Method for isolating plasminogen from a plasma fraction - Patents.com

Abstract

The present invention relates to a method for isolating plasminogen from a plasma fraction, comprising dispersing a precipitate of the plasma fraction containing plasminogen in a basic aqueous buffer, separating the solid portion to remove at least a portion of other proteins, and extracting the plasminogen with an acidic aqueous buffer.

Description

[Technical field]

[0001] The present invention relates to a method for isolating plasminogen from a plasma fraction, comprising dispersing a precipitate of the plasma fraction containing plasminogen in a basic aqueous buffer, separating the solid portion to remove at least a portion of other proteins, and extracting the plasminogen with an acidic aqueous buffer. [Background technology]

[0002] Thrombotic events can cause serious health problems. For example, coronary infarction, stroke and pulmonary embolism are some of the leading causes of death in developed countries. There is a great need for treatment and prevention of thrombotic events.

[0003] Many cases of thrombotic events are known. The establishment of thrombotic events may occur, for example, during surgical interventions such as vascular surgery (e.g., coronary artery bypass surgery), the presence of endoprostheses (i.e., the insertion of foreign bodies / implants or grafts, e.g., blood vessels / endoprostheses for blood vessels), or in stenosis traumatic injury. Various further causes are known.

[0004] Treatment of thrombotic events is usually based on the administration of medicines. Anticoagulant therapy is required to prevent the expansion of the clot. Heparin preparations or inhibitors of factor Xa are used. 4-hydroxycoumarins such as phenprocoumon, warfarin or ethyl biscoumarate are used for about 3-6 months. Coumarins prevent thrombosis, but also increase the tendency to bleed, so the use of this drug typically requires regular blood tests and special caution. Once the growth of the clot has stopped, the body can start cleaning up the damage. The body typically breaks down the clot and tries to regain free flow in the vein (revascularization). This typically takes days, weeks, or even months. In the breakdown of the clot and regeneration of the vein, substances that increase the coagulability of the blood may be released. Most anticoagulants known in the art have major drawbacks and often cause significant health risks. Problems with clotting can be caused, for example, by unexplained liver failure or by overdosing on anticoagulants. The site of fibrinolysis is not clear at present. Bilateral observation may help to improve the outcome of the treatment. Improved treatment is desirable.

[0005] As described in US Pat. No. 5,999,233 and US Pat. No. 5,999,249, it has been experimentally found that plasminogen, particularly Glu-plasminogen, can be effectively used to prevent or treat thrombotic events, particularly microthrombotic events, in patients (US Pat. No. 5,999,249). This may be of particular interest in patients suffering from congenital or acquired plasminogen deficiency. Glu-plasminogen is a natural circulating precursor in human plasma. Glu-plasminogen may be cleaved to Lys-plasminogen due to different isolation and purification processes. Treatment with Lys-plasminogen may result in undesirable side effects such as bleeding. Glu-plasminogen has a significantly longer half-life and well-predictable activity. Therefore, purification of Glu-plasminogen is preferred.

[0006] There is a great interest in obtaining plasminogen, especially Glu-plasminogen, in good yield and purity. Plasminogen is naturally present in blood. Blood and blood fractions also serve as important sources of plasminogen and its subspecies. Therefore, there is a need for a method for isolating plasminogen, especially Glu-plasminogen, from blood fractions.

[0007] For decades, blood fractionation has been used to obtain blood proteins, such as albumin, immunoglobulin fractions, and hormones, as well as other components of blood, such as platelets. Well-established blood fractionation methods are the Cohn method or the Kistler-Nitschmann method. However, these methods do not typically provide plasminogen, much less in high concentrations and yields. For technical and economic reasons, it would be beneficial to be able to obtain plasminogen, especially Glu-plasminogen, from such established methods.

[0008] US Patent No. 5,399, 667 describes the preparation of plasminogen from frozen Cohn fraction III or II / III paste by suspending the thawed material in phosphate buffered saline (PBS) containing 1 μM p-nitrophenyl-p-guanidinobenzoate, followed by centrifugation and filtration steps, followed by chromatography through lysine-agarose material. Elution from the column is achieved by increasing the aminocaproic acid content in the PBS. Alternatively, US Patent No. 5,399, 667 refers to a similar process performed at pH 8.0. No significant and deliberate modification of the pH appears to have been performed in the method of US Patent No. 5,399, 667.

[0009] US Pat. No. 5,399,633 describes several stationary phases for the chromatographic purification of plasmin and plasminogen from frozen cryoprecipitates of plasma in contact with a buffer solution of pH 7.0. Here, tranexamic acid (TEA) immobilized on agarose was found to be a suitable column to use, which also allows the simultaneous isolation of plasmin(ogen) and fibrinogen. The pH range used is neutral to basic.

[0010] US Patent No. 5,399,663 teaches a particular affinity chromatography material that can be used to purify plasminogen from plasma and its use at pH 7.5. US Patent No. 5,399,663 teaches a pharmaceutical composition comprising plasminogen having a pH of about 3 to about 10, and an osmolality adjusting agent and a stabilizer. The plasminogen is obtained from extracting the plasminogen at a pH that resembles a neutral pH.

[0011] US Pat. No. 5,399,663 describes mixing the caprylate precipitate obtained from Cohn fractions II and III with a buffer having a pH of about 3.5 to about 10.5. In a more specific example of the above mentioned patent, the caprylate precipitate has a pH of 5. Extraction is taught to be carried out at a pH between neutral and pH 10.5, further adjusted to pH 7.5. Extraction at pH 3.5 is shown to be relatively inefficient under the conditions used in US Pat. No. 5,399,663. Furthermore, the addition of lysine or epsilon aminocaproic acid to such a mixture, followed by cation exchange chromatography, is also taught. The final plasminogen is eluted and buffered at a pH of about 2.5 to about 4.

[0012] Patent document 1 describes a method for isolating Glu-plasminogen from plasma or a plasma fraction, which comprises contacting the plasma or a plasma fraction with an anion exchanger and optionally adjusting the pH to a desired range.

[0013] The above-mentioned methods at least partially allow the isolation of plasminogen from plasma fractions, some of which even have a high Glu-plasminogen content. However, it is desirable to further improve the yield and purity, and to provide efficient methods that allow the obtaining of high-quality plasminogen from plasma fractions, or even from by- or waste fractions of conventional plasma fractionation methods. [Prior art documents] [Patent documents]

[0014] [Patent Document 1] International Publication No. 2018 / 162754 [Patent Document 2] International Publication No. 2020 / 152322 [Patent Document 3] U.S. Patent No. 5,288,489 [Patent Document 4] International Publication No. 2002 / 095019 [Patent Document 5] European Patent No. 1885485 [Patent Document 6] International Publication No. 2016 / 095013 [Patent Document 7] U.S. Patent No. 8,268,782 Summary of the Invention [Problem to be solved by the invention]

[0015] Surprisingly, it has been found that a process comprising dispersing a precipitate of a plasma fraction containing plasminogen in a basic aqueous buffer, separating the solid portion thereof, and extracting the plasminogen preferably with an acidic aqueous buffer containing at least one compound of formula (I) or a salt or combination thereof results in plasminogen. [Means for solving the problem]

[0016] A first aspect of the present invention relates to a method for isolating plasminogen from a plasma fraction, comprising the steps of: (i) dispersing a precipitate of the plasma fraction containing plasminogen in a basic aqueous buffer of pH 7-10; (ii) incubating the dispersion from step (i) to dissolve at least a portion of the proteins and other impurities soluble in the basic aqueous buffer; (iii) separating the solid and liquid portions of the incubated dispersion of step (ii) from each other; (iv) Reconstituting the solid portion from step (iii) with dissolved lysine and / or a compound of formula (I): (H 2 N) n -R-(A) m (I), (In the formula: n is an integer of 1 or 2; m is an integer of 0, 1 or 2; A is, independently in each occurrence, a carboxyl group or an amino group; R is a straight or branched chain C 3 ~C 12 -Alkylene, linear or branched C 3 ~C 12 -heteroalkylene, one or more halogens or one or more C 1 ~C 4 -C optionally substituted with (hetero)alkyl residues 6 ~C 12 -arylene, one or more halogens or one or more C 1 ~C 4 -C optionally substituted with (hetero)alkyl residues 3 ~C 12 -heteroarylene, one or more halogens or one or more C 1 ~C 4 -C optionally substituted with (hetero)alkyl residues 3 ~C 12 -Alkylene-C 6 ~C 12-arylene, one or more halogens or one or more C 1 ~C 4 -C optionally substituted with (hetero)alkyl residues 3 ~C 12 -Alkylene-C 3 ~C 12 -heteroarylene) or a salt thereof, Optionally incubating the mixture to dissolve the plasminogen and optionally removing the solid portion; and (v) Obtaining a solution containing isolated plasminogen from step (iv). The present invention relates to a method comprising the steps of:

[0017] It has surprisingly been found that the claimed method achieves high yields of highly pure plasminogen, particularly Glu-plasminogen. The method can be carried out with relatively little labor effort. Thus, the present invention is of technical advantage. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] As used herein, plasminogen can be any plasminogen or its more species (e.g., Glu-plasminogen, Lys-plasminogen and / or plasmin) derivative or combination. The structure of such proteins and the function of the plasminogen / plasmin system are generally well known. Plasminogen in the sense of the present invention can be plasminogen of any species of interest. Preferably, the plasminogen is of human or mammalian origin.

