Preparing and use of Glu-plasminogen from blood fractions

Abstract

The present invention relates to a method for isolating Glu-plasminogen, said method comprising the anion exchange chromatography of blood plasma or a plasma fraction comprising Glu-plasminogen. Furthermore, the present invention relates to Glu-plasminogen obtainable from the method of the present invention and its use in a method for treating a patient suffering from or being at risk of developing a disorder selected from the group consisting of organ failure, a thrombotic event, arterial obstructive disease, microcirculation, disseminated intravascular coagulation (DIC), and a combination of two or more thereof.

Claims

1. A method for isolating Glu-plasminogen, the method comprising the following steps: (i) providing a plasma fraction comprising Glu-plasminogen, wherein the plasma fraction is selected from the group consisting of: (a) cryo-poor plasma; and (b) fractions I+III of the Cohn or Kistler-Nitschmann process, fractions I+II+III of the Cohn or Kistler-Nitschmann process, or a combination thereof, wherein, optionally, octanoic acid is added to the fractions I+III or the fractions I+II+III; (ii) contacting the plasma fraction with an anion exchanger based on a resin comprising cationic groups; (iii) washing the anion exchanger obtained from step (ii) loaded with the plasma fraction with a first buffer B1 having a pH of 8.5 to 11 not comprising cations competing with the cationic groups of the resin of the anion exchanger; (iv) eluting the Glu-plasminogen from the washed anion exchanger of step (iii) with a second buffer B2 having a pH of 8.5 to 11 comprising cations competing with the cationic groups of the resin of the anion exchanger, thereby obtaining a solution comprising the second buffer B2 and Glu-plasminogen; (v) adjusting the pH of the solution obtained from step (iv) to a pH in a desired range of pH 4.5 to 6.5; (vi) stabilizing the Glu-plasminogen by adding one or more stabilizers that prevent the Glu-plasminogen from maturing into plasmin and Lys-plasminogen to the solution obtained from step (iv) and/or step (v); (vii) optionally subjecting the solution from any of steps (iv) to (vi) to one or more antiviral treatments; and (viii) optionally drying or freeze-drying the solutions comprising Glu-plasminogen obtained from any of steps (iv) or (vii).

2. The method of claim 1, wherein the resin of the anion exchanger bears amino groups or salts thereof.

3. The method of claim 1, wherein the resin of the anion exchanger bears primary amino groups or salts thereof.

4. The method of claim 1, wherein the second buffer B2 comprises, as a cation competing with the cationic groups of the anion exchanger, a soluble amine or a salt thereof.

5. The method of claim 1, wherein the one or more stabilizers of step (vi) are selected from the group consisting of aprotinin, alpha-2-antiplasmin, D-phenylalanyl-L-prolyl-arginine chloromethyl ketone, small molecule stabilizers, and combinations thereof.

6. The method of claim 1, wherein the solution obtained from any of steps (iv) to (vi) is subjected to the one or more antiviral treatments of step (vii), wherein the one or more antiviral treatments are selected from the group consisting of: (vii-a) adding one or more detergents; (vii-b) adding one or more other antiviral agents other than detergents; (vii-c) ultrafiltration; and (vii-d) combinations of two or more thereof.

7. The method of claim 1, wherein the resin of the anion exchanger comprises amino groups having the structure moiety —R—NH.sub.2 or —R—NH.sub.3.sup.++A.sup.−, wherein R is a C.sub.1-C.sub.10-alkylene residue and A.sup.− is an anionic counterion; wherein the cations in the second buffer B2 comprise a primary C.sub.1-C.sub.10-amine or a salt thereof competing with the amino groups of the anion exchanger; wherein the pH in the adjusting step (v) is in the range from 4.5 to 5.5; and wherein the method comprises subjecting the solution from any of steps (iv) to (vi) to the one or more antiviral treatments of step (vii).

8. The method of claim 7, wherein the one or more antiviral treatments comprise: (vii-I) adding one or more detergents and one or more other antiviral agents; (vii-II) removing the solution of step (vii-I); and (vii-III) ultrafiltration.

9. The method of claim 1, wherein the resin of the anion exchanger comprises amino groups having the structure moiety —R—NH.sub.2 or —R—NH.sub.3++A.sup.−, wherein R is a C.sub.1-C.sub.10-alkylene residue and A.sup.− is an anionic counterion; wherein the first buffer B1 comprises a buffer agent at a concentration from 0.01 to 0.1M; and wherein the second buffer B2 comprises a buffer agent at a concentration from 0.01 to 0.1M, and the cations in the second buffer B2 comprises a primary C.sub.1-C.sub.10-amine or a salt thereof competing with the amino groups of the anion exchanger.

Description

EXAMPLES

(1) Method of Production of Glu-Plasminogen Preparation

(2) Purification Process of Glu-Plasminogen

(3) For the isolation of Glu-plasminogen, plasma, cryo-poor plasma, factions from Cohn/Kistler-Nitschmann (KN) process or optional flow through eluate from the 4-PCC (prothrombin complex concentrate) process can be used. A usable process may be summarized as follows: 1. Plasma or cryo-poor plasma 2. optional: capture of 4-PCC complex 3. isolation of Glu-plasminogen and stabilization 4. first virus inactivation (solvent/detergent (SD) treatment) 5. SD removal 6. final purification of Glu-plasminogen complex 7. second virus inactivation (ultra/nanofiltration) 8. formulation (ultrafiltration (nanofiltration), stabilization, freezing, drying) 9. obtaining the Glu-plasminogen product

(4) In this process, step 2 can lead to a next generation 4-PCC product. A fraction of step 3 can be introduced into the Cohn/KN (Kistler-Nitschmann) process.

(5) Steps 1-3 can be designated as plasminogen capture step. Steps 4 and 5 can be designated as SD treatment/virus removal. Steps 6 and 7 can be designated as final plasminogen purification.

(6) The process described herein provides (general comments):

(7) Use of modern chromatographic technologies (new resin and bead structure, used in licensed product process already) can handle sanitization standards (1M NaOH) with a maximum of reusability.

(8) Lysine modified gels were used for the isolation process of Glu-plasminogen. But other gels containing free amino groups are usable, too, like other natural or synthetic amino acids, natural and synthetic compounds which containing a free amino group with different spacers.

(9) The process can be integrated in an established fractionation process with minimal regulatory efforts and changes in the cryo-poor plasma stream. The cryo-poor plasma stream was used directly without any changes in the Cohn/KN (Kistler-Nitschmann)-Process for the isolation of IgG, albumin and other proteins.

(10) The capture step achieves higher yields of Glu-plasminogen because of minimized activation of plasminogen due to modern chromatographic steps and resins.

(11) The following Glu-plasminogen yields (step yield/overall yield) were obtained [in % (w/w)]: 1. plasma (100/100) 2. cryo-poor plasma (90/90) 3. isolation of Glu-plasminogen and stabilization (80/72) 4. first virus inactivation (solvent/detergent (SD) treatment) (98/71) 5. SD removal (95/67) 6. final purification of Glu-plasminogen complex (95/64) 7. second virus inactivation (ultra/nanofiltration) (98/62) 8. formulation (ultrafiltration, stabilization, freezing, drying) (90/56) 9. obtaining the Glu-plasminogen product, overall yield: 56% (w/w)

(12) Directly after the isolation, the pH of the Glu-plasminogen was changed with citric acid to pH 7.5 and stabilizer, like aprotinin or alpha-2-antiplasmin (A2AP) were added.

