阅读说明：本技术 源自血液的谷氨酸-纤溶酶原的制备和使用 (Preparation and use of glutamic acid-plasminogen from blood ) 是由 R·格鲁伯 H·R·格尔丁 M·马楚尔 S·T·基西希 H·E·卡热斯 于 2018-03-09 设计创作，主要内容包括：本发明涉及一种分离Glu-纤溶酶原的方法,所述方法对包括血浆或包含Glu-纤溶酶原的血浆级分进行阴离子色谱交换。此外,本发明还涉及用本发明的方法获得的Glu-纤溶酶原及其在治疗方面的用途。所述治疗用途是针对患有或有风险患有以下疾病的患者：器官衰竭,血栓形成事件,动脉阻塞性疾病,微循环,弥散性血管内凝血(DIC)及其中两种或更多种的组合。(The present invention relates to a method for isolating Glu-plasminogen by anion chromatographic exchange of a plasma fraction comprising or comprising Glu-plasminogen. Furthermore, the invention relates to Glu-plasminogen obtained with the method of the invention and to its use in therapy. The therapeutic use is for a patient suffering from or at risk of suffering from: organ failure, thrombotic events, arterial occlusive disease, microcirculation, Disseminated Intravascular Coagulation (DIC), and combinations of two or more thereof.)

1. A method of isolating Glu-plasminogen, the method comprising the steps of:

(i) providing plasma or a plasma fraction comprising Glu-plasminogen;

(ii) contacting the plasma or plasma fraction with an anion exchanger based on a resin comprising cationic groups;

(iii) (iii) washing the plasma or plasma fraction loaded anion exchanger obtained from step (ii) with a first buffer B1, which first buffer does not comprise cations competing with the cationic groups of the resin of the anion exchanger;

(iv) (iv) eluting Glu-plasminogen from the washed anion exchanger of step (iii) with a second buffer B2, said second buffer B2 comprising cations competing with the cationic groups of the resin of the anion exchanger, thereby obtaining a solution comprising buffer B2 and Glu-plasminogen;

(v) (iii) optionally, adjusting the pH of the solution obtained from step (iv) to within a desired range;

(vi) (viii) optionally, stabilizing Glu-plasminogen by adding to the solution obtained in any of steps (iv), (v) or (vii) one or more stabilizers which prevent the maturation of Glu-plasminogen to plasmin or Lys-plasminogen;

(vii) (vi) optionally, subjecting the solution of any one of steps (iv) to (vi) to one or more antiviral treatments; and

(viii) optionally, drying or freeze-drying the Glu-plasminogen-containing solution obtained from any of steps (iv) or (vii).

2. The method of claim 1, wherein the plasma fraction is selected from the group consisting of:

(a) low temperature lean plasma, typically obtained by freeze-thawing a supernatant of plasma;

(b) waste fractions of pastes I + II + III or I + III of the Cohn or Kistler-Nitschmann process, or combinations of two or all three of these fractions; and

(c) paste I + II + III or paste I + III of the Cohn or Kistler-Nitschmann process, or any Glu-plasminogen containing fraction or waste fraction thereof.

3. Process according to any of claims 1 or 2, wherein the resin of the anion exchanger carries an amino group or a salt thereof, preferably a primary amino group or a salt thereof, more preferably a resin having the structure-R-NH₂or-R-NH₃ ⁺+A^-Wherein R is C₁-C₁₀Alkylene residue, A^-Are anionic counterions, in particular residues in which the anion exchanger bears lysyl groups.

4. The process according to any one of claims 1 to 3, wherein the first buffer B1 and/or the second buffer B2 is an alkaline buffer having a pH of 7.1 to 11.5, preferably 8.5 to 11, in particular 10 to 11.

5. The process according to any one of claims 1 to 4, wherein the second buffer B2 comprises a soluble amine or a salt thereof, preferably primary C₁-C₁₀Primary amines or salts thereof, in particular lysine or salts thereof, as the cation which competes with the cationic group of the anion exchanger.

6. The method according to any one of claims 1 to 5, wherein the stabilizer of step (vi) is selected from aprotinin, alpha-2-antiplasmin, D-phenylalanyl-L-prolyl-arginine chloromethyl ketone, a small molecule stabilizer, and combinations thereof.