[0019] The terms "protein", "polypeptide" and "peptide" are understood in the broadest sense throughout the present invention interchangeably as any chemical entity that is composed primarily of amino acid residues and contains at least 20 amino acid residues that are consecutively linked to another amino acid residue via amide bonds. It will be understood that a protein in the sense of the present invention may or may not be subjected to one or more post-translational modifications and / or may or may not be conjugated to one or more non-amino acid moieties. The termini of the protein may optionally be capped by any means known in the art, such as, for example, amidation, acetylation, methylation, acylation, etc.

[0020] Post-translational modifications are well known in the art and may be, but are not limited to, lipidation, phosphorylation, sulfation, 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, non-metal ions, peptides or small molecules, and addition of iron-sulfide clusters. In addition, cofactors, particularly cyclic guanosine monophosphate (cGMP), and optionally further, e.g., ATP, ADP, NAD, + , NADH+H + , NAD + , NADPH+H + Cofactors such as metal ions, anions, lipids, etc. may be bound to proteins independent of the biological effects of these cofactors. In the context of plasminogen, in particular, glycosylation and disulfide bonds may occur.

[0021] In a preferred embodiment, in the context of the present invention, plasminogen is Glu-plasminogen. Therefore, throughout the present invention, plasminogen may be defined as Glu-plasminogen. The advantage of the method of the present invention is that two glycosylation patterns of Glu-plasminogen can be observed in the isolated product. This can reflect native Glu-plasminogen.

[0022] In an alternative embodiment, the plasminogen is Lys-plasminogen. In an alternative embodiment, the plasminogen is a combination of Glu-plasminogen and Lys-plasminogen, where the Lys-plasminogen and Glu-plasminogen can be combined in any variation (preferably predominantly Glu-plasminogen). In an alternative preferred embodiment, the plasminogen is a combination of Glu-plasminogen, Lys-plasminogen and one or more other plasminogen derivatives, where the Lys-plasminogen, Glu-plasminogen and one or more other plasminogen derivatives can be combined in any variation (preferably predominantly Glu-plasminogen).

[0023] Glu-plasminogen is a plasma-derived enzyme precursor. It is known that Glu-plasminogen has (essentially) no proteolytic activity. Preferably, the plasminogen composition (preferably containing Glu-plasminogen) administered to the patient has (essentially) no proteolytic activity. Such (essentially) absence of proteolytic activity is understood in the broadest sense generally understood by those skilled in the art. Preferably, the enzyme activity of the Glu-plasminogen and / or plasminogen composition (preferably containing Glu-plasminogen) administered to the patient is less than 70 units (U, i.e. μmol substrate / min) per 1.0 g / L of total protein content, or less than 50 U, less than 25 U, less than 10 U, less than 9 U, less than 8 U, less than 7 U, less than 6 U, less than 5 U, less than 2 U, less than 1 U, less than 0.5 U, less than 0.1 U, or less than 0.01 U per 1.0 g / L of total protein content. In this context, proteolytic activity can be determined by any means. For example, it can be the activity determined by S-2288 (Chromogenix) proteolytic activity assay. Alternatively, it can be determined as the degradation of fibrin resulting in the generation of D-dimers. The specific enzymatic activity of plasmin can be determined by measuring the generation of D-dimers from the degradation of fibrin. Preferably, the proteolytic activity of Glu-plasminogen and / or plasminogen composition (preferably containing Glu-plasminogen) administered to the patient is below the detection limit of the assay.

[0024] In a preferred embodiment, the plasminogen composition administered to the patient contains plasminogen, in particular 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 protein content.

[0025] In a preferred embodiment, the resulting plasminogen composition (preferably containing a Glu-plasminogen composition) contains no endotoxin or only a low endotoxin content of less than 1 EU / mL, less than 0.5 EU / mL, less than 0.1 EU / mL, less than 0.05 EU / mL, or less than 0.01 EU / mL (as determined in the Limulus Amebocyte Extract (LAL) endosafe endochrome assay as described in Chapter 2.6.14 of the European Pharmacopoeia (Version 5.0)).

[0026] In a preferred embodiment, the plasminogen composition (preferably containing Glu-plasminogen) administered to the patient contains no immunoglobulin or only a low immunoglobulin content of less than 5 g / L, less than 2 g / L, less than 1 g / L, less than 0.5 g / L, or less than 0.1 g / L of immunoglobulin (as determined in a nephelometric assay).

[0027] As used herein, other impurities soluble in basic aqueous buffer may be any impurity, such as, for example, one or more low molecular weight impurities, such as octanoic acid and / or one or more excipients, and / or one or more high molecular weight impurities.

[0028] Plasma (also referred to as "plasma," "plasm," or "blood plasma") can be obtained from any source. It can be obtained, for example, from blood storage fluid from which cells have been removed. Plasma is also commercially available from a variety of suppliers.

[0029] In the context of the present invention, the term "plasma fraction" is understood in the broadest sense as any part separated from plasma, containing Glu-plasminogen. Several methods for preparing plasma fractions from plasma are known to those skilled in the art. One commonly known example is the Cohn method (also called Cohn's method), which is based on freeze-thaw cycles and gradually increasing the ethanol concentration in the solution.

[0030] As used herein, the plasma fraction precipitate may be plasma or any fraction thereof that contains plasminogen. Preferably, the plasma fraction is obtained from a well-established blood fractionation method, such as, for example, the Cohn or Kistler-Nitschmann method. In a preferred embodiment, the plasma fraction is: (a) Poor cryoplasmic supernatant or poor cryoplasmic precipitate; (b) any fraction of Cohn or Kistler-Nitschmann paste I, II or III, or a combination of two or all of them; (c) any fraction of Cohn or Kistler-Nitschmann paste I, II or III, or a combination of two or all of them; and (d) A combination of two or all of these. is selected from the group consisting of:

[0031] In a preferred embodiment, the plasma fraction is selected from the group consisting of a fraction of Cohn paste I, a fraction of Cohn paste II, a fraction of Cohn paste III, a fraction of Cohn paste I and II, a fraction of Cohn paste I and III, a fraction of Cohn paste II and III, a fraction of Cohn paste I, II and III. In a preferred embodiment, the plasma fraction is selected from the group consisting of a secondary or waste fraction of Cohn paste I, a secondary or waste fraction of Cohn paste II, a secondary or waste fraction of Cohn paste III, a secondary or waste fraction of Cohn paste I and II, a secondary or waste fraction of Cohn paste I and III, a secondary or waste fraction of Cohn paste II and III, a secondary or waste fraction of Cohn paste I, II and III.

[0032] In a preferred embodiment, the plasma fraction is a fraction of Cohn process pastes II and III. In a preferred embodiment, the plasma fraction is a fraction of Cohn process pastes I and II. In a preferred embodiment, the plasma fraction is a side or waste fraction of Cohn process pastes II and III. In a preferred embodiment, the plasma fraction is a side or waste fraction of Cohn process pastes I and II.

[0033] In another preferred embodiment, the plasma fraction is selected from the group consisting of a fraction of Kistler-Nitschmann paste I, a fraction of Kistler-Nitschmann paste II, a fraction of Kistler-Nitschmann paste III, a fraction of Kistler-Nitschmann pastes I and II, a fraction of Kistler-Nitschmann pastes I and III, a fraction of Kistler-Nitschmann pastes II and III, a fraction of Kistler-Nitschmann pastes I, II and III. In another preferred embodiment, the plasma fraction is selected from the group consisting of: a secondary or waste fraction of Kistler-Nitschmann paste I, a secondary or waste fraction of Kistler-Nitschmann paste II, a secondary or waste fraction of Kistler-Nitschmann paste III, a secondary or waste fraction of Kistler-Nitschmann pastes I and II, a secondary or waste fraction of Kistler-Nitschmann pastes I and III, a secondary or waste fraction of Kistler-Nitschmann pastes II and III, a secondary or waste fraction of Kistler-Nitschmann pastes I, II and III. In a preferred embodiment, the plasma fraction is Kistler-Nitschmann Precipitate A (PPT-NA).