(13) Isolation Process of Glu-Plasminogen from Cryo-Poor Plasma/Plasma Fractions—Process Steps

(14) Capture Step: The human plasma or cryo-poor plasma was directly captured on a Lysine Gel (9 CV loadability used). The raw Glu-plasminogen was isolated and stabilized (see example 1.1).

(15) SD-Treatment/1.sup.st virus removal: To the raw Glu-plasminogen was added 1% Tween-20 and 0.3% TnBP. The conditions used: 22° C., 2 h and treatment with gentle shaking.

(16) SD-Removal: On a anionic exchanger, Fractogel M TMEA, Merck the SD-solution (diluted 1:10 (10 mM citrate buffer pH 7.6)) was injected (gel: EQ with 25 mM acetate buffer pH 5.75) and all SD-reagents were washed below their specifications). The Glu-plasminogen was eluted with 25 mM acetate buffer pH 5.75 with 0.5 M NaCl).

(17) Final Purification: Same step as Capture Step.

(18) Isolation Process of Glu-Plasminogen—Out of Paste I−III Waste Fractions Obtained from Cohn/KN Process

(19) The waste fractions paste I+II+III (i.e., I−III) or paste I+III obtained from Cohn/KN Process can be used for the isolation of Glu-plasminogen. 1. obtaining paste I+II+III (i.e., I−III) or paste I+III from the Cohn/KN (Kistler-Nitschmann) process 2. thawing, dilution, pH adjustment, filtration 3. isolation of Glu-plasminogen 4. stabilization 5. obtaining the Glu-plasminogen product

(20) Herein, steps 2-4 can also be considered as referring to the Glu-plasminogen capture step.

(21) Experimental Data

(22) Assay Methods

(23) Detection of Glu-Plasminogen Product

(24) Detection was performed as follows (data not shown): Chromogenic assay detection of plasminogen Siemens Healthcare diagnostic Inc. Newark, Del. 19714 U.S.A, Berichrom Plasminogen.

(25) Detection of Glu-plasminogen-by TECHNOZYMGlu Glu-plasminogen ELISA Kit 96T (Ref: TC12040 Technoclone GmbH, Austria) Technozym: The TC Glu-plasminogen test is a solid phase enzyme immunoassay to determine the amount of Glu- and not Lys-plasminogen. The assay measures Glu-plasminogen in a range from 0.06-0.5 μg/mL. Normal plasma levels are 60-250 μg/mL. The inter- and intra-assay variations are less than 10% and 5%, respectively.

(26) The 96 well plate is precoated with a monoclonal anti-plasminogen antibody and blocked with 1% bovine serum albumin (BSA), lyophilized. (TC-Code GX)). The samples and the standard (lyophilized Normal Plasma, (TC-Code BJ)) are diluted with the incubation buffer (PBS; pH 7.3); containing stabilizer protein; 0.05% proclin; and blue dye. The standard curve contains Glu-plasminogen concentrations of 0.5 μg/mL, 0.25 μg/mL, 0.125 μg/mL, 0.063 μg/mL and 0.0 μg/mL.

(27) Pipette 0.1 mL of the diluted samples/standard into separate wells. Running standard/sample in duplicate is recommended. Cover the plate with a plastic foil and incubate overnight at 4° C. 3. Reconstitute (required) strips by adding 0.25 mL of wash buffer (Concentrate—(Predilution 1+11.5) (PBS; pH 7.3) containing detergent; 0.01% merthiolat) to the wells and tip out the contents. Wash the strips four times further with wash buffer. Tap strips on absorbent paper and make sure the wells are completely dry. Add 0.1 mL of the diluted PDX anti-plasminogen antibody to all wells, preferably with a multichannel pipette. Cover and incubate the plate for 1 hour at 37° C. Wash five times as described before. Pipette 0.1 mL of TMB substrate to all wells. Incubate for 15 minutes at room temperature. Pipette 0.1 mL of stop solution to all wells. Measure absorbances at 450 nm (with 620 nm reference filter if available). Read absorbances within one hour after the addition of the stop solution. Construct a graph of standard curve. Locate the absorbance for each sample on the curve and read the corresponding value from the horizontal axis. Do not forget to multiply by the dilution factor for the samples.

(28) SDS-PAGE

(29) To determine the purity of the Glu-plasminogen, SDS-PAGE was used with an additional Coomassie staining. The BioRad Mini-Protean TGX Stain free gels 4-20% (Cat: 456-9093) were used in combination with Precision Plus Protein Standards all blue (BioRad Cat: 161-0373), Glu-plasminogen standard (Coachrom Cat: HPGG) and Lys-plasminogen Standard (Coachrom Cat: HPGL). The proteins were stained with Bio Safe™ Coomassie G-250 Stain (BioRad Cat: 161-0786). The background was destained with destilled H.sub.2O.

(30) Bradford Method—Determination of Total Protein

(31) The Quick Start™ Bradford protein assay is a simple and accurate procedure for determining the concentration of protein in solution. The assay supplies ready-to-use dye reagent at 1× concentration (BioRad: Cat #500-0205). Protein concentration is determined in one step. Quick Start Bradford protein assay kits offer bovine serum albumin standard sets (BioRad: Cat #500-0206). From Samples and from the prediluted standard concentrations (0.125, 0.25, 0.5, 0.75, 1.0, 1.5, and 2.0 mg/ml) 5 μL were added into a polypropylen 96 well plate F-bottom (Eppendorf: Lot: G171297G). Finally, 250 μL Dye Reagent is added to each well. Mix wells and incubate the 96-Well Plate at 37° C. for 5 min (max 60 min). The absorption kinetics were measured at 595 nm at 37° C. using a spectrophotometer; locate the absorbance for each sample on the curve and read the corresponding value from the horizontal axis.

(32) Distribution of molecular size analyzed by HPLC Size Exclusion Chromatography (SEC) for Glu-plasminogen product.

(33) The below method can be utilized to determine the percentage of aggregates in Glu-plasminogen preparations (as used in Example 1.4, 1.6).

(34) Test Solution:

(35) Samples were injected undiluted at approx. 1 g/L with an injection volume of 100 μL. As reference solution Glu-plasminogen (e.g. Coachrom HPPG) was used. The standard solution was from Bio-Rad (gel filtration standard, Art.-No. 151-1901)

(36) A Column (size: 1=30 mm, Ø=7.8 mm) was used with the stationary phase from Tosoh Bioscience TSK-Gel G4000 SWXL. As mobile phase, a buffer was generated containing 4.873 g of disodium hydrogen phosphate dihydrate, 1.741 g of sodium dihydrogen phosphate monohydrate, 11.688 g of sodium chloride and 50 mg of sodium azide, dissolved in 1 liter of water. Flow rate was at 0.5 mL/min. The detection was carried outspectrophotometrically at 280 nm. The chromatograms obtained were compared with the reference solution. The chromatogram was integrated according to the following scheme and the peaks were identified: Polymer (>1200 kD), 10-13 min Proteins high molecular weight (150-900 kD), 13-22 min Glu-plasminogen (92 kDa): 22-24 min Albumin (66 kD), 25-27 min Fragments (<100 kD), 26-40 min

(37) Determination of Proteolytic Activity

(38) The proteolytic activity was assessed by mixing a chromogenic substrate (in particular those sensitive to at least one serine protease) and a sample of the Glu-plasminogen preparation (usually diluted in buffer to meet the linear range of the assay) at 37° C. and monitoring the absorption kinetics using a spectrophotometer. The proteolytic activity of the sample is calculated from the initial absorption (extinction E) difference (ΔE/min) by using the equation C (U/L)=S×ΔE/min×F (C=proteolytic activity; S=conversion factor relating to specific adsorption change of the chromogenic substrate; and F=dilution factor). Use of the substrate is according to manufacturer's instructions. The proteolytic activity can in particular be assessed via the following steps: (a) 25 mg of the substrate S-2288 (Chromogenix) is dissolved in 7.2 mL of water-for-injection; (b) a sample of the Glu-plasminogen preparation is diluted into buffer (100 mM Tris-HCl pH 8.4, 106 mM NaCl) to meet the linear range of the assay and temperature is adjusted to 37° C.; (c) equal amounts (e.g. 100 μl) of the diluted Glu-plasminogen preparation and the dissolved substrate were mixed; (d) the absorption kinetics were measured at 405 nm for 1 to 3 minutes at 37° C. using a spectrophotometer; (e) the proteolytic activity of the sample is calculated from the initial absorption difference (ΔE/min) by using the equation C (U/L)=313×ΔE/min×F (C=proteolytic activity, F=dilution factor)

(39) The limit of quantitation of this method is 8 U/L, and using a sample of the Glu-plasminogen preparation proteolytic activity is undetectable. As such the level of the proteolytic activity in the final product was found below 8 U/L.