7. The method according to any one of claims 1 to 6, wherein the solution obtained in any one of steps (iv) to (vi) is subjected to an antiviral treatment of step (vii), wherein the antiviral treatment is selected from:

(vii-a) adding one or more detergents, preferably one or more detergents selected from Tween-20, Tween-80 and Triton-X-100;

(vii-b) adding one or more other antiviral agents, such as phosphate esters, in particular tri-n-butyl phosphate;

(vii-c) ultrafiltration, in particular nanofiltration; and

(vii-d) a combination of two or more of the foregoing.

8. The method according to any one of claims 1 to 7, wherein the method comprises the steps of:

(i) providing plasma or a plasma fraction comprising Glu-plasminogen;

(ii) contacting the plasma or plasma fraction with an anion exchanger based on a resin with the structure-R-NH₂or-R-NH₃+A^-Wherein R is C₁-C₁₀Alkylene radical, A^-Is an anionic counterion, particularly a lysyl residue;

(iii) (iii) washing the plasma or plasma fraction loaded anion exchanger obtained from step (ii) with a first buffer B1, said first buffer B1 having a pH of 8.5 to 11 and not comprising cations competing with the cationic groups of the resin of the anion exchanger;

(iv) (iv) eluting Glu-plasminogen from the washed anion exchanger of step (iii) with a second buffer B2, pH of said second buffer B2 being between 8.5 and 11, comprising primary C₁-C₁₀A primary amine or a salt thereof, in particular lysine or a salt thereof, and competes with the amino group of the anion exchanger, thereby obtaining a solution comprising Glu-plasminogen;

(v) (iii) adjusting the pH of the solution obtained in step (iv) to 7 to 8 or 4.5 to 5.5, in particular 7 to 8;

(vi) (viii) adding to the solution obtained from any of steps (iv), (v) or (vii) one or more stabilizers that prevent the maturation of Glu-plasminogen to plasmin or Lys-plasminogen for stabilizing Glu-plasminogen, in particular wherein said stabilizers are selected from the group consisting of aprotinin, α -2-antiplasmin, D-phenylalanyl-L-prolyl-arginine chloromethyl ketone, small molecule stabilizers and combinations thereof; and

(vii) (vi) subjecting the solution of any one of steps (iv) to (vi) to an antiviral treatment, in particular wherein the antiviral treatment is:

(vii-I) adding thereto one or more detergents, preferably one or more detergents selected from Tween-20, Tween-80 and Triton-X-100, and one or more other antiviral agents, such as phosphate esters, in particular tri-n-butyl phosphate;

(vii-II) removing the solution of step (vii-I); and

(vii-III) ultrafiltration, in particular nanofiltration; and

(viii) optionally, drying or freeze-drying the Glu-plasminogen containing solution obtained from any of steps (iv) or (vii), in particular freeze-drying.

9. The method according to any one of claims 1 to 8, wherein the method comprises the steps of:

(i) a plasma fraction comprising Glu-plasminogen selected from the group consisting of:

(a) low temperature lean plasma, which is typically obtained after freezing and then thawing of the plasma supernatant; and

(b) waste fractions of pastes I + II + III or I + III of the Cohn or Kistler-Nitschmann process, or combinations of two or three of these fractions,

in particular paste I + II + III or I + III waste fractions of the Cohn or Kistler-Nitschmann process;

(ii) contacting the plasma or plasma fraction with an anion exchanger based on a resin having the structure-R-NH₂or-R-NH₃ ⁺+A^-Wherein R is as defined above, in particular wherein R is C₁-C₁₀An alkylene radical, and A^-Is an anionic counterion, particularly a lysyl group;

(iii) (iii) washing the plasma or plasma fraction loaded anion exchanger obtained from step (ii) with 0.01 to 0.1M first buffer B1 having a pH of 8.5 to 11, wherein the first buffer B1 does not contain cations competing with the cationic groups of the resin of the anion exchanger;

(iv) (iv) eluting Glu-plasminogen from the anion exchanger washed in step (iii) with 0.01 to 0.1M second buffer B2 having a pH of 8.5 to 11, wherein the second buffer B2 comprises C₁-C₁₀A primary amine or a salt thereof, in particular lysine or a salt thereof, which competes with the amino group of the anion exchanger, thereby obtaining a solution comprising Glu-plasminogen;

