be reasonably and securely distinguished from one another by nonreduced SDS-PAGE. It was found that Lys-Plasminogen as well as Plasmin should be removed from the composition to obtain pure Glu-Plasminogen. Furthermore, SDS-PAGE was also conducted at reductive conditions of which results are not shown herein. The findings confirmed the results as shown below for non-reduced SDS-PAGE. In addition, Western-Blots based on anti-Plasminogen antibodies (sheep IgG (CoaChrom Diagnostica GmbH) and secondary antibodies (anti-sheep IgG-horseradish peroxidase conjugated (Seramun Diagnostica GmbH)) were conducted of which results are not shown herein. Results As noted above, the elution efficacy and purity of the composition obtained from the CEX step were compared for different elution buffers. The results are depicted in Table 1 below. Table 1. Elution efficacy and purity of the composition obtained from the CEX step as assessed from non-reduced SDS-PAGE band intensities. Ex. Concentration of NaCI in elution buffer1 PH Eluted content in relation to the loaded content Glu- Plasminogen Lys-Plasminogen / Plasmin 12 100 mM Gradient of 5.0 to 7.0 >90% >90% 2 200 mM 6.0 >90% ca. 80% 3 100 mM 6.0 ca. 50% <10% 4 100 mM 6.5 >90% <10% 5 100 mM 7.0 >90% >90% 1 The elution buffers are each aqueous containing 50 mM citrate and 50 mM glycine and NaCI in the indicated concentration. 2 In this example, the pH was altered over time during CEX chromatography. The subsequent step of chromatography with lysine-conjugated Sepharose as performed in the other Examples 2 to 5 was omitted in Example 1. The pH gradient was applied to determine the exact pH value at which Glu-Plasminogen and Lys-Plasminogen / Plasmin start to elute. It was found that Glu-Plasminogen starts to elute at pH 5.8. The faction of Lys-Plasminogen / Plasmin starts to elute at pH 6.8. The Example (Ex.) 4 was successfully tested for two different octanoic acid (OA) pastes and a paste from fractions l / lll. The indicated values are the mean values that were observed for all the fractions. It was found that elution at pH 6.5 leads to particularly beneficial results. The balance of purity of Glu-Plasminogen (indicated as essential absence of Lys-Plasminogen and Plasmin) and yield was particularly good. The elution buffer as used in Example (Ex.) 1 was investigated further. The absorption at 280 nm, the pH and the conductivity were monitored during cation exchange chromatography (CEX) and lysine affinity chromatography. In particular, in the chromatogram indicating the absorption at 280 nm, the CEX chromatography showed different peaks, including the main peaks indicating the wash of other components, elution of Glu-Plasminogen fraction and elution of waste fraction containing Lys-Plasminogen and other proteases. The absorption at 280 nm monitored during the lysine affinity chromatography showed a sharp peak of Glu-Plasminogen. As shown in Figure 1, the present invention allows the preparation of pure Glu-Plasminogen in good yields from a blood plasma fraction such as an octanoic acid (OA) precipitation. There is virtually no Lys-Plasminogen found. Thus, the mass ratio of Glu-Plasminogen : Lys-Plasminogen is very high. Furthermore, in the Western Blot, the CEX feed showed bands of Glu-Plasminogen and Lys-Plasminogen, whereas the CEX elution shows essentially only a pure band of Glu-Plasminogen. 5 In summary, the purity of Glu-Plasminogen as obtained by the method of the present invention was determined by reduced and non-reduced SDS-PAGE and confirmed by Western Blots.

Claims

1. A method for isolating Glu-Plasminogen from a solution containing Glu-Plasminogen and one or more other polypeptides, wherein the method comprises the following steps:(i) contacting the solution with a cation exchange solid phase at a pH below 5.5 and thereby immobilizing the Glu-Plasminogen on the cation exchange solid phase;(ii) washing the cation exchange solid phase on which Glu-Plasminogen is immobilized obtained from step (i) with a washing buffer of a pH below 5.5; and(iii) eluting the Glu-Plasminogen from the cation exchange solid phase with an elution buffer of a pH in the range of 6.2 to 6.5.

2. The method of claim 1, wherein the step (iii) is conducted with an elution buffer of a pH of 6.5.

3. The method of any of claims 1 or 2, wherein the washing buffer of step (ii) is a citrate buffer, in particular a citrate buffer containing 25 to 100 mM citric acid.

4. The method of any of claims 1 to 3, wherein the elution buffer of step (iii) is a citrate buffer, in particular a citrate buffer containing 25 to 100 mM citric acid.

5. The method of any of claims 1 to 4, wherein the washing buffer of step (ii) comprises 10 to 200 mM sodium chloride.

6. The method of any of claims 1 to 5, wherein the elution buffer of step (iii) comprises 10 to 200 mM sodium chloride.

7. The method of any of claims 1 to 6, wherein the one or more other polypeptides comprise at least one polypeptide selected from the group consisting of Lys-Plasminogen and Plasmin.

8. The method of any of claims 1 to 7, wherein the solution of step (i) originates from blood or a fraction thereof, preferably from a blood plasma fraction, in particular a blood plasma fraction selected from the group consisting of: (a) cryo-poor plasma supernatant or cryo-poor plasma precipitate;(b) a fraction of any of paste I, II or III of the Cohn process, or a combination of two or all thereof;(c) a fraction of any of paste I, II or III of the Kistler-Nitschmann process, or a combination of two or all thereof; and(d) a combination of two or all thereof.