Experimental Example 1—Isolation Process of Glu-Plasminogen

(40) Material and Methods

(41) Chromatography experiments were performed using a 1 cm internal diameter, 5 cm bed height chromatography column (Gotec Labortechnic) together with a Bio-Rad NGC Chromatography system. The column (Lysine Hyper D Resin) was equilibrated with PBS Buffer pH 6.6 (5×) at a contact time of 5 min. Cryo-poor plasma/flow through PCC was then loaded at a contact time of 5 min.

(42) Post-load wash was with 0.05 M Na-acetate/0.05 M glycine buffer pH 10.3 to baseline absorbance.

(43) The column was then eluted with 0.05 M Na-acetate/0.05 M glycine buffer/0.025 M Lysine pH 10.3 at a contact time of 5 min, and regenerated with 0.5 M sodium hydroxide. Load, non-bound, and elution fractions were analyzed by nephelometry to determine IgG and albumin content. SDS PAGE was carried out to determine purity. ELISA was carried out to determine plasminogen content.

(44) Results

(45) Plasminogen was isolated with a yield of 84% (recovery 84%) from the flow through fraction of the 4-PCC product.

(46) TABLE-US-00001 Plasminogen Plasminogen Yield Sample [IU/dL] [IU] [%] Feed 84.4 [66 mL] 55.7 100 Flow Through <17.7 [85 mL] 15.0 0 Capture Fraction 316.4 [10.0 mL] 31.6 83.6

(47) Total Protein

(48) The Total Protein (TP) yield of the flow through fraction was quantitative within the error range. The plasminogen capture fraction shows a TP content of 1.0 g/L, which demonstrates a very pure product. No interaction of albumin, IgG, with the column resulted in a 100% flow through. The eluted Glu-Plasminogen had very low total protein concentration which resulted in a minimized impurity.

(49) TABLE-US-00002 Total Protein TP Absolut Yield Sample [mg/mL] [mg] [%] Feed 55.313 [66 mL] 3651 100.0 Flow Through 43.219 [85 mL] 3673 100.6 Capture Fraction 1.031 [10.0 mL] 10.3 0.28

(50) Albumin

(51) The albumin content in the flow through fraction was 104% (quantitative, within error ranges).

(52) TABLE-US-00003 Albumin Albumin Absolut Yield Sample [mg/mL] [mg] [%] Feed 31.971 [66 mL] 2110.1 100.0 Flow Through 25.800 [85 mL] 2193.0 104.0 Capture Fraction — [10.0 mL] — —

(53) IgG

(54) The IgG content in the flow through fraction was 99.5% (quantitative, within error ranges).

(55) TABLE-US-00004 IgG IgG Absolut Yield Sample [mg/mL] [mg] [%] Feed 8.79 [66 mL] 580.14 100.0  Flow Through 6.79 [85 mL] 577.15 99.5 Capture Fraction — [10.0 mL] — —

Experimental Example 1.1—Isolation Process of Glu-Plasminogen from Cryo-Poor Plasma

(56) Glu-Plasminogen can be purified from cryopoor plasma with the method mentioned in example 1. As contact time 10 min was used. The load ability of cryo-poor plasma was 8 CV (40 mL cryo-poor plasma). The eluted Glu-Plasminogen gained comparable yields as in example 1. The yield was 76% with a recovery of Glu-Plasminogen of 85%.

(57) Product Specification and Administration of Glu-Plasminogen

(58) The purified naturally occurring protein Glu-plasminogen will be used for multiple occasions on several days.

(59) Target purity of the plasminogen preparation is ≥90%, containing at best exclusively Glu-plasminogen and not Lys-plasminogen. Activation of inactivated Glu-plasminogen molecules can be initiated by Lys-plasminogen. Lys-plasminogen was already enzymatically cleaved and has an open conformation for a faster activation process to plasmin. Therefore, Lys-plasminogen is not usable for the application into a human body because of the unspecific activation, followed by strong adverse effects. The usage of modern technology of chromatographic resins and SD-treatment resulted in minimal losses of plasminogen during the total process and high yields of inactivated Glu-plasminogen. Potential activation from Glu- to Lys-plasminogen was balanced by Aprotinin, which captured the activation process (Lys-plasminogen and Plasmin). Additionally, several inhibitors such as the natural inhibitor A2AP and pPack were tested for stabilizing the inactivated Glu-plasminogen product.

Experimental Example 2—Comparison of Buffers and Resins for Capture-Step-Of Glu-Plasminogen from PCC Flow Through

(60) Studies were performed to determine efficacy of two different resins for a first Glu-plasminogen capture step. The Resin Lysine Hyper D (Pall) and the ECH-Lysine Sepharose™ 4 Fast Flow (GE Healthcare) were analyzed. The Glu-plasminogen was purified from the feed stream flow through Pro Thrombin Complex (PCC). This feed contains IgM 0.8 g/L, IgG 8.83 g/L, albumin 32.49 g/L, total protein (TP) 57 g/L and Glu-plasminogen 74 μg/mL. The column chromatography was performed with a öeGötec column (d 1 cm h: 20 cm) using a Biorad NGC Chromatography System.

(61) A column was used with a column volume (CV) of 4-5 mL. A constant contact time of 7- or 8 min was applied in each experimental approach. The feed passed the column with a flow rate of 0.5-0.6 mL/min. The feed loadability was constant at 10 CV.

(62) The column was equilibrated (4 CV) with 0.05 M phosphate buffer pH 6.6 (Method 1) or with 0.01 M Tri-Na-citrate/1 mM CaCl.sub.2/0.12 M NaCl pH 7.0 (Method 2) or 0.1 M phosphate buffer pH 7.4 (Method 3). The pure flow through PCC (pH 7.3 13.5 mS/cm) passed the column. As a wash buffer 0.05 M acetate/0.05 M glycine pH10.3 (Method 1) or with 0.01 M Tri-Na-citrate/1 mM CaCl.sub.2/0.12 M NaCl pH 7.0 (Method 2) or 0.1 M phosphate buffer pH 7.4 (Method 3) was used. The flow though was collected in bottles and frozen at −35° C. For elution of Glu-plasminogen either 0.05 M acetate/0.05 M glycine/0.025 M Lysine pH 10.3 (Method 1), 0.05 M Tris/0.025M Lysine/1 M NaCl pH 9.0 (Method 2) or 0.1 M phosphate buffer 0.2 M {acute over (ε)}-aminocaproic pH 7.4 (Method 3) as was used. As cleaning in place (CIP) program 0.1 M NaOH (4 CV), 0.1 M HCl (4CV) was used and the column was stored in 20% ethanol. The final Glu-plasminogen product was frozen at −35° C. A Glu-plasminogen ELISA Technozym was used to determine Glu-plasminogen concentration. The immunoglobulin concentration was analyzed nephelometrically. The albumin concentration was determined by polychromatic endpoint determination and TP by Bradford method.