(v) (iii) adjusting the pH of the solution obtained in step (iv) to within a desired range;

(vi) optionally, adding to the solution obtained in step (iv), (v) or (vii) one or more stabilizers that prevent the maturation of Glu-plasminogen to plasmin or Lys-plasminogen for stabilizing Glu-plasminogen. In particular, wherein the stabilizer is selected from the group consisting of aprotinin, alpha-2-antiplasmin, D-phenylalanyl-L-prolyl-arginine chloromethyl ketone, small molecule stabilizers, and combinations thereof; and

(vii) optionally, subjecting the solution of step (vi) to an antiviral treatment.

10. A Glu-plasminogen obtained with the method of any of claims 1 to 9.

11. A protein composition comprising at least 80 wt.% Glu-plasminogen based on total polypeptide mass, in particular wherein said Glu-plasminogen is obtainable by the method of any one of claims 1 to 9.

12. A pharmaceutical composition comprising Glu-plasminogen, preferably Glu-plasminogen of claim 10, and at least one pharmaceutically acceptable carrier.

13. Use of glutamine plasminogen for treating a patient suffering from or at risk of a disease selected from organ failure, a thrombotic event, an arterial occlusive disease, microcirculation, Disseminated Intravascular Coagulation (DIC), and combinations of two or more thereof, particularly wherein the disease is organ failure.

14. Use of Glu-plasminogen according to claim 13, wherein the patient is characterized in that:

(a) the ratio of alpha-2-antiplasmin to Glu-plasminogen in the patient's blood is at least 1.1-fold greater than the average for the same population of species; and/or

(b) Glu-plasminogen levels are at least 1% (mol/mol) lower in the patient's blood than the mean of the same species population.

15. Use of Glu-plasminogen according to any of claims 13 or 14, wherein the organ failure is or is associated with the following conditions: pathologic acute renal failure, acute transplant rejection, hypercoagulability, Disseminated Intravascular Coagulation (DIC), and thrombotic events of organs, particularly wherein the organs are selected from the group consisting of heart, lung, and vein.

16. Use of Glu-plasminogen according to any of claims 13 to 15, wherein the Glu-plasminogen is the Glu-plasminogen of claim 10 and/or forms part of a pharmaceutical composition of claim 12.

17. Use of Glu-plasminogen according to any of claims 13 to 16, wherein the risk of developing organ failure or thrombotic events is caused by acquired plasminogen deficiency.

18. Use of Glu-plasminogen according to any of claims 13 to 17, wherein the risk of developing organ failure or thrombotic events is caused by microgel disorders.

19. Use of Glu-plasminogen according to any of claims 13 to 18, wherein said patient suffers from, or is at risk of, deep vein thrombosis and/or pulmonary embolism.

20. Use of Glu-plasminogen according to any of claims 13 to 19, wherein the risk of developing organ failure or thrombotic events is caused by an increase in acquired plasmin inhibitor, in particular wherein said plasmin inhibitor is α -2-antiplasmin.

21. Use of Glu-plasminogen according to any of claims 13 to 20, wherein the disease is selected from organ failure, deep vein thrombosis, chronic or acute organ embolism, organ infarction, acute or chronic inflammation causing an imbalance of the local fibrinolytic system such as acute transplant rejection, hypercoagulation, Disseminated Intravascular Coagulation (DIC) and thrombotic events in organs, in particular the organs are selected from heart, lung and vein.

Examples

Production method of glutamine plasminogen preparation and purification process of glucose plasminogen

Glu-plasminogen can be isolated from plasma, cryo-barren plasma, fractions of the Cohn/Kistler-Nitschmann (KN) process, or optional eluents of the 4-PCC (prothrombin complex concentrate) process. The separation process can be summarized as follows:

1. providing plasma or cold plasma

2. Optionally: capture of 4-PCC complexes

3. Isolation and stabilization of Glu-plasminogen

4. Performing a first viral inactivation (solvent/detergent (SD) treatment)

5. Removal of SD

6. Final purification of Glu-plasminogen complexes

7. Performing a second viral inactivation (ultrafiltration/nanofiltration)

8. Preparation (Ultrafiltration (nanofiltration), stabilization, freezing, drying)

9. Obtaining a glutamate plasminogen product

In this process, step 2 may be further used to produce the next generation 4-PCC product. The fractionation of step 3 can be introduced into the Cohn/KN (Kistler-Nitschmann) process.