9. The method of any of claims 1 to 8, wherein the solution contacted with the cation exchange solid phase in step (i) is obtained from the following steps: (0-a) dispersing the octanoic acid precipitate, in particular an octanoic acid precipitate of blood or a fraction thereof according to claim 8, in a basic aqueous buffer of pH 8 to 10 more preferably pH 8.5 to 9.5, and optionally incubating the obtained dispersion to allow the dissolution of at least parts of the polypeptides and other impurities which are soluble in the basic aqueous buffer;(0-b) separating the solid parts of the dispersion from the liquid parts of step (i-a), in particular by filtration, centrifugation dialysis, phase separation, sedimentation, centrifugation, or a combination thereof, and optionally washing the solid parts;(0-c) dissolving a fraction containing Glu-Plasminogen from the solid parts obtained from step (i-b), in particular a buffer of pH 2 to 6.6, preferably pH 4.5 to pH 5; and(0-d) obtaining the solution containing Glu-Plasminogen.

10. The method of any of claims 1 to 9, wherein the solution of step (i) comprises dissolved lysine and / or at least one other compound of formula (I) or a salt thereof:(H2N)n-R-(A)m(I),wherein:n is an integer of 1 or 2;m is an integer of 0, 1 or 2;A is at each occurrence independently from each other a carboxyl group, or an amino group;R is a linear or branched C3-Ci2-alkylene, a linear or branched C3-C12-heteroalkylene, a C6-Ci2-arylene optionally substituted by one or more halogens or one or more Ci-C4-(hetero)alkyl residues, a C3-C12-heteroarylene optionally substituted by one or more halogens or one or more Ci-C4-(hetero)alkyl residues, a C3-Ci2-alkylene-C6-Ci2-arylene optionally substituted by one or more halogens or one or more C1-C4-(hetero)alkyl residues, a C3-Ci2-alkylene-C3-Ci2-heteroarylene optionally substituted by one or more halogens or one or more Ci-C4-(hetero)alkyl residues, preferably wherein the compound of formula (I) is selected from the group consisting of aminohexanoic acid, aminopentanoic acid, aminoheptanoic acid, aminooctanoic acid, aminononanoic acid, 1,6-diaminohexane, aminodecanoic acid, ornithine, aminomethyl benzoic acid, oxalysine, and a salt thereof, in particular wherein the at least one compound of formula (I) is 6-aminohexanoic acid or a salt thereof.

11. The method of any of claims 1 to 10, wherein the solution containing isolated Glu-Plasminogen obtained in step (iii) is further subjected to a further step of increasing the purity and / or the concentration of the Glu-Plasminogen by means of performing chromatography based on a solid phase comprising immobilized lysine and / or at least one immobilized compound of formula (I) wherein preferably first a buffer allowing the interaction of the Glu-Plasminogen with the solid phase, preferably of a pH in the range of 4.4 to 5.5, is used which is followed by eluting the Glu-Plasminogen by means of a buffer, preferably of a pH in the range of 2 to 4, that decreases the interaction of the Glu-Plasminogen with the Glu-Plasminogen.

12. The method of any of claims 1 to 11, wherein the method further comprises a step of filtration, including dead-end filtration, tangential flow filtration, or a combination thereof, dialysis, and / or phase separation, preferably by sedimentation, and / or centrifugation, optionally comprising addition of a filtration aid, preferably wherein the filtration aid is selected from the group consisting polymers, preferably high-molecular weight polyethylene glycol (PEG), cellulose, or cellulose derivatives, diatomaceous earth, perlite, detergents, and combinations of two or more thereof.

13. The method of any of claims 1 to 12, wherein the method comprises the following steps:(I) dispersing an octanoic acid precipitate containing Glu-Plasminogen, in particular an octanoic acid precipitate of blood or a fraction thereof according to claim 8, in a basic aqueous buffer of pH 8 to 10, more preferably pH 8.5 to 9.5;(II) incubating the dispersion obtained from step (I) to allow the dissolution of at least parts of the polypeptides and other impurities which are soluble in the basic aqueous buffer;(III) separating solid parts and liquid parts of the incubated dispersion of step (II) from each other by means of filtration, including dead-end filtration, tangential flow filtration, or a combination thereof, dialysis, phase separation, sedimentation, and / or centrifugation, or a combination thereof;(IV) mixing the solid parts obtained from step (III) with an acidic aqueous buffer of pH 2 to 6.6, preferably pH 4.5 to pH 5, wherein the acidic aqueous buffer further comprises dissolved lysine, at least one compound of formula (I) or a salt or combination thereof, and optionally incubating the mixture to allow the Glu-Plasminogen to dissolve, and removing solid parts; and(V) conducting a method of any one of claims 1 to 5, wherein the solution contacted with a cation exchange solid phase in step (i) is obtained in step (IV), and obtaining a solution containing isolated Glu-Plasminogen;(VI) optionally increasing the purity and / or the concentration of the Glu-Plasminogen obtained from step (V) by means of performing chromatography based on a solid phase comprising immobilized lysine and / or at least one immobilized compound of formula (I), wherein first a buffer allowing the interaction of the Glu-Plasminogen with the solid phase is used which is followed by eluting the Glu-Plasminogen by means of a buffer that decreases the interaction of the Glu-Plasminogen with the solid phase; and obtaining a solution containing isolated Glu-Plasminogen.

14. The method of any of claims 1 to 13, further comprising:(a) a virus inactivation step of the obtained composition containing isolated Glu-Plasminogen;(b) adjusting the pH of the solution containing isolated Glu-Plasminogen to a desired range, in particular to a pH below 5.5;(c) freeze drying or drying of the Glu-Plasminogen; or (d) a combination of two or more thereof.

515. A composition comprising Glu-Plasminogen obtainable from a method of any of claims 1 to 14, wherein the mass ratio of Glu-Plasminogen : Lys-Plasminogen is at least 10 : 1, in particular wherein Glu-plasminogen makes up at least 70% (w / w) of the total polypeptide content of the composition.10

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