[0034] In a preferred embodiment, the plasma fraction is a fraction of pastes II and III of the Kistler-Nitschmann method. In a preferred embodiment, the plasma fraction is a fraction of pastes I and II of the Kistler-Nitschmann method. In a preferred embodiment, the plasma fraction is a side or waste fraction of pastes II and III of the Kistler-Nitschmann method. In a preferred embodiment, the plasma fraction is a side or waste fraction of pastes I and II of the Kistler-Nitschmann method.

[0035] In another preferred embodiment, the plasma fraction is selected from the group consisting of a fraction of Cohn paste I, a fraction of Cohn paste II, a fraction of Cohn paste III, a fraction of Cohn paste I and II, a fraction of Cohn paste I and III, a fraction of Cohn paste II and III, a fraction of Cohn paste I, II and III.

[0036] In a preferred embodiment, the plasma fraction is Cohn paste II and III fraction.In a preferred embodiment, the plasma fraction is Cohn paste I and II fraction.

[0037] In another preferred embodiment, the plasma fraction is selected from the group consisting of a fraction of Kistler-Nitschmann paste I, a fraction of Kistler-Nitschmann paste II, a fraction of Kistler-Nitschmann paste III, a fraction of Kistler-Nitschmann pastes I and II, a fraction of Kistler-Nitschmann pastes I and III, a fraction of Kistler-Nitschmann pastes II and III, a fraction of Kistler-Nitschmann pastes I, II and III.

[0038] In a preferred embodiment, the plasma fraction is the fraction of pastes II and III of the Kistler-Nitschmann method.In a preferred embodiment, the plasma fraction is the fraction of pastes I and II of the Kistler-Nitschmann method.

[0039] It will be understood that the precipitate of step (i) contains plasminogen, in particular Glu-plasminogen. Such a precipitate may be obtainable (obtainable) by any means. In a preferred embodiment, the precipitate is obtained by contacting a blood fraction containing plasminogen with an organic solvent (e.g., octanoic acid, methanol, ethanol, propanol, butanol, pentanol, hexanol, heptanol, octanol, phenol, acetone, etc.). In another preferred embodiment, the precipitate is obtained by contacting a blood fraction containing plasminogen with a chaotropic compound (e.g., a chaotropic salt (e.g., a barium salt, a calcium salt, a magnesium salt, a chlorate), a thiocyanate such as guanidinium hydrochloride, guanidinium thiocyanate, a perchlorate, an iodide, urea, thiourea, a high concentration of sodium chloride, etc.). Such an organic solvent or a chaotropic salt may also be referred to as a precipitating agent. In another preferred embodiment, the precipitate is obtained from cooling a blood fraction containing plasminogen or from subjecting the fraction to freeze-thaw cycles.

[0040] The precipitation can be obtained by any means known in the art. In a preferred embodiment, the precipitation of the plasma fraction containing plasminogen is obtained by freezing and thawing the plasma or a fraction thereof (even by subjecting the plasma or a fraction thereof to freeze-thaw cycles), by changing the temperature, by changing the pH, by adding at least one precipitating agent, or by a combination of two or more of these. In a preferred embodiment, the precipitating agent is selected from the group consisting of octanoic acid, ethanol, salt, or polyethylene glycol, or by a combination of two or more of these to the plasma fraction.

[0041] In a preferred embodiment, the precipitation of the plasma fraction containing plasminogen is obtained by adding octanoic acid to the plasma fraction. It will be understood that the term "octanoic acid" as used herein is understood in the broadest sense as any compound containing the octanoate anion (i.e., including the free acid) and salts thereof.

[0042] In a preferred embodiment, the precipitation of the plasma fraction containing plasminogen comprises: (a) Poor cryoplasmic supernatant or poor cryoplasmic precipitate; (b) any by- or waste fraction of Cohn or Kistler-Nitschmann process pastes I, II or III, or any combination of two or all thereof; (c) any fraction of Cohn or Kistler-Nitschmann paste I, II or III, or a combination of two or all of them; and (d) A combination of two or all of these. The plasma fraction is obtained by adding octanoic acid to a plasma fraction selected from the group consisting of:

[0043] The precipitate can be provided in any form. In a preferred embodiment, the precipitate of step (i) is obtained by filtration, preferably dead-end filtration. In this case, the precipitate can also be referred to as a 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 containing plasminogen with octanoic acid and then filtering. Alternatively, the precipitate of step (i) is obtained by centrifugation.

[0044] The precipitate obtainable from the plasma fractionation process may be used directly when obtained from the plasma fractionation process or may be stored. Optionally, the method of the invention may be combined with the plasma fractionation process and performed as part of a procedure flow (which may also be designed as online). Alternatively, a stored precipitate may be used for the method of the invention. Such a stored precipitate may be optionally cooled or frozen and thawed when performing the method of the invention.

[0045] Dispersion can be performed by any means, for example, dispersion can be performed by adding a basic aqueous buffer and shaking, mixing (manually or via a stirrer, mixer or blender), or a combination thereof.

[0046] As used herein, the term "aqueous buffer" is understood in the broadest sense as any buffer solution that contains primarily (i.e., more than 50% by weight) water.

[0047] In a preferred embodiment, the basic aqueous buffer solution does not contain dissolved lysine or at least one compound of formula (I), a salt or a combination thereof.

[0048] The dispersion of step (i) can be obtained from any precipitate:basic aqueous buffer ratio. In a preferred embodiment, the precipitate:basic aqueous buffer weight ratio is in the range of 20:1 to 1:20, 10:1 to 1:10, 2:1 to 1:20, 1:1 to 1:20, 1:2 to 1:10, 1:3 to 1:7, 1:4 to 1:6, or in the range of about 1:5.

[0049] Step (i) can be carried out under any conditions suitable for dispersing the precipitate in a basic aqueous buffer. Preferably, this step is carried out at a temperature ranging between 0°C and 30°C, preferably between 4°C and 25°C, between 4°C and 10°C, or between 17°C and 22°C.

[0050] As mentioned above, the basic aqueous buffer in step (i) may have a pH in the range of pH 7 to 10. In preferred embodiments, the basic aqueous buffer in step (i) is of pH 8 to 10, pH 8.1 to 9.9, pH 8.2 to 9.8, pH 8.3 to 9.7, pH 8.4 to 9.6, pH 8.5 to 9.5, pH 8.6 to 9.4, pH 8.7 to 9.3, pH 8.8 to 9.2, pH 8.9 to 9.1, or pH 9.

[0051] As mentioned above, 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 of pH 2 to 6, pH 2.5 to 5.9, pH 3 to 5.7, pH 3.5 to 5.6, pH 4 to 6, pH 4.5 to 5.5, pH 4.6 to 5.4, pH 4.7 to 5.3, pH 4.8 to 5.2, pH 4.9 to 5.1, or pH 5. Preferably, the pH of the acidic aqueous buffer in step (iv) is pH 6.6, i.e., below the isoelectric point of Glu-plasminogen, more preferably below it.

[0052] In a preferred embodiment, the basic aqueous buffer in step (i) is of pH 8.1-9.9, pH 8.2-9.8, pH 8.3-9.7, pH 8.4-9.6, pH 8.5-9.5, pH 8.6-9.4, pH 8.7-9.3, pH 8.8-9.2, pH 8.9-9.1, or pH 9; The acidic aqueous buffer in step (iv) is of pH 2.5-5.9, pH 3-5.7, pH 3.5-5.6, pH 4-6, pH 4.5-5.5, pH 4.6-5.4, pH 4.7-5.3, pH 4.8-5.2, pH 4.9-5.1, or pH 5.

[0053] In preferred embodiments, the pH difference between the basic aqueous buffer of step (i) and the aqueous buffer of 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 preferred embodiments, the pH difference between the basic aqueous buffer of step (i) and the aqueous buffer of 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.

[0054] It will be understood that the numerical values ​​taught herein are understood in the broadest sense as commonly understood, including any subsequent decimal places, as rounded values, for example, pH 5 includes values ​​from pH 4.5 to 5.4, as commonly understood.

[0055] The incubating step (ii) can be carried out under any conditions that dissolve at least a portion of the proteins soluble in the basic aqueous buffer. Preferably, this step is carried out at a temperature in the range between 0° C. and 30° C., preferably between 4° C. and 25° C., between 4° C. and 10° C., or between 17° C. and 22° C. The temperature may also be optionally varied over time.

[0056] The incubation time in step (ii) can be any range that dissolves at least a portion of the proteins soluble in the basic aqueous buffer and does not essentially destroy the plasminogen of interest. In a preferred embodiment, the incubation in step (ii) is carried out for a time range of 1 minute to 48 hours, 5 minutes to 24 hours, 10 minutes to 12 hours, 15 minutes to 6 hours, 20 minutes to 2 hours, 25 minutes to 1 hour, 25 minutes to 45 minutes, or approximately 30 minutes.