Steps 1-3 may be referred to as plasminogen capture steps. Steps 4 and 5 may be referred to as SD processing/virus removal steps. Steps 6 and 7 may be referred to as final plasminogen purification steps.

A general review of the process is here:

modern chromatographic techniques (new resin and bead structures, already used in licensed product manufacturing processes) can be used to process the disinfection standard (1M NaOH) to maximize its reusability.

Lysine-modified gels were used in the isolation of Glu-plasminogen. However, other gels containing free amino groups, such as other natural or synthetic amino acids, as well as other natural and synthetic compounds, containing free amino groups with different spacers, may also be used.

This process can be integrated into a given fractionation process, which requires little adjustment and variation to the process of processing low temperature lean plasma. The cold temperature-depleted plasma was used directly in the Cohn/KN (Kistler-Nitschmann) process without any modification to isolate IgG, albumin and other proteins.

Since activation of plasminogen is minimized using modern chromatographic and resin techniques, the capture step can achieve higher Glu-plasminogen yields. The Glu-plasminogen yields (fractional/total, weight percent) obtained were as follows:

1. blood plasma (100/100)

2. Low temperature barren plasma (90/90)

3. Isolation and stabilization of Glu-plasminogen (80/72)

4. First viral inactivation (solvent/detergent (SD) treatment) (98/71)

5. Removing SD (95/67)

Final purification of Glu-plasminogen Complex (95/64)

7. Second viral inactivation (ultra/nanofiltration) (98/62)

8. Preparation (Ultrafiltration, stabilization, freezing, drying) (90/56)

9. Obtaining a Glu-plasminogen product, overall yield: 56% by weight

After isolation, the pH of Glu-plasminogen is adjusted to 7.5 with citric acid and a stabilizer, such as aprotinin or alpha-2-antiplasmin (A2AP) is added.

Process step for the isolation of Glu-plasminogen from a cryo-poor plasma/plasma fraction

A capturing step: human plasma or cryo-depleted plasma was directly captured on lysine gels (using 9CV loading capacity) and the original Glu-plasminogen was then isolated and stabilized (see example 1.1).

SD treatment/first virus removal: to the original Glu-plasminogen was added 1% Tween-20 and 0.3% TnBP. The conditions used were: 22 ℃ for 2h with gentle shaking.

Removal of SD: SD solution (diluted 1:10 with 10mM citrate buffer pH 7.6; equilibration gel with 25mM acetate buffer pH 5.75) was injected onto anion exchanger (Fractogel M TMEA, Merck) and the SD-reagent was washed to the specified level). Glu-plasminogen was then eluted with 25mM acetate buffer (containing 0.5M NaCl, pH 5.75).

And (3) final purification step: same as the capture step.

Glu-plasminogen isolation procedure (paste I-III waste fraction from Cohn/KN Process)

Waste fraction paste I + II + III (i.e., I-III) or paste I + III during the Cohn/KN process can be used to isolate Glu-plasminogen.

1. Obtaining waste fraction pastes I + II + III pastes (i.e. I-III) or pastes I + III from the Cohn/KN (Kistler-Nitschmann) process

2. Thawing, diluting, adjusting pH, filtering

3. Isolation of Glu-plasminogen

4. Stabilization of Glu-plasminogen

5. Obtaining a Glu-plasminogen product

Here, steps 2-4 may also be referred to as Glu-plasminogen capture steps.

Experimental data

Measurement method

Detection of glutamate plasminogen product

The assay was performed as follows (data not shown): chromogenic determination of Plasminogen was carried out using the Berichrom Plasminogen product from Siemens Healthcare diagnostic (Newark, DE 19714U.S. A.).

Detection of Glu-plasminogen (TECHNOZYMGlu method)

Glu-plasminogen ELISA kit 96T (product number: TC12040 Technoclone GmbH, Austria) was used. The TECHNOZYMGlu assay is a solid-phase enzyme immunoassay for detecting Glu-plasminogen, and is used for measuring the amount of Glu-plasminogen instead of Lys-plasminogen. The assay determines Glu-plasminogen in the range of 0.06-0.5. mu.g/mL. Normal plasma levels were 60-250. mu.g/mL. The inter-and intra-batch differences were less than 10% and 5%, respectively.