(63) The results indicate, that the usage of different resins and different buffer conditions leads to variable results. The aim is to first bind Glu-plasminogen and then elute Glu-plasminogen with a high yield. The other proteins should flow through with 100%. This flow through is mostly used for further purification in in other processes. The results indicate that IgG and albumin flow through with 100% by the usage of both resins. The IgM molecules interact with the column depending on different buffer conditions. The best flow through result could be achieved by the usage of the method 1 with both resins (Table 1).

(64) TABLE-US-00005 TABLE 1 Glu-plasminogen purification on two different resins using Method 1, 2 and 3, Analysis of fflow-through. Flow-through 10 CV- total total Volume- total IgM total IgG Albumin Protein Method Resin used FT [mL] recovery recovery recovery recovery 1 Lysine Hyper-D 57.3 103%  110% 108% 106% Lysine-Sepharose- 63.6 104%  102%  98% 103% 2 Lysine Hyper-D 59.9 74%  90% 102% 103% Lysine-Sepharose- 65.4 96%  98%  99% 102% 3 Lysine Hyper-D- 58.5 90% 103% 104% 100% Lysine-Sepharose- 65.9 85%  98% 108% 103% * ±10% Inter- and 5% intra-assay variations.

(65) The yield of Glu-plasminogen varies with the usage of the resin Lysine Hyper D in combination with the three methods. The highest yield of Glu-plasminogen can be achieved by method 1. The plasminogen yield is 90% and respectively the recovery (Glu-Plasminogen yield+Glu-Plasminogen in flowthrough) at around 95%. The intended application of the resin ECH-Lysine Sepharose results in comparable Glu-plasminogen yields for each of the three methods.

(66) The usage of method 1 with the Lysine Hyper D and Lysine Sepharose Resins resulted in higher purities of the Glu-plasminogen preparation in comparison to the other methods 2 and 3.

(67) TABLE-US-00006 TABLE 2 Glu-plasminogen purification on two different resins using Method 1, 2 and 3, Analysis of eluates. Eluate Plasminogen Volume-Eluate Glu-plasminogen Plasminogen yield recovery Method Resin used [mL] [μg/mL] (Eluate) (E + FT) 1 Lysine Hyper-D- 5.73 527 90% 95% Lysine-Sepharose- 3.99 753 83% 83% 2 Lysine Hyper-D- 7.33 253 69% 77% Lysine-Sepharose- 6.86 519 81% 81% 3 Lysine Hyper-D- 4.07 392 50% 65% Lysine-Sepharose 4.29 680 81% 81% * ±10% Inter- and 5% intra-assay variations

Experimental Example 3—P Purification of Glu-Plasminogen with 10 mM Acetate Buffers and Loadability Up to 25 CV

(68) This example was performed to determine the binding capacity the resin ECH-Lysine Sepharose™ 4 Fast Flow (GE Healthcare) in a first Glu-plasminogen Capture Step. The Glu-plasminogen was purified from the feed stream flow through Pro Thrombin Complex (PCC). This feed contains IgM 0.8 g/L, IgG 8.83 g/L, albumin 32.49 g/L, Total protein (TP) 57 g/L and Glu-plasminogen 74 μg/mL. The column chromatography was performed with aGötec column (d 1 cm h: 20 cm) using a Biorad NGC Chromatography System.

(69) A column was used with a column volume (CV) of 5.02 mL and the chromatographic purification was performed with a constant contact time of 7.8 min. The feed passed the column with a flow rate of 0.64 mL/min. The feed loadability was up to 25 CV.

(70) The column was equilibrated (4 CV) with 0.05 M phosphate buffer pH 6.6 (Method 1). The pure flow through PCC (pH 7.3 13.5 mS/cm) passed the column. The 0.01 M acetate/0.05 M glycine pH10. was used as a wash buffer. The flow-through and the wash fraction was collected in bottles and frozen at −35° C. The buffer 0.01 M acetate/0.05 M glycine/0.025 M lysine pH 10.3 was used to eluate Glu-plasminogen. More acidic or neutral pH values resulted in a reduction of Glu-plasminogen yields. For the CIP program 0.1 M NaOH (4 CV), 0.1 M HCl (4 CV) was used and the column was stored in 20% ethanol.

(71) The pH value of eluted Glu-plasminogen product was adjusted with 1M acetic acid to pH 5.0. To produce a stable Glu-plasminogen without additives the product was diafiltrated (Pall 10 kDa Centrifugal device Number MCP010C41) into a 0.32 M glycine buffer and was frozen at −35° C. A Glu-plasminogen ELISA Technozym was used to determine Glu-plasminogen concentration. The immunoglobulin concentration was analyzed nephelometrically. The albumin concentration was determined by polychromatic endpoint determination and TP by Bradford method.

(72) This example demonstrates the advantages to use the ECH-Lysine Sepharose with an 0.01 M acetate buffer for washing and Glu-plasminogen elution. The resin offers a high loadability >25CV of the feed and a high Glu-plasminogen yield of 93% under the mentioned conditions. The ECH-Lysine Sepharose can be used with a high binding capacity of min. 1.85 mg plasminogen/mL Resin. Especially the purity of Glu-plasminogen product is extremely high with >90%. Coomassie-stained SDS-PAGE detects only albumin as an impurity with <10%. However, the presence of albumin offers an additional stabilizing effect (Table 3).

(73) TABLE-US-00007 TABLE 3 Step Yields of the Glu-plasminogen purification process with the use of 10 mM acetate buffers andand a loadability up to 25 CV. Exp: Number: Glu-PLG IgM IgG Albumin Total protein 02_PLG_01_CH_028.2 [μg/mL] [g/L] [g/L] [g/L] [g/L] Feed (25 CV/125 mL) — — — — — volume [L] 0.1256 concentration [g/L] 0.064 0.743 8.650 30.482  53.258  total protein [g] 0.008 0.093 1.086 3.829 6.689 Flow Through — — — — — volume [L] 0.125 concentration [g/L] 0.726 8.220 31.132  52.439  total protein [g] 0    0.091 1.028 3.892 6.555 Wash-Fraction — — — — — volume [L] 0.01656 concentration [g/L] <0.053  3.180 12.234  20.354  total protein [g]  0.00000 0.001 0.053 0.203 0.337 Eluate - Glu-plasminogen — — — — — volume [L] 0.00547 concentration [g/L] 1.36  <0.053  <0.350  0.412 1.452 total protein [g] 0.007 0.000 0.002 0.002 0.008 Yield-Glu-plasminogen 93%  0%  0%  0%  0% Recovery of Plasma 93% 98% 100% 107% 103% proteins * ±10% Inter- and 5% intra-assay variations

Experimental Example 4 Capture of Glu-Plasminogen from Fraction I+II+III and Resulting Waste Fractions

(74) The fraction I+II+III (140 g) originating from cold ethanol fractionation of human plasma was suspended in 760 mL Buffer (0.1 M acetate buffer pH 5.05). The suspension was mixed for 15-30 minutes after the suspension temperature is reached (22° C.). In the suspension, a Glu-plasminogen concentration of 73 μg/mL was measured. After filtration by depth filtration or centrifugation, Glu-plasminogen might be purified from the resulting filtrate/supernatant according to example 3.