[0057] Once the incubation of step (ii) is completed, the solid and liquid portions of the incubated dispersion of step (ii) are separated from each other. This can be carried out by any means. In a preferred embodiment, the separation of the solid and liquid portions from each other in step (iii) is obtained by a means selected from the group consisting of filtration, including dead-end filtration, tangential flow filtration, or a combination thereof, dialysis, phase separation, preferably by sedimentation, and / or centrifugation. In a preferred embodiment, the separation of step (iii) is achieved by filtration, preferably dead-end filtration. In this case, the separated solid portion obtained can also be referred to as a filter cake.

[0058] In a preferred embodiment, the step (iii) of separating the solid part and the liquid part from each other is obtained by filtration, where at least one filter aid is added to the dispersion before step (iii).The filter aid can be any filter aid known in the art.In a preferred embodiment, the filter aid is selected from the group consisting of polymers, preferably high molecular weight polyethylene glycol (PEG), cellulose or cellulose derivatives, diatomaceous earth, perlite, surfactants (e.g. Tween-20), and combinations of two or more of these.It will be understood that combinations of two or more filter aids can also be used.

[0059] Step (iii) can be carried out under any conditions suitable for this purpose. Preferably, this step is carried out at a temperature in the range between 0° C. and 30° C., preferably between 4° C. and 25° C., between 4° C. and 10° C., or between 17° C. and 22° C.

[0060] Each of steps (i) to (iii) may be carried out once or repeated two or more times.

[0061] Step (iv) of mixing the solid portion from step (iii) with an acidic aqueous buffer can be carried out by any means, for example, mixing can be carried out by adding the acidic aqueous buffer and shaking, mixing (manually or via a stirrer, mixer or blender), or a combination thereof.

[0062] The one or more buffering agents used in any of the buffer solutions used herein may be of any chemical nature. Preferably, the buffering agent is a pharma- ceutically acceptable buffering agent. In a preferred embodiment, the acidic aqueous buffer solution comprises formic acid, acetate, carbonate, hydrogen carbonate, and / or citrate, preferably sodium formate, sodium acetate or sodium citrate, in particular sodium acetate. In a preferred embodiment, the acidic aqueous buffer solution is an acetate buffer solution, in particular a sodium acetate buffer solution. For example, the acidic aqueous buffer solution used herein may comprise a sodium acetate / glycine buffer system.

[0063] It will be appreciated that the acidic aqueous buffer may also optionally comprise one or more further components. In a preferred embodiment, it comprises a water-soluble or emulsifiable polymer, such as, for example, polyethylene glycol (PEG), preferably having an average molecular weight (determinable by size exclusion chromatography) of 200-35000 Da, 1000-100000 Da, 200-1000 Da, 500-5000 Da, 1000-10000 Da, 5000-50000 Da, or 10000-100000 Da.

[0064] The mixture of step (iv) can be obtained from any solid portion:acidic aqueous buffer ratio. In a preferred embodiment, the solid portion:acidic aqueous buffer weight ratio is in the range of 20:1 to 1:20, 10:1 to 1:10, 2:1 to 1:20, 1:1 to 1:20, 1:1 to 1:10, 1:2 to 1:7, 1:2 to 1:5, or in the range of about 1:3.

[0065] Step (iv) can be carried out under any conditions suitable for this purpose. Preferably, this step is carried out at a temperature ranging between 0°C and 30°C, preferably between 4°C and 25°C, between 4°C and 10°C, or between 17°C and 22°C.

[0066] 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, aminomethylbenzoic acid, and oxalizine, in particular the at least one compound of formula (I) is 6-aminohexanoic acid, in particular aminohexanoic acid.

[0067] In a preferred embodiment, the acidic aqueous buffer in step (iv) contains lysine, in particular L-lysine.

[0068] In a preferred embodiment, the acidic aqueous buffer in step (iv) comprises 6-aminohexanoic acid.

[0069] Optionally, the mixture of step (iv) comprises one or more further components. Optionally, one or more types of anion exchange resins may be added to the mixture of step (iv).

[0070] Optionally, step (iv) comprises incubating the mixture to dissolve the plasminogen and optionally removing the solid portion.

[0071] In a preferred embodiment, the solid portion obtained in step (iv) is separated from the liquid fraction mainly containing dissolved plasminogen. This can be carried out by any means. In a preferred embodiment, step (iv) comprises separating the solid portion and the liquid portion from each other by means selected from the group consisting of filtration, including dead-end filtration, tangential flow filtration or a combination thereof, dialysis, phase separation, preferably by sedimentation, and / or centrifugation. In a preferred embodiment, the optional separation in step (iv) is achieved by filtration, preferably dead-end filtration. In this case, the separated solid portion obtained can also be referred to as a filter cake. Optionally, the washed solid portion is further washed with an acidic aqueous buffer. For example, half of the volume used for the previous mixing can be used. This can be done one or more times. The obtained solutions can be combined (also designated as a pool).

[0072] From the above procedural steps, a solution containing isolated plasminogen can be obtained, which already has particularly advantageous technical properties, such as particularly high yield and purity. Nevertheless, the method can be improved even further by adding further steps.

[0073] In a preferred embodiment, step (v) further comprises one or more means for reducing the concentration of dissolved precipitant in the solution, which can be carried out by any means including multi-phase separation.

[0074] In a preferred embodiment, step (v) further comprises one or more means for reducing the concentration of the precipitant dissolved in the solution, contacting the solution with at least one chromatographic resin which interacts with at least a portion of the octanoate anions, more preferably said chromatographic resin being an ion exchanger, in particular an anion exchanger. In a preferred embodiment, contacting the dispersion with at least one anion exchanger which interacts with at least a portion of the octanoate anions.

[0075] In a preferred embodiment, the solution containing isolated plasminogen obtained in step (v) comprises: contacting the solution with at least one ion exchanger (e.g., an anion exchanger or a cation exchanger) with or without size exclusion properties that interacts with at least a portion of the lysine, the compound of formula (I), or both; contacting the solution with at least one size exclusion resin that interacts with at least a portion of the lysine, the compound of formula (I), or both; contacting the solution with at least one hydrophobic or mixed-mode interaction chromatography resin that interacts with at least a portion of the lysine, the compound of formula (I), or both; subjecting the solution to diafiltration to remove at least a portion of the lysine, the compound of formula (I), or both; 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 plasminogen; molecular size chromatography; dialysis; Ultrafiltration, including dead-end ultrafiltration, tangential flow ultrafiltration, or a combination thereof; and A combination of two or more of these The process is further subjected to at least one further step selected from the group consisting of:

[0076] In a preferred embodiment, the solution containing isolated plasminogen obtained in step (v) is subjected to at least the following further steps: (vi) removing at least a portion of the compounds containing one or more amino groups, in particular lysine, the compound of formula (I), or both, from the solution obtained in step (v), preferably by contacting the solution with at least one chromatographic resin, in particular an ion exchanger (e.g. an anion or cation exchanger, in particular an anion exchanger), which interacts with at least a portion of the lysine, the compound of formula (I), or both; and (vii) increasing the purity and / or concentration of plasminogen, preferably by carrying out a chromatography based on a stationary phase comprising immobilized lysine and / or at least one immobilized compound of formula (I), preferably using a buffer that first allows the interaction of plasminogen with the stationary phase, followed by elution of the plasminogen with a buffer that reduces the interaction of the plasminogen with the stationary phase; and (viii) Obtaining a solution containing isolated plasminogen from step (vii). is further provided.

[0077] Steps (vi) and (vii) can be carried out in any buffer and at any temperature available for this purpose.

[0078] Preferably, the buffer usable in step (vi) is an acidic aqueous buffer. For example, such an acidic aqueous buffer may be of pH 2.5-5.9, pH 3-5.7, pH 3.5-5.6, pH 4-6, pH 4.5-5.5, pH 4.6-5.4, pH 4.7-5.3, pH 4.8-5.2, pH 4.9-5.1, or pH 5. Preferably, this step (vi) is carried out at a temperature ranging between 0°C and 30°C, preferably between 4°C and 25°C, between 4°C and 10°C, or between 17°C and 22°C. Elution from the column can be carried out by using a high salt buffer, for example having a molar concentration of salt, such as sodium chloride, between 0.5 and 2 M, such as approximately 1 M.