The 96-well plates were pre-coated with monoclonal anti-plasminogen antibody and blocked with lyophilized 1% Bovine Serum Albumin (BSA). (TC code GX). Samples and standards (lyophilized normal plasma, (TC code BJ)) were diluted with incubation buffer (PBS; pH 7.3). Contains a stabilizer protein; 0.05% propulin (proclin); and a blue dye. The Glu-plasminogen concentrations of the standard curve were 0.5. mu.g/mL, 0.25. mu.g/mL, 0.125. mu.g/mL, 0.063. mu.g/mL, and 0.0. mu.g/mL, respectively.

Pipette 0.1mL of diluted sample/standard into individual wells. It is recommended to run the standard/sample in duplicate. Cover the plate with plastic film and incubate overnight at 4 ℃. The necessary reconstituted bars (Reconstituted strips) were obtained by adding 0.25mL of wash buffer (concentrate-prediluted 1+11.5 fold) (PBS; pH 7.3, detergent; 0.01% thiolate) to the wells and pouring the contents. The reconstituted bars were further washed four times with wash buffer. Gently tap the reconstitution strips on the absorbent paper to ensure the wells are completely dry. To all wells 0.1mL of diluted POX anti-plasminogen antibody was added, preferably using a multichannel pipettor. The lid was covered and incubated at 37 ℃ for 1 hour. Washed five times as before. Add 0.1mL TMB substrate to all wells. Incubate at room temperature for 15 minutes. Add 0.1mL stop solution to all wells. The absorbance at 450nm was measured (using a 620nm reference filter, if any). Within one hour after addition of the stop solution, the absorbance was read. A standard curve is plotted, the absorbance of each sample is found on the curve, and the corresponding value is read from the horizontal axis. This value needs to be multiplied by the dilution factor of the sample.

DS-PAGE electrophoresis

The purity of Glu-plasminogen was determined by SDS-PAGE and Coomassie staining techniques. BioRad Mini-Protean TGX non-fouling gel 4-20% (Cat No.: 456-9093), Precision Plus all blue protein standard (BioRad Cat No.: 161-0373), Glu-plasminogen standard (Coachrom Cat No.: HPGG) and Lys-plasminogen standard (Coachrom Cat No.: HPGL) were used. The protein was stained with Bio Safe TM Coomassie G-250 stain (BioRad cat # 161-. The background was decolorized with distilled water.

Determination of Total protein (Bradford method)

Quick Start^TMThe Bradford protein assay cartridge is a simple and accurate assayMethod of protein concentration in solution. The assay kit provides 1 Xconcentration of ready-to-use dye reagent (BioRad: Cat. 500-. The assay kit provides a bovine serum albumin standard set (BioRad: cat # 500-. From the samples and from pre-diluted standards (concentrations 0.125, 0.25, 0.5, 0.75, 1.0, 1.5 and 2.0mg/ml), 5. mu.L were added to the wells of a polypropylene 96-well plate (Eppendorf: batch No.: G171297G). Finally, 250 μ L of Dye Reagent was added to each well. The well contents were mixed and incubated in 96-well plates at 37 ℃ for 5 minutes (up to 60 minutes). The dynamic absorption values were measured at 595nm and 37 ℃ using a spectrophotometer. The absorbance of each sample was found on the curve and the corresponding value was read from the horizontal axis.

Molecular size distribution determination (HPLC size exclusion chromatography) of Glu-plasminogen product

The percentage of aggregates in the Glu-plasminogen formulation (as used in examples 1.4, 1.6) can be determined using the following method.

Test solutions: the sample was injected undiluted at about 190 ℃. The sample volume was 100. mu.L, and the concentration was about 1 g/L. The reference solution used Glu-plasminogen (e.g., HPPG from Coachrom). The standard solution was from Bio-Rad (gel filtration Standard, cat No. 151-1901)

A Tosoh Bioscience TSK-Gel G4000 SWXL stationary phase chromatography column (size: l 30mm, ). Buffer as mobile phase, preparation of the buffer: 4.873g of disodium hydrogen phosphate dihydrate, 1.741g of sodium dihydrogen phosphate