(75) In a second option, the suspension was further treated by addition of octanoic acid (0.110 kg per kg fraction I+II+III used) at room temperature and the protein solution was further mixed for 80 minutes, using a vibrating mixer (Vibromixer®, Size 4) The octanoic acid was added slowly over 30 min. Approx. 0.015× amount of fraction I+II+III of tri-calcium phosphate (Ca.sub.3PO.sub.4).sub.2) was added and the protein solution was further mixed for 15-30 min. Filter aid Celpure® P100 (4.3-5.7 g/kg protein solution) was added into the suspension and incubated for 15 min. Additionally, Filter aid Harbolite® 900 (10 g/kg protein solution) was added into the suspension and incubated for 15 min.

(76) The precipitate was separated from the filtrate by depth filtration. The filtrate contains 80% of IgG, IgM and albumin of the starting suspension, Glu-plasminogen could not be detected. However, proteins remain in the filter cake (waste-fraction), which is usually removed and discarded, but seems to have a high potential to regenerate new proteins (Glu-Pasminogen). Isolation of Glu-plasminogen from the precipitate (filter cake) by several washing steps was successful with a concentration of ˜28 μg/mL, resulting in a yield of 38% from the starting material. As the filter cake, being a waste fraction, is usually discarded, the yield is subordinate to the fact, that indeed native Glu-plasminogen can be obtained by recycling of waste fractions. Therefore, also other waste fractions of the Cohn/KN process can be used for isolation and further purification of Glu-plasminogen.

(77) The Glu-plasminogen isolated from the filter cake may be further purified: The Glu-plasminogen solution was diluted in 0.05M phosphate buffer at pH 6.6. The final Glu-plasminogen capture step was performed according to example 3.

(78) In general, the Glu-plasminogen can be isolated from each fraction I+II+III and resulted waste fraction. The Glu-plasminogen concentration differs of each individual Cohn or Kistler-Nitschmann process.

Experimental Example 5 Purity of Glu-Plasminogen Preparation

(79) The Glu-plasminogen product was purified by a selective affinity chromatography. Although the Lysine residues have a high affinity, impurities can be captured as well. Furthermore, the purification process of Glu-plasminogen can generate aggregates or fragments. The Glu-plasminogen should have a high purity without presence of aggregates or fragments. In Table 4, SEC analysis of the Glu-plasminogen products purified by Example 2 are shown. The different methods show similar results. Only the Glu-plasminogen product purified by Method 2 with Lysine Hyper D shows molecules with high molecular weight. The other Glu-plasminogen products contain no fragments and no aggregates. The albumin content in the Glu-plasminogen products varies minimally between 10-20%.

(80) TABLE-US-00008 TABLE 4 SEC-Analysis of Glu-plasminogen (PLG) product (left column Method No. 1-3) SEC-Relative-Area Total Protein molecules high Content Aggregates molecular weight PLG Albumin Fragments Resin used [μg] >1200 kDa >630 kDa (92 kDa) (66 kDa) <100 kDa 1 Lysine Hyper-D 46.9 n.d. n.d. 84% 16% n.d. Lysine-Sepharose 37.2 n.d. n.d. 89% 11% n.d. 2 Lysine Hyper-D 25.3 n.d. 9% 81% 19% n.d. Lysine-Sepharose 51.9 n.d. n.d. 88% 12% n.d. 3 Lysine Hyper-D 39.2 n.d. n.d. 81% 19% n.d. Lysine-Sepharose 68.0 n.d. n.d. 87% 13% n.d. PLG Standard 104.4 n.d. n.d. 78% 22% n.d. Coachrom * ±10% Inter- and 5% intra-assay variations

Experimental Example 6—Determination of Residual Proteolytic Activity of Purified Glu-Plasminogen Products

(81) The Glu-plasminogen purification was performed according to example 2. The proteolytic activity in samples of Glu-plasminogen products (e.g. c: 250 μg/mL) purified by the before mentioned purification methods was determined using the chromogenic substrate S-2288 (Chromogenix), following the manufacturer's instructions. 25 mg of the substrate S-2288 (Chromogenix) were dissolved in 7.2 mL water-for-injection. Samples were diluted into buffer (100 mM Tris/HCl pH 8.4, 106 mM NaCl) to meet the linear range of the assay, 100 μL buffer were mixed with 100 μL sample (mixing and temperature adjustment to 37° C.). 100 μL of the prediluted sample were mixed with 100 μL chromogenic substrate solution into a 96-well plate. The absorption kinetics are measured at 405 nm (1-3 min) at 37° C., using a spectrophotometer. The proteolytic activity of the sample was calculated from the initial absorption difference (ΔE/min) by using the equation C (U/L)=313*ΔE/min*F (C=proteolytic activity, F=dilution factor).

(82) TABLE-US-00009 TABLE 5 Proteolytic activity of Glu-plasminogen products. Determination of proteolytic activity Mean residual proteolytic Starting material (U/L) activity in Glu-plasminogen 30 Product (U/L) Lysine Hyper D Lysine Sepharose Method 1 50.6 <8 Method 2 486.8 <8 Method 3 32.5 <8 Method 1-DF Glycine r <8 * ±10% Inter- and 5% intra-assay variations

(83) The purified Glu-plasminogen products show different proteolytic activity levels depending on the purification method. In some samples an increased proteolytic activity could be determined due to buffer conditions or resin characteristics. To compare the different purification methods, Glu-plasminogen concentration was similar in each sample. Glu-plasminogen products purified by the usage of ECH-Lysine-Sepharose had no proteolytic activity.

(84) This result indicates, that the purification method does not activate Glu-plasminogen and does not purify other contaminating proteases.

(85) After purification with Lysine Hyper D resin higher proteolytic activities were measured, varying with the use of different buffer conditions. Saturating the Lysine Hyper D column with aprotinin minimizes the proteolytic activity of the generated Glu-plasminogen preparations. Furthermore, proteolytic activity was drastically reduced after diafiltration into 0.32M glycine buffer.

Experimental Example 7—Storage Stability Studies with Liquid Glu-Plasminogen Product

(86) Glu-plasminogen products prepared according to examples 2 and 3 were incubated at 37° C. over a time course of 48 h and analyzed afterwards for the presence of degradation products (e.g. Lys-plasminogen) according to SDS-PAGE. Furthermore, the remaining content of Glu-plasminogen after 48 h of incubation was determined by Glu-plasminogen ELISA.

(87) According to Coomassie-stained SDS-PAGEs, the stability of Glu-plasminogen in the preparations depend on the chromatographic resin used for purification as well as on buffers used for elution and storage of Glu-plasminogen. The Glu-plasminogen content of a preparation generated using method 1 on Lysine-sepharose, that was diafiltrated into 0.32 M glycine buffer pH 4.3, stayed constant over a time course of 48 h at 37° C. No degradation to Lys-plasminogen could be detected in the sample. In some of the other Glu-plasminogen products prepared according to example 2, degradation processes of Glu-plasminogen were visible.

(88) This tendency can also be observed by measuring the remaining Glu-plasminogen content after 48 h at 37° C. of the different Glu-plasminogen preparations using a Glu-plasminogen ELISA (table 6). The Glu-plasminogen concentration of each sample at time point t=0 was defined as 100%.