[0079] Preferably, the buffer usable in step (vii) is an acidic aqueous buffer. For example, such an acidic aqueous buffer may be of pH 2.5-5.9, pH 3-5.7, pH 3.5-5.6, pH 4-6, pH 4.5-5.5, pH 4.6-5.4, pH 4.7-5.3, pH 4.8-5.2, pH 4.9-5.1, pH 3, or pH 4, or pH 5. Preferably, this step (vii) is carried out at a temperature in the range between 0°C and 30°C, preferably between 4°C and 25°C, between 4°C and 10°C, or between 17°C and 22°C. The stationary phase in step (vii) may be, for example, lysine-conjugated sepharose.

[0080] Optionally, the pH may be maintained fairly low during elution, for example in the pH range between pH 2 and pH 5. This can stabilize plasminogen, especially Glu-plasminogen, against undesired degradation and increase its shelf life.

[0081] In a preferred embodiment, the method of the present invention comprises the steps of: (i) dispersing a precipitate of a plasma fraction containing plasminogen in a basic aqueous buffer solution having a pH of 7 to 10, preferably 8 to 10, and more preferably 8.5 to 9.5; (ii) incubating the dispersion from step (i) to dissolve at least a portion of the proteins that are soluble in the basic aqueous buffer; (iii) separating the solid and liquid portions of the incubated dispersion of step (ii) from each other by filtration, including dead-end filtration, tangential flow filtration, or a combination thereof, dialysis, phase separation, preferably by sedimentation, and / or centrifugation; (iv) mixing the solid portion from step (iii) with an acidic aqueous buffer of pH 2 to 6.6, preferably pH 4.5 to pH 5, further comprising dissolved lysine, at least one compound of formula (I) or a salt or combination thereof, and optionally incubating the mixture to dissolve the plasminogen and remove the solid portion; and (v) obtaining a solution containing isolated plasminogen from step (iv); (vi) removing at least a portion of the compounds containing one or more amino groups, in particular lysine, the compounds of formula (I), or both, from the solution obtained in step (v), preferably by contacting the solution with at least one chromatographic resin, in particular an ion exchanger, which interacts with at least a portion of the lysine, the compounds of formula (I), or both; and (vii) increasing the purity and / or concentration of plasminogen by carrying out chromatography based on a stationary phase comprising immobilized lysine and / or at least one immobilized compound of formula (I), using a buffer that first allows the interaction of plasminogen with the stationary phase, followed by elution of the plasminogen with a buffer that reduces the interaction of the plasminogen with the stationary phase; and (viii) Obtaining a solution containing isolated plasminogen from step (vii). Includes.

[0082] The resulting plasminogen, particularly Glu-plasminogen, can optionally be formulated, stored or used directly for any purpose.

[0083] In a preferred embodiment, step (iv) and / or step (vi) comprises contacting the solution with at least one small pore anion exchange resin which interacts with at least a portion of the precipitant, in particular octanoic acid, if present.

[0084] In a preferred embodiment, such at least one small porosity anion exchange resin that interacts with at least a portion of the precipitant is a resin having a microporous structure, e.g., pores in the range of 0.1-10 nm, 1-100 nm, 0.1-10 μm, or 1-100 μm. In a preferred embodiment, such at least one small porosity anion exchange resin that interacts with at least a portion of the precipitant is a resin based on one or more styrene-based (co)polymers, which may contain quaternary ammonium groups and / or sulfonic acid groups.

[0085] In a preferred embodiment, such at least one small porosity anion exchange resin that interacts with at least a portion of the precipitant is a Dowex resin, in particular Dowex 1x8 having 50-100 mesh (average particle size of 0.15-0.3 mm), 100-200 mesh (average particle size of 0.08-0.15 mm), or 200-400 mesh (average particle size of 0.04-0.08 mm).

[0086] For example, it can be used for therapeutic use. In this case, plasminogen, particularly Glu-plasminogen, can be administered to an individual for use in a method for treating or preventing one or more thrombotic events. In this case, preferably, the pH of the solution is changed to a desired range before administration. Optionally, one or more additional pharmacologic acceptable components may be added. The pharmaceutical and medical uses of plasminogen, particularly Glu-plasminogen, are described in detail in WO 2018 / 162754 and WO 2020 / 152322.

[0087] If storage is desired, the plasminogen obtained, particularly Glu-plasminogen, may optionally be lyophilized (freeze-dried) or frozen.

[0088] It will be appreciated that the method of the present invention allows not only for the preparation of plasminogen, but also for the preparation of one or more further components as a desired by-product.

[0089] In a preferred embodiment, the method of the present invention comprises the steps of: (a) preferably recovering the proteins at least partially dissolved in step (ii), including one or more fractions of one or more immunoglobulins, in particular immunoglobulin G, optionally isolated; (b) the method further comprises a step of viral inactivation of the fraction of interest; (c) the pH of the solution containing the isolated plasminogen is further adjusted to a desired range, optionally by exchanging the aqueous buffer; or (d) the method further comprises freeze-drying or desiccating the plasminogen; (e) A combination of two or more of these The present invention is further characterized by the following.

[0090] As mentioned above, the plasminogen obtainable (obtainable) by the method of the invention, in particular Glu-plasminogen, has as such special structural features, such as a particularly high purity and possibly trace residues of the agents used in the procedural steps.

[0091] Thus, a further aspect of the present invention relates to a composition comprising plasminogen obtainable from the method of the present invention, wherein plasminogen, in particular Glu-plasminogen, constitutes at least 70% (w / w) of the total protein content.

[0092] It will be understood that the embodiments and definitions explained in the context of the above method apply mutatis mutandis to the plasminogen obtainable therefrom.

[0093] In preferred embodiments, plasminogen, in particular Glu-plasminogen, constitutes at least 75% (w / w), at least 80% (w / w), at least 85% (w / w), at least 90% (w / w), or at least 95% (w / w) of the total protein content.

[0094] In one embodiment of the present invention, the (Glu-)-plasminogen obtainable (or obtainable) from the method of the present invention is provided in a pharmaceutical composition. It may therefore be admixed with one or more pharma- ceutically acceptable carriers. Thus, a further aspect of the present invention refers to a pharmaceutical composition comprising the (Glu-)-plasminogen obtainable (or obtainable) from the method of the present invention and one or more pharma- ceutically acceptable carriers.

[0095] The terms "pharmaceutical composition" and "pharmaceutical formulation" are understood interchangeably. As used herein, the terms "pharmaceutical acceptable carrier", "pharmaceutical acceptable excipient", "carrier" and "excipient" are understood interchangeably in the broadest sense as any substance capable of supporting the pharmacological tolerability of plasminogen and at least one plasminogen activator, respectively. Such pharmaceutical compositions may be ready-made, preferably liquid formulations, in particular injectable portions.

[0096] The storage form may be a liquid, but also a dry form (e.g. a powder, e.g. a powder comprising dried or freeze-dried plasminogen and at least one plasminogen activator, respectively), a paste, a syrup, etc. Optionally, the dry form, the paste or the syrup may be dissolved or emulsified before administration to the patient.

[0097] The pharma- ceutically acceptable carrier may be illustratively selected from the list consisting of aqueous buffer, saline, water, dimethylsulfoxide (DMSO), ethanol, vegetable oil, paraffin oil, or a combination of two or more thereof. Additionally, the pharma- ceutically acceptable carrier may optionally contain one or more surfactants, one or more foaming agents (e.g., sodium lauryl sulfate (SLS), sodium dodecyl sulfate (SDS)), one or more coloring agents (e.g., food colorings), one or more vitamins, one or more salts (e.g., sodium, potassium, calcium, zinc salts), one or more humectants (e.g., sorbitol, glycerol, mannitol, propylene glycol, polydextrose), one or more enzymes, one or more preservatives (e.g., benzoic acid, methylparaben), one or more antioxidants, one or more herbal and botanical extracts, one or more stabilizers, one or more chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)), and / or one or more uptake mediators (e.g., polyethyleneimine (PEI), cell penetrating peptides (CPPs), protein transduction domains (PTDs), antimicrobial peptides, and the like).

[0098] The present invention also relates to dosage units of the pharmaceutical compositions that can be used in the context of the treatment or prevention of the present invention. Exemplarily, the present invention may refer to single-dose containers or multi-dose forms.

[0099] The present invention also relates to a plasminogen according to the invention, in particular Glu-plasminogen, or a pharmaceutical composition according to the invention, for use as a medicament.

[0100] The present invention also relates to the plasminogen of the present invention, in particular Glu-plasminogen, or the pharmaceutical composition of the present invention for use in a method for treating or preventing thrombotic events. Such treatments with plasminogen, in particular Glu-plasminogen, are described in detail in WO 2018 / 162754 and WO 2020 / 152322.

[0101] As mentioned above, the use of a basic aqueous buffer prior to purifying the plasminogen has been found to be particularly beneficial to the purity of the plasminogen that can be obtained.

[0102] Thus, a further aspect of the present invention relates to the use of a basic aqueous buffer having a pH of 8 to 11 for removing proteins other than plasminogen from a precipitate of a plasma fraction containing plasminogen, preferably including the method of the present invention.

[0103] It will be understood that the embodiments and definitions explained in the context of the method above apply mutatis mutandis to this use.

[0104] The following examples and figures are intended to provide exemplary embodiments of the invention described and claimed herein. These examples are not intended to provide limitations on the scope of the inventive subject matter. The following figures, examples and claims further illustrate the invention. [Brief description of the drawings]

[0105] [Figure 1]A flow chart of an example of the procedure of the invention is shown. The precipitate of the plasma fraction containing plasminogen (e.g. octanoic acid precipitate (OA-PPT)) can be provided as a filter cake and can be subjected to a resuspension step (A). This can provide for the resuspension of the filter cake (4) in an acidic aqueous buffer. The resulting solution can be subjected to a cation exchange chromatography (CEX) step (B) in a column (7) suitable for this purpose (e.g. Eshmuno CPX, Merck, Germany). Furthermore, an affinity chromatography step (C) is applied. For this purpose, a chromatography column (11) based on a stationary phase comprising immobilized lysine and / or at least one immobilized lysine analogue (e.g. based on lysine-conjugated sepharose) can be used. A plasminogen preparation solution (12) can be obtained. [Diagram 2]FIG. 1 shows an overview of another example of the procedure of the present invention, in which a precipitate (1) of a plasma fraction (e.g., an octanoic acid precipitate (OA-PPT) resuspension) containing plasminogen can be dispersed in a basic aqueous buffer (2). For example, the weight ratio of (1):(2) can be 1:5 and the pH can be set at pH 9 (e.g., pH 9.0). The dispersion can be incubated and filtered. This separates the filtrate (total protein concentration can be, for example, 3-6 g / L) containing the major impurities from the precipitate. The remaining filter cake (4) can be dissolved in an acidic aqueous buffer containing lysine and / or a lysine analogue (e.g., 0.2 M sodium acetate containing 0.2 M 6-aminohexanoic acid (AHA)) having a pH of 5 (e.g., pH 5.0) in a filter cake:buffer weight ratio of 1:3. The resulting solution (5) contains solubilized plasminogen and can have a total protein content of less than 1 g / L. This solution (5) can be further diluted (e.g., in a 1:3 ratio with an acidic buffer, such as a buffer containing 10 mM sodium acetate and 50 mM glycine at pH 5 (e.g., pH 5.0). The resulting solution (6) can be subjected to a cation exchange chromatography (CEX) column (7) (e.g., Eshmuno CPX, Merck, Germany), thereby effectively removing lysine and / or lysine analogs (8) (e.g., 6-aminohexanoic acid) from the solution. This allows obtaining a solution (9) containing the desired plasminogen. Here, a high-salt buffer can be used as the elution buffer used for elution (e.g., 50 mM sodium acetate, 50 mM glycine, and 1 M sodium chloride, pH 5 (e.g., 5.0)). The solution (9) can be further diluted with a preferably acidic buffer (10) (e.g., 50 mM sodium acetate, 50 mM glycine, pH 5 (e.g., 5.0)). The resulting solution can be subjected to a chromatography column (11) based on a stationary phase containing immobilized lysine and / or at least one immobilized lysine analogue (e.g., based on lysine-conjugated Sepharose), to which the plasminogen of interest can bind.The eluate (13) preferably does not contain detectable plasminogen content. The plasminogen preparation solution (12) can be obtained, for example, with an elution buffer containing 50 mM citrate and 50 mM glycine at pH 3 (e.g., pH 3.0). [Diagram 3] Figure 1 shows SDS-PAGE under non-reducing conditions with a total protein load of 2.5 μg / well. Lane A shows Glu-plasminogen standard (0.6 μg / well). Lane B shows All Blue Precision Standard. Lane C shows Lys-plasminogen standard (0.6 μg / well). Lane D shows Lys-plasmin standard (0.6 μg / well). Lane E shows the eluate from the lysine-conjugated Sepharose column. [Figure 4] Chromatogram of cation exchange (CEX) chromatography performed with resin Eshumuno CPX (Merck, Germany), as described in the experimental section below. No significant peak was present before peak A / 1. [Diagram 5] 3 shows the elution shows a chromatogram of affinity chromatography performed using an immobilized lysine-based column, as described in the experimental section below. Two peaks, A / 1 and B / 1, are present. [Figure 6]FIG. 1 shows an SDS-PAGE gel provided as described in the Experimental section below, with lanes as follows: (1) filtrate from the first resuspension step, 2.5 μg / well; (2) filtrate from the first wash step of the resuspension step, 2.5 μg / well; (3) filtrate from the second wash step of the resuspension step, 2.5 μg / well; (4) filtrate from the second wash step of the resuspension step, original concentration; (5) Glu-plasminogen standard, 6 μg / well; (6) All Blue Precision Protein Standard; (7) Lys-plasminogen standard, 6 μg / well; (8) Lys-plasmin standard, 0.6 μg / well; (9) CEX chromatography feed before loading the column, second resuspension filtrate diluted 1:3; (10) CEX chromatography feed after loading the column, second resuspension filtrate diluted 1:3; (11) affinity chromatography feed before loading the column, CEX eluate diluted 1:2, 2.5 μg / well; (12) affinity chromatography feed after loading the column, CEX eluate diluted 1:2, 2.5 μg / well; (13) affinity chromatography eluate, 2.5 μg / well; and (14) affinity chromatography flow-through, 2.5 μg / well. [Figure 7]FIG. 1 shows a Western blot gel provided as described in the Experimental section below, with lanes as follows: (1) filtrate from the first resuspension step, 1 μg / well; (2) filtrate from the first wash step of the resuspension step, 1 μg / well; (3) filtrate from the second wash step of the resuspension step, 1 μg / well; (4) filtrate from the second wash step of the resuspension step, 1 μg / well; (5) Glu-plasminogen standard, 0.09 μg / well; (6) All Blue Precision Protein Standard; (7) Lys-plasminogen standard, 6 μg / well; (8) Lys-plasmin standard, 0.9 μg / well; (9) CEX chromatography feed before loading the column, second resuspension filtrate diluted 1:3, 1 μg / well; (10) CEX chromatography feed after loading the column, second resuspension filtrate diluted 1:3, 1 μg / well; (11) affinity chromatography feed before loading the column, CEX eluate diluted 1:2, 1 μg / well; (12) affinity chromatography feed after loading the column, CEX eluate diluted 1:2, 1 μg / well; (13) affinity chromatography eluate, 0.15 μg / well; and (14) affinity chromatography flow-through, 1 μg / well. [Figure 8] 1 shows SDS-PAGE gel bands showing Glu-plasminogen compared to Lys-plasminogen under non-reducing (A) and reducing (B) conditions, where it is clear that Lys-plasminogen is more clearly visible. EXAMPLES

[0106] Example of a method for isolating (Glu-)-plasminogen from a plasma fraction I) Materials and Methods The precipitate obtained from the precipitation of the plasma fraction containing Glu-plasminogen with octanoic acid (OA) (OA-precipitate, OA-PPT) was obtained from a standard blood fractionation method. For this purpose, the OA-PPT from the Kistler-Nitschmann method (Kistler-Nitschmann precipitate A, PPT-NA) was used.

[0107] In a further comparative example, OA-precipitate from Kistler-Cohn process (Kistler-Cohn fraction I+II+III) was used, which gave comparable results. OA-precipitate 180g filter cake was used. In a further comparative example, OA-precipitate 360g was used, which gave comparable results.

[0108] a) Dispersion of the precipitate in a basic aqueous buffer The OA-precipitate filter cake was dispersed in a basic aqueous buffer (containing 0.2 M sodium acetate and 3.5% (w / v) PEG2000) at a filter cake:buffer mass ratio in the preferred range of 1:5 (ranges from 1:1 to at least 1:10 were also illustratively used in alternative examples) (the resulting suspension was adjusted to a pH of 9.0).

[0109] b) Incubation of the dispersion The dispersion was incubated and stirred for 1 h under moderate cooling conditions (Tempering apparatus, Lauda, ​​Germany). Filter aid (5 g / kg Harborlite 900, Imerys Filtration France SAS) was added and further incubated and stirred for 30 min at room temperature.

[0110] c) Separation of the solid portion from the liquid portion of the incubated dispersion The dispersion containing the filter aid was filtered through a standard filter (PuraFix CH 9P, Filtrox AG, Switzerland). The filter cake was washed with up to half the volume of buffer used to disperse the precipitate. The filtration time was adjusted to the individual experiment. It was followed until the filtration was complete. Typically, it was 73 kg / m3 The range was approximately 22 minutes.