(89) TABLE-US-00010 TABLE 6 Stability-study of liquid Glu-plasminogen product. purified Glu-plasminogen content Storage in hours at 37° C. Method Resin used 0 3 7 18 24 48 1 Lysine Hyper-D 100% 100%  82%  4%  0% 0% Lysine Hyper-D- 100% 100% 100% 100% 100% 100%  Glycine- Diafiltrated Lysine-Sepharose 100% 100% 100% 100% 100% 80%  Lysine- 100% 100% 100% 100% 100% 100%  Sepharose- Glycine- diafiltrated 2 Lysine Hyper 100%  70%  5%  0%  0% 0% Lysine-Sepharose 100% 100% 100%  92%  18% 2% 3 Lysine Hyper-D 100% 100%  75%  3%  0% 0% Lysine-Sepharose 100% 100% 100% 100% 100% 70%  * ±10% Inter- and 5% intra-assay variations

(90) Generally, it can be observed, that Glu-plasminogen products show a higher stability, if purified by ECH-Lysine Sepharose in contrast to Lysine Hyper D. However, independent of the resin used for purification, Glu-plasminogen products eluted with acetate buffer could be further stabilized by diafiltration into glycine buffer, showing similar high stabilities afterwards. The stability analysis indicates, that the usage of buffer conditions from method 1 resulted in a highly stable purified Glu-plasminogen product.

(91) After incubation of this preparation at 37° C. for 48 h, the product remained within the specifications defined to show stability of the Glu-plasminogen preparation: presence of degradation products (e.g. Lys-plasminogen) according to SDS-PAGE, aggregate and fragment content measured with high performance size exclusion chromatography (HPSEC), proteolytic activity (PA) and determination of the remaining content of Glu-plasminogen after 48 h incubation by Glu-plasminogen ELISA (see table 7).

(92) Other parameters like coloration, opalescence, pH value were also determined and stayed unchanged over the whole study period.

(93) In the ongoing stability study, also after 96 h the preparations show the same stability profile, confirming the hypothesis, that the stability of a Glu-plasminogen product depends on buffer compositions, used chromatographic resin, pH value and conductivity.

(94) Additionally, in a second stability study over 48 h at 37° C., it was demonstrated, that addition of rec. aprotinin (0.019 μg rec. aprotinin/μg Glu-plasminogen) further increased stability of Glu-plasminogen products, independent of the storage buffers used.

(95) TABLE-US-00011 TABLE 7 Stability study of a liquid Glu-plasminogen product (Method 1. Lysine-Sepharose_glycine diafiltrated) with specific product specifications. Parameters Requirement Storage in hours at 37° C. tested (Tolerance) 0 3 7 18 24 48 TP content 0.8-1.2 1.1 1.1 1.2 0.9 0.9 1.1 [g/L] HPSEC % aggre- <5 <5 n.t. <5 n.t. <5 <5 gates >1200 kD % frag- <5 <5 n.t. <5 n.t. <5 <5 ments <100 kD proteolytic <8 <8 n.t. <8 n.t. <8 <8 activity (U/L) Glu- >80% 93% 92% 93% 91% 93% 92% plasminogen content (%) * ±10% Inter- and 5% intra-assay variations

Experimental Example 8—Mode-Of-Action Study

(96) A new diagnostic study of patients with acute kidney failure demonstrates that a significant increase in the ratio of A2AP and PLG was detected in a control population (55 patients) and patients (25 patients) in the University Hospital of Mannheim.

(97) Post-mortem studies of patients with sepsis demonstrate microvascular thrombi in many organs including the kidney, liver, lung, gut, adrenals and brain, and the degree of organ injury is related to the quantity of thrombi. For the analysis of organ failure, animal models of sepsis are used to demonstrate therapies, that inhibit coagulation or promote fibrinolysis, which reduce organ failure and mortality.

(98) However, the usage of these sepsis-models leads to inconcrete analysis of the positive initiation of fibrinolysis. The mechanism of plasminogen cannot be defined in a complex disease model.

(99) Four potential mode of action (MoA) models in pre-clinical experiments can define the treatment potential of Glu-plasminogen.

(100) The question was, if Glu-plasminogen can initiate the fibrinolysis and will stabilize the balance between protein and inhibitor. It is proven which MoA is suitable.

(101) 2.1. Total Occlusion of V. Cava in a Murine Model (Prof. Anders University Munich).

(102) Hypothesis (Found by Previous Experiments)

(103) Intravenous injection of Glu-plasminogen has the potential of shifting the balance towards fibrinolysis to resolve the existing vein thrombus.

(104) Short Overview

(105) Process: During 3 days a deep vein thrombosis was built.

(106) Analytic: The endpoint of the study.fwdarw.measurement of the clot size

(107) TABLE-US-00012 Advantages Disadvantages Established MoA model Model for testing of coagulation Easy to handle the occlusion of and not for fibrinolysis cava Potential Lethal outcome Fast injury for many mice per day before dosage optimization Defined endpoint of clot size

(108) Material and Methods

(109) In Vivo Experiment

(110) Seven to eight-week-old male C57BL/6N mice were procured from Charles River Laboratories, Sulzfeld, Germany. They were maintained under standard housing conditions with free access to food and water. All animals underwent IVC ligation surgery and monitored for 72 h after surgery. All mice were sacrificed by cervical dislocation at the end of the study. All animal experiments were performed in accordance with the European protection laws of animal welfare, and with approval by the local government authorities Regierung von Oberbayern (reference number: 55.2-1-54-2532-54-2017).

(111) TABLE-US-00013 Test system Species Mice. Strain C57BL/6N Source Charles River, Germany Number on study 12 mice in vehicle group and 9 in Glu-plasminogen group Age/gender 7 weeks male mice Identification Animals were identified by marks on the tail. system Individual cage cards were affixed to each cage, displaying details such as the animal number, the study number, the initiation dates and the experimenter Justification for The animal model is a well-established, suitable model selection model for the study of venous thrombosis model. Husbandry Standard laboratory conditions. Air conditioned with Conditions target ranges for room temperature of about 22 ± 4° C., for relative humidity of about 30-70% and approximately 10-15 air changes per hour. There was a 12-hour light/12-hour dark cycle. Accommodation Mice were kept in a group of 4 in cages with filter tops and standardized softwood bedding as enrichment as well as red-transparent houses. Diet Animals were fed on normal chow diet (Ssniff, Soest, Germany). The diet was available ad libitum. Drinking water Community tap water was supplied ad libitum by an automatic water dispenser. The quality of the drinking water was pursuant to the “Trinkwasserverordnung” (Directive in Potable Water) dated May 22, 1986, promulgation of the revised Directive on Jan. 1, 1991 in the German Federal Law Gazette I, no. 66, dated Dec. 12, 1990, pp 2 613-2 629 and amended in the German Federal Law Gazette I, no. 7, dated Feb. 8, 1991, p 227.

(112) Animal Receipt, Acclimatization and Monitoring

(113) Qualified personnel inspected each animal upon receipt. Animals judged to be in good health and suitable as test animals were placed in quarantine for at least 1 week.

(114) During acclimatization and biological phase of the study, animals were observed once daily for changes in general appearance and behavior.

(115) Grouping and Treatment

(116) Mice were randomized and assigned into two groups. Vehicle/PBS group (n=12) and Glu-plasminogen group (n=9 per group).

(117) Surgery

(118) All surgical procedures were carried out in sterile and designated area. Before surgery each animal was injected with 100 μl of narcosis and allowed the animals for 5-10 minutes into a 37° C. breeding chamber to undergo deep sleep. After 10 minutes, animal was taken out and placed on pre-heated (40° C.) heating plate so that the body temperature of the animal will be maintained during the surgery. 1-2 cm abdominal incision was made opened and inferior vena-cava (IVC) was located. Using 7-0 (8.0 mm0 mm 3/8 c8c) polypropylene monofilament non-absorbable suture (Prolene #8735H, Ethicon, Norderstedt, Germany) IVC was ligated (100% stenosis). After successful ligation, abdominal incision was closed and injected with 200 μL of antagonists and buprenorphine and place back again into a 37° C. breeding chamber and monitored for another one hour for any surgical complications.

(119) Treatment Schedule

(120) TABLE-US-00014 Route of administration Intravenous injection. Frequency One-time injection 15 h after surgery Application volume 180 μL total volume with 4.755 μg/mL concentration.

(121) Sacrifice

(122) All animals were placed back into the animal facility and monitored and injected with 200 μl of Buprenorphin for every 12 h. All animal's stress conditions were recorded in a prescribed score sheets (Table 2). 72 hours after surgery all animals were sacrificed with cervical dislocation method. IVCs were dissected out and thrombus clot weights were measured and recorded.

(123) Observations

(124) Mortality and Clinical Signs

(125) Animals were observed twice a day for any abnormal clinical signs and serious adverse events such as mortality.

(126) Thrombus Formation and Resolution

(127) Upon sacrifice individual animals were evaluated for thrombus formation (control animals) and resolution (treatment animals). Each individual animal thrombus weights were recorded and assessed for efficacy drug.

(128) Results

(129) Mortality

(130) In the control group two animals and in Glu-plasminogen group one animal died due to surgical complications.

(131) Thrombus Formation and Resolution

(132) All vehicle-/PBS-treated group animals developed significant amount of thrombus clots, while almost all Glu-plasminogen-treated group animals showed no clot or maximum resolution of clot (Table 1 and FIGS. 1-5).

(133) Discussion and Conclusions

(134) The Glu-plasminogen produced according to this invention has surprisingly a high and excellent fibrinolytic activity. Therefore, we hypothesized that the product according to this invention will resolve the existing thrombus.

(135) To test the hypothesis, we have used 100% stenosis of IVC in murine model venous thrombosis. Control mice with vehicle injection have shown development of thrombus clot formation after 72 h of stenosis. Compared with thrombus weights of vehicle mice, Glu-plasminogen group animals had no thrombus or significantly reduced thrombus weight after the administration of Glu-plasminogen extract. Therefore, the injection of Glu-plasminogen leads to no negative effect and shift the imbalance of coagulation and fibrinolysis to an increased fibrinolytic activity. In both groups mortality was observed due to surgical complications.

(136) Therefore, we conclude that Glu-plasminogen developed has capabilities to initiate the fibrinolytic activity on this experimental venous thrombosis model.

(137) TABLE-US-00015 TABLE 8 Table 1 showing the thrombus weight (in grams) Mouse No Vehicle Glu-plasminogen 1 0.0019 0.0012 2 0.0025 0.0019 3 0.0018 0.0000 4 0.0026 0.0000 5 0.0017 0.0000 6 0.0026 0.0000 7 0.0018 0.0000 8 0.0021 0.0000 9 0.0019 dead 10 0.0023 11 Dead 12 Dead

(138) Representative Graph of Thrombus Weights in Both Groups.

(139) Thrombus weights were measured in grams. Data represent mean±SEM. p<0.0001 (n=10 vehicle group; n=8 treatment group). These data represent a highly significant (****) reduction of the already built thrombus after the injection of Glu-plasminogen.

Experimental Example 8.1. Transient Singular Ischemia-Reperfusion of the Kidney in a Murine Model

(140) Process: Ischemia-necrosis of renal tubules

(141) Analytic: Glomerular filtration rate (day 1 and 30), delta kidney weight (day 30), dimension of kidney fibrosis (day 30). Prof. Anders University Munich

(142) TABLE-US-00016 Advantages Disadvantages Kidney-model for further indication Focus on ischemia of tubules Renal health and general recovery No experience with injury of after a time period glomerular and clot formation No definition of primary endpoint formation Long time period Not comparable with a disease or indication of a human patient

Experimental Example 8.2. Kidney-Transplantation in a Murine-Model

(143) Process: Analysis of the kidney rejection due to necrosis of kidney tissue and micro clot formation in capillaries.

(144) Analytic: Glomerular filtration rate (day 1 and 30), delta kidney weight (day 30), dimension of kidney fibrosis (day 30) and clot formation. In contact.

(145) TABLE-US-00017 Advantages Disadvantages Established mode of action model Difficult to transplanted Similar biochemical mechanism as 1 or 2 mice per day, Long time a human transplantation period Excellent comparable High costs No definition of primary endpoint formation

Experimental Example 8.3. Graft Versus Host Disease (GvHD) in Murine Model

(146) Process: bone marrow transplantation, leads to (GvHD) due to Von Willebrand factor activation and multimeric thrombocyte aggregation.

(147) Analytic: The endpoint of the study.fwdarw.longer life time or not.

(148) TABLE-US-00018 Advantages Disadvantages Established mode of action model Complex disease Easy for transplantation Lethal outcome Fast injury Difficult to analyze the success Simulate most likely DIC of treatment Many mechanism are influenced in the disease

Experimental Example 9. Indication Opportunities for Glu-Plasminogen

(149) In publications 30 years ago, it has been published that an increased ratio of alpha-2-antiplasmin (A2AP) and plasminogen (PLG) was detected in patients with cadaver kidney transplantation.

(150) Furthermore, it was shown that the administration of plasminogen improved the physical conditions of patients with sepsis.

(151) The imbalance of the high amount of alpha-2-antiplasmin and plasminogen shuts down the fibrinolytic activity and coagulation is not balanced. In a new diagnostic study of patients with acute kidney failure was shown that a significant ratio was detected between control population (55 patients) and patients (25 patients).

(152) Raw-data: measurement of PLG and A2AP in patients with acute kidney failure. Normal range in the control population (55 healthy blood donors):

(153) PLG: 82.7%-144.5%

(154) A2AP: 96.9%-118.9%

(155) ratio: 0.80-1.26

(156) Results of the non-statistical normal distribution of 25 patients with acute renal failure: 1. 12/25 (48%) patients <82.67% PLG 2. 14/25 (56%) patients <96% A2AP 3. 6/25 (24%) patients >1.26 ratio (A2AP/PLG) 4. 4/25 (16%) patients >1.26 ratio and <82% PLG

(157) TABLE-US-00019 No. Plasminogen [%] α-2-Antiplasmin [%] Ratio 1 119.61 101.8 0.85 2 103.2 93.5 0.91 3 82.3 90.1 1.09 4 114.2 113 0.99 5 133.7 97.6 0.73 6 65.5 92.5 1.41 7 82.1 92.8 1.13 8 74.6 89.9 1.21 9 94.9 93.7 0.99 10 137.6 105.5 0.77 11 109.9 117.4 1.07 12 74.9 50.8 0.68 13 67.8 96.5 1.42 14 85.6 115.6 1.35 15 90.9 107.3 0.18 16 80.2 77.3 0.96 17 9.8 55.5 5.66 18 80.4 76.2 0.95 19 41.3 41.8 1.01 20 77.3 99.3 1.28 21 125.5 115.7 0.92 22 92.1 84.8 0.92 23 123.1 114.3 0.93 24 77.8 98.5 1.27 25 108.1 67.5 0.62 Mean 90.10 91.56 1.21 Std 28.81 20.45 0.95 Min 9.80 41.80 0.62 Max 137.60 117.40 5.66 Range 127.80 75.60 5.04 VK 31.98% 22.34% 78.57% Out of <82.67 <96.925 <0.8 Normal Range Number of 12 14 4 Patients Out of >144.45 >118.9 >1.26 Normal Range Number of 0 0 6 Patients Rest of 13 11 15 Patients

(158) The study of acute kidney failure resulted in a significant difference between patients (Pat, suffering from acute kidney failure) and control group (NP, healthy individuals) in different parameters (rounded values):

(159) TABLE-US-00020 control group Patient [55 individuals] [25 individuals] alpha-2-antiplasmin (A2AP) 107.7   ± 7.2 .sup.   83.1 ± 4.1  Glu-plasminogen (PLG) 108.3 ± 18.8 78.2 ± 28.8 A2AP/PLG [P = 0.16]  1.02 ± 0.21 1.26 ± 0.95

(160) Ongoing analysis of patients with acute kidney failure (AKI) resulted into a significant acquired plasminogen deficiency.

(161) Study-Outline-Acute Kidney Failure (AKI) Measurement of alpha-2-antiplasmin (A2AP) and Glu-plasminogen (PLG) 77 patients with acute renal failure (AKI) 53 control population (CP)

(162) Result of t-Test-Mann Whitney Analysis: A significant (**) acquired plasminogen deficiency in Patients with AKI A significant (**) acquired alpha-2-antiplasmin deficiency in Patients with AKI No significant difference of the ratio (A2AP/PLG)

(163) Conclusion:

(164) Patients with acute kidney failure (AKI) have in a high percentage an acquired plasminogen deficiency, i.e., an indication for a Glu-plasminogen substitution therapy.

(165) TABLE-US-00021 TABLE 9 Mann Whitney analysis of plasminogen concentration. Table Analyzed Data 1 Column A CP-plasminogen Vs vs Column B AKI-plasminogen Mann Whitney test P value 0.0031 Exact or approximate P value? Gaussian approximation P value summary ** Are medians signif. different? (P < 0.05) Yes One- or two-tailed P value? Two-tailed Sum of ranks in column A, B 4096, 4420 Mann-Whitney U 1417

(166) TABLE-US-00022 TABLE 10 Mann Whitney analysis of alpha-2-antiplasmin concentration. Table Analyzed Data-2 Column A CP-alpha-2-antiplasmin vs vs Column B AKI-alpha-2-antiplasmin Mann Whitney test P value 0.0011 Exact or approximate P value? Gaussian approximation P value summary ** Are medians signif. different? (P < 0.05) Yes One- or two-tailed P value? Two-tailed Sum of ranks in column A, B 4160, 4355 Mann-Whitney U 1352

(167) TABLE-US-00023 TABLE 11 Mann Whitney analysis of the ratio (A2AP/PLG). Table Analyzed Data-3 Column A CP-ratio vs vs Column B AKI-ratio Mann Whitney test P value 0.1241 Exact or approximate P value? Gaussian approximation P value summary ns Are medians signif. different? (P < 0.05) No One- or two-tailed P value? Two-tailed Sum of ranks in column A, B 3147, 5369 Mann-Whitney U 1716

Experimental Example 9.1 Indication Disseminated Intravascular Coagulation (DIC)

(168) Study-Outline-DIC:

(169) Measurement of alpha-2-antiplasmin (A2AP), Glu-plasminogen (PLG), D-Dimer 21 Patients with DIC 53 control population

(170) Result of Mann Whitney Analysis: A significant (**) acquired plasminogen deficiency in patients with DIC No acquired alpha-2-antiplasmin deficiency in patients with DIC A significant (***) difference of the ratio (A2AP/PLG)

(171) Conclusion:

(172) Patients with DIC have in a high percentage an acquired plasminogen deficiency, i.e., an indication for a Glu-plasminogen substitution therapy.

(173) TABLE-US-00024 TABLE 12 Mann Whitney analysis analysis of plasminogen concentration. Table Analyzed PLG Column A CP-plasminogen Vs vs Column B DIC-plasminogen Mann Whitney test P value 0.0013 Exact or approximate P value? Gaussian approximation P value summary ** Are medians signif. different? (P < 0.05) Yes One- or two-tailed P value? Two-tailed Sum of ranks in column A, B 2256, 519 Mann-Whitney U 288.0

(174) TABLE-US-00025 TABLE 13 Mann Whitney analysis analysis alpha-2-antiplasmin concentration. Table Analyzed A2AP Column A CP-alpha-2-antiplasmin vs vs Column B DIC-alpha-2-antiplasmin Mann Whitney test P value 0.0730 Exact or approximate P value? Gaussian approximation P value summary ns Are medians signif. different? (P < 0.05) No One- or two-tailed P value? Two-tailed Sum of ranks in column A, B 2138, 637.5 Mann-Whitney U 406.5

(175) TABLE-US-00026 TABLE 14 Mann Whitney analysis analysis of the ratio (A2AP/PLG). Table Analyzed Ratio-DIC Column A CP-ratio vs vs Column B DIC-ratio Mann Whitney test P value <0.0001 Exact or approximate P value? Gaussian approximation P value summary *** Are medians signif. different? (P < 0.05) Yes One- or two-tailed P value? Two-tailed Sum of ranks in column A, B 1634, 1141 Mann-Whitney U 203.0

Experimental Example 9.2 Indication Sepsis

(176) Study-Outline-Sepsis:

(177) Measurement of alpha-2-antiplasmin (A2AP), Glu-plasminogen (PLG), PCTP 9 Patients 53 control population

(178) Result of t-Test-Mann Whitney Analysis: A significant (*) acquired plasminogen deficiency in Patients with Sepsis No acquired Alpha-2-antiplasmin deficiency in Patients with Sepsis No significant difference of the ratio (A2AP/PLG)

(179) Conclusion:

(180) Patients with sepsis have in a high percentage an acquired plasminogen deficiency, i.e., an indication for a Glu-plasminogen substitution therapy.

(181) TABLE-US-00027 TABLE 15 Mann Whitney analysis analysis of plasminogen concentration. Table Analyzed Sepsis-PLG Column A CP-plasminogen vs vs Column B Sepsis-plasminogen Mann Whitney test P value 0..0377 Exact or approximate P value? Gaussian approximation P value summary * Are medians signif. different? (P < 0.05) Yes One- or two-tailed P value? Two-tailed Sum of ranks in column A, B 1774, 179 Mann-Whitney U 134.0    

(182) TABLE-US-00028 TABLE 16 Mann Whitney analysis analysis alpha-2-antiplasmin concentration. Table Analyzed Sepsis-A2AP Column A CP-alpha-2-antiplasmin vs vs Column B Sepsis-alpha-2-antiplasimin Mann Whitney test P value 0.0704 Exact or approximate P value? Gaussian approximation P value summary ns Are medians signif. different? (P < 0.05) No One- or two-tailed P value? Two-tailed Sum of ranks in column A, B 1761 , 192.5 Mann-Whitney U 147.5

(183) TABLE-US-00029 TABLE 17 Mann Whitney analysis analysis of the ratio (A2AP/PLG). Table Analyzed Sepsis-ratio Column A CP-ratio vs vs Column B Sepsis-ratio Mann Whitney test P value 0.2182 Exact or approximate P value? Gaussian approximation P value summary ns Are medians signif. different? (P < 0.05) No One- or two-tailed P value? Two-tailed Sum of ranks in column A, B 1608, 345.5 Mann-Whitney U 176.5

(184) Overall Conclusion:

(185) Acquired plasminogen deficiency is a highly complex disease, currently underdiagnosed due to fact that patients on risk are not tested for plasminogen and/or alpha-2-antiplasmin. The testing of these patients on risk may result in an indication for a Glu-plasminogen substitution therapy. Also, the testing of both parameters within the course of the disease should result in an indication for Glu-plasminogen.