[0111] Surprisingly, it was found that the use of a basic aqueous buffer to disperse the precipitate, followed by filtration, allowed for the efficient and substantial removal of impurities such as, for example, many proteins other than (Glu-)-plasminogen, fatty acids, PEG2000, and residues of octanoic acid. The impurities could be removed in the filtrate. The remaining filter cake contained (Glu-)-plasminogen.

[0112] d) Solubilization of (Glu-)-plasminogen The filter cake containing (Glu-)plasminogen was mixed with an acidic aqueous buffer (containing 0.2 M sodium acetate and 0.2 M lysine analog 6-aminohexanoic acid, adjusted to pH 5.0) in a filter cake:buffer weight ratio in the preferred range of 1:3 (ranges from 1:1 to at least 1:10 were also illustratively used in alternative examples). 30 g of Dowex 1x8 (DuPont Dow Chemicals, USA) was added per 180 g of OA-precipitate.

[0113] The mixture was incubated for 1.5 h under moderate cooling conditions (Tempering apparatus, Lauda, ​​Germany). The residue was then removed by filtration through a standard filter (PuraFix CH 9P, Filtrox AG, Switzerland). The filtration time was adjusted to the individual experiment. It was required until the filtration was complete. Typically, it ranged around 6 min, but was modified to suit the filtration volume. The filter cake was washed to remove the residue dissolved fraction. (Glu-)plasminogen remained essentially in solution. To improve the yield, the filter residue was washed with up to half the volume of acidic aqueous buffer used for solubilization. The two solutions were then combined and then further processed.

[0114] e) Cation Exchange (CEX) Chromatography The (Glu-)plasminogen-containing solution obtained above was diluted in acidic aqueous buffer (10 mM sodium acetate, 50 mM glycine, adjusted to pH 5.0) in a volume ratio of 1:3 (wider ranges were used in further comparative experiments).

[0115] Strong cation exchange (CEX) chromatography resin Eshumuno CPX (Merck, Germany) was used as stationary phase. It was used in 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 (contact time of 5 min). The column was equilibrated with equilibration buffer (200 mM sodium acetate, adjusted to pH 5.0). The dilution solution containing (Glu-)-plasminogen was loaded. The column was then washed with wash buffer (50 mM sodium acetate, 50 mM glycine, adjusted to pH 5.0). Finally, the fraction containing (Glu-)-plasminogen was eluted with elution high salt buffer (50 mM sodium acetate, 50 mM glycine, 1 M NaCl, adjusted to pH 5.0).

[0116] It was found that the lysine analog 6-aminohexanoic acid could be effectively removed from solution.

[0117] f) Chromatography with Lysine-Conjugated Sepharose The (Glu-)plasminogen-containing solution obtained above was diluted in an acidic aqueous buffer (50 mM sodium acetate, 50 mM glycine, adjusted to pH 5.0) in a volume ratio of 1:2 (wider ranges were used in further comparative experiments).

[0118] ECH-Lysine Sepharose 4 Fast-Flow (based on 4% cross-linked agarose, GE Healthcare / Cytiva, USA) was used as stationary phase. It was used in an NGC chromatography system (BioRad, USA) with a 12 mL chromatography column (126 x 11 mm) using a flow-through of 1.2 mL / min (10 min contact time) or 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 with 50 mM sodium acetate buffer at pH 5.0. A solution containing (Glu-)plasminogen was loaded. The column was washed with a first wash buffer (50 mM sodium acetate, 50 mM glycine, 1 M NaCl, adjusted to pH 5.0). The column was then washed with a second wash buffer (10 mM sodium acetate, 50 mM glycine, adjusted to pH 5.0). Finally, the fraction containing Glu-plasminogen was eluted with a citrate-containing elution salt buffer (50 mM citrate, 50 mM glycine, adjusted to pH 3.0).

[0119] g) SDS-PAGE and Western Blot SDS-PAGE was performed using a Mini-PROTEAN Tetracel system (BioRad, USA) and 10% precast polyacrylamide gels. For non-reduced SDS-PAGE, samples were diluted 1:4 with Laemmli buffer (non-reduced) and then loaded onto the gel. For reduced SDS-PAGE, protein samples were diluted 1:4 in Laemmli buffer containing 10% dithiothreitol (DTT) and incubated at 95°C for 5 min. For SDS-PAGE, lanes were loaded with 3 μg protein per well, and for Western blots, 1.2–2.4 μg protein per well were used. Each gel contained four standard lanes: Glu-plasminogen, Lys-plasminogen, Lys-plasmin, and Precision Plus Protein All Blue Pre-Stained Protein standard (BioRad, USA). Electrophoresis was performed at 80-200 V with 1x running buffer for approximately 1 h in a 4°C refrigerator. Transfer onto nitrocellulose membranes was performed by wet blotting in 1x transfer buffer (10x buffer diluted 1:10 with 20% (v / v) methanol) using a Mini Trans-Blot® electrophoretic transfer system (BioRad, USA) at 350 mA for 1 h. Because the transfer system was prepared outside the refrigerator, a sealed ice unit was added to the buffer tank to cool the system and prevent overheating and temperature fluctuations. The transfer membrane was then blocked in 5% (w / v) nonfat dry milk in TBST buffer for 1 h and incubated with primary antibody (1:700) in 5% (w / v) nonfat dry milk in TBST buffer (Tris-buffered saline containing Tween-20) overnight at 4°C. After a washing step with TBST buffer (3 x 5 min), the membrane was incubated with a secondary antibody conjugated to horseradish peroxidase (1:2000) (in 5% (w / v) nonfat dry milk in TBST buffer) for 1 h at room temperature. Protein bands were detected using SeramunBlau prec® membrane substrate (Seramun Diagnostica GmbH, Germany).

[0120] h) Determination of proteolytic activity Total proteolytic activity: Proteolytic activity (PA) was assessed by monitoring the absorption kinetics at 450 nm of samples (100 μL + 100 μL sample) mixed with the chromogenic substrate S-2288 for 1-3 min at 37 °C using a spectrophotometer. Samples in the pH range of 7-9 were pre-diluted 1:4 with PA Assay Buffer I (0.1 M Tris-HCl, 0.106 M NaCl, pH 8.7). For acidic resuspended samples with a pH below 7, the samples were pre-diluted 1:8 with PA Assay Buffer I to achieve a pH value at which the protease becomes active again. A dilution ratio of at least 1:2 with PA Assay Buffer I was used to fill the linear range of the assay. All buffers used were evaluated with respect to the required dilution ratio with PA Buffer I to achieve a target pH of 8.4 appropriate for total proteolytic activity.

[0121] Prevalent proteolytic activity: To assess the predominant proteolytic activity of acidic samples in a resuspension environment, an alternative protocol was used. Samples were mixed 1:2 with PA assay buffer II (0.025 M Tris-HCl, 0.026 M NaCl, pH 8.7). The mixture was then mixed 1:2 with the chromogenic substrate S-2288, and the absorbance kinetics at 450 nm was measured using a spectrophotometer for 1-3 min at 37 °C.

[0122] II) Results Surprisingly, it has been found that it is possible to obtain very pure Glu-plasminogen in high yields.

[0123] In one example, the solubilized protein content (filtrate and wash) after the first dispersion and filtration was 17.6 g, the total proteolytic activity of the first filtrate was 38 IU / g, and the total proteolytic activity after the first wash was 62 IU / g.

[0124] In this example, the solubilized protein content (filtrate) after solubilization with acidic buffer was 153 mg. It contained 19.2 mg Glu-plasminogen. The total proteolytic activity in the filtrate was 59 IU / g, and the predominant proteolytic activity was below the detection level. In this context, the predominant proteolytic activity describes the measurement of the converted substrate to measure proteases that are now active but inhibited at the low processing pH. For the total proteolytic activity, the sample pH was adjusted back to an optimal value for the evaluation of all the proteases present that may harm the substrate conversion and the product.

[0125] The recovery rate of the cation exchange (CEX) chromatography was 81% (a wider range was used in further comparative experiments). The total proteolytic activity was reduced from 59 IU / g before CEX to 37 IU / g after CEX. There was no observable change in the product composition (Glu / Lys-plasminogen). On SDS-PAGE / Western blot (WB), the upper two bands represent the Glu form (two different glycosylations) and the lower band represents the Lys form with two different glycosylations. The lower band of the Glu form overlaps with the upper band of the Lys form, so that only three bands are visible. For the Western blot, a commercial anti-plasminogen antibody "sheep anti-human plasminogen (Pg)" ("SAPG-IG") from CoaChrom Diagnostica GmbH (Austria) was fused. Anti-sheep IgG antibody was used as secondary antibody.

[0126] [Table 1]

[0127] In this experiment, some variation in values ​​was observed at higher protein concentrations. We focused on the comparison of samples of different orders of magnitude in a single experiment, i.e., Bradford assay or ELISA.

[0128] The eluate after chromatography on lysine-conjugated Sepharose contained 601 μg / mL plasminogen, of which 587 μg / mL was Glu-plasminogen, a purity of >97% (w / w) in terms of plasminogen content. The chromogenic activity of plasminogen was 164%. The total proteolytic activity was 145 IU / g, with the predominant proteolytic activity below the level of detection.

[0129] In the SDS-PAGE gel (see FIG. 6) and Western blot (see FIG. 7), no plasminogen was detected in the wash buffer flow-through before elution (see lanes (1) and (2), respectively).

[0130] In the eluate of affinity chromatography (see lane (13) in Figures 6 and 7), a significant amount of pure (Glu-)-plasminogen was obtained. The identity was shown by Western blot and the purity in an SDS-PAGE gel. All visible impurities were essentially removed. The molecular weight met the mass of a Glu-plasminogen standard (see lane (5)).

[0131] Notably, the affinity chromatography flow-through did not show detectable amounts of (Glu-)plasminogen (see lane (14) in Figures 6 and 7), indicating that the affinity chromatography step did not significantly reduce the yield obtained.

[0132] The eluate purity of plasminogen showed a peak of 90.78%. Potential impurities detected were 0.7% (w / w) albumin, 1.0% (w / w) immunoglobulin IgG, 0.8% (w / w) immunoglobulin IgG, and 1.1% (w / w) immunoglobulin IgM.

[0133] [Table 2]

[0134] 6-Aminohexanoic acid (6-AHA) was added to the samples, i.e. equivalent amounts of 6-AHA were added to improve comparability between samples.

[0135] Thus, the method of the present invention can be used with different sources of octanoic acid (OA) and provides highly reliable and pure Glu-plasminogen.

Claims

1. A method for isolating plasminogen from a plasma fraction, comprising the following steps: (i) dispersing a precipitate of the plasma fraction containing plasminogen in a basic aqueous buffer at pH 7-10; (ii) incubating the dispersion obtained from step (i) to dissolve at least a part of the proteins and other impurities soluble in the basic aqueous buffer; (iii) separating the solid part and the liquid part of the incubated dispersion of step (ii) from each other; (iv) dissolving the solid part obtained from step (iii) in lysine, formula (I): (H ２ N) 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 independently of each other a carboxyl group or an amino group in each occurrence; R is a linear or branched C ３ - C １２ - alkylene, linear or branched C ３ - C １２ - heteroalkylene, one or more halogens or one or more C １ - C ４ - (hetero)alkyl residue optionally substituted by a C ６ - C １２ - arylene, one or more halogens or one or more C １ - C ４ - (hetero)alkyl residue optionally substituted by a C ３ - C １２ - heteroarylene, one or more halogens or one or more C １ - C ４ - (hetero)alkyl residue optionally substituted by a C ３ - C １２ - alkylene - C ６ - C １２- an arylene, one or more halogens or one or more C １ ~C ４ - (hetero)alkyl residue optionally substituted C ３ ~C １２ - alkylene-C ３ ~C １２ - heteroarylene), further mixing with an acidic aqueous buffer solution of pH 2 to 6.6 containing at least one other compound of or both of or a salt thereof; and (v) obtaining a solution containing the isolated plasminogen from step (iv) A method comprising.

2. The method according to claim 1, wherein the plasminogen is Glu-plasminogen.

3. The plasma fraction is: (a) cryoprecipitate-poor supernatant or cryoprecipitate-poor precipitate; (b) a fraction of any one of Cohn method pastes I, II or III, or a combination of two or all of them; (c) a fraction of any one of Kistler-Nitschmann method pastes I, II or III, or a combination of two or all of them; and (d) a combination of two or all of these The method according to claim 1 or 2, selected from the group consisting of.

4. The basic aqueous buffer solution in step (i) has a pH of 8.5 to 9.5; The acidic aqueous buffer solution in step (iv) is a formic acid, acetic acid or citric acid buffer solution, has a pH of 4.5 to pH 5.5, or both; or both, The method according to claim 1.

5. The step (iii) of separating the solid part and the liquid part from each other is obtained by means selected from the group consisting of filtration, dialysis, and phase separation, including dead-end filtration, tangential flow filtration, or a combination thereof, according to the method of claim 1.

6. The step (iii) of separating the solid part and the liquid part from each other is obtained by filtration, where at least one filter aid is added to the dispersion before step (iii), according to the method of claim 1.

7. Steps (i) to (iii) are each performed once or repeated two or more times, according to the method of claim 1.

8. 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, aminomethylbenzoic acid, and oxalazine, according to the method of claim 1.

9. The solution containing the isolated plasminogen obtained in step (v) is: Contacting the solution with at least one ion exchanger with or without size exclusion properties that interacts with at least a part of lysine, the compound of formula (I), or both; Contacting the solution with at least one size exclusion resin that interacts with at least a part of lysine, the compound of formula (I), or both; Contacting the solution with at least one hydrophobic or mixed-mode interaction chromatography resin that interacts with at least a part of lysine, the compound of formula (I), or both; Subjecting the solution to diafiltration to remove at least a part of lysine, the compound of formula (I), or both; Subjecting the solution to at least one precipitation-washing cycle; Chromatography based on a stationary phase containing immobilized lysine, at least one immobilized compound of formula (I), or both; Selective affinity chromatography for plasminogen; Molecular size chromatography; Dialysis; Ultrafiltration including dead-end ultrafiltration, tangential flow ultrafiltration, or a combination thereof; and At least one additional step selected from the group consisting of combinations of two or more of these The method according to claim 1, further subjected to at least one additional step selected from the group consisting of combinations of two or more of these.

10. The solution containing the isolated plasminogen obtained in step (v) is subjected to at least the following additional steps: (vi) Removing at least a part of the compound containing one or more amino groups from the solution obtained in step (v); and (vii) Increasing the purity, concentration, or both of plasminogen; and (viii) Obtaining a solution containing the isolated plasminogen from step (vii) The method according to claim 1, further subjected to this.

11. The following steps: (i) Dispersing the precipitate of the plasma fraction containing plasminogen in a basic aqueous buffer at pH 7-10; (ii) Incubating the dispersion obtained in step (i) to dissolve at least a part of the proteins and other impurities soluble in the basic aqueous buffer; (iii) Separating the solid part and the liquid part of the incubated dispersion of step (ii) from each other by filtration including dead-end filtration, tangential flow filtration, or a combination thereof, dialysis, phase separation; (iv) Mixing the solid part obtained in step (iii) with an acidic aqueous buffer at pH 2-6.6 further containing dissolved lysine, at least one compound of formula (I) or a salt or combination thereof; and (v) Obtaining a solution containing the isolated plasminogen from step (iv); (vi) removing at least a portion of the compound(s) containing one or more amino groups from the solution obtained in step (v); and (vii) increasing the purity, concentration, or both of the plasminogen by performing chromatography based on a stationary phase comprising immobilized lysine, at least one immobilized compound of formula (I), or both, using first a buffer that allows interaction of plasminogen with the stationary phase and subsequently eluting the plasminogen with a buffer that reduces interaction of plasminogen with plasminogen; and (viii) obtaining a solution containing the isolated plasminogen from step (vii). The method according to claim 1, comprising the above steps.

12. Step (iv), step (vi), or steps (iv) and (vi) of the method according to claim 1 comprise contacting the solution with at least one small pore anion exchange resin that interacts with at least a portion of the precipitating agent.

13. (a) Is the protein that is at least partially dissolved in step (ii) recovered? (b) Does the method further comprise a virus inactivation step for the fraction of interest? (c) Is the pH of the solution containing the isolated plasminogen further adjusted to a desired range? (d) Does the method further comprise freeze-drying or drying of the plasminogen? Or (e) Is it a combination of two or more of these? The method according to claim 1.

14. A composition comprising plasminogen obtainable from the method according to claim 1, wherein the plasminogen constitutes at least 70% (w / w) of the total protein content.

15. Step (iv) of the method according to claim 1 further comprises incubating the mixture to dissolve the plasminogen.

16. The method according to claim 15, wherein step (iv) further comprises removing a solid portion.

17. The method according to claim 11, wherein step (iv) further comprises incubating the mixture to dissolve plasminogen.

18. The method according to claim 17, wherein step (iv) further comprises removing a solid portion.

19. The method according to claim 1, wherein the pH of the solution containing the isolated plasminogen is further adjusted to a desired range by exchanging an aqueous buffer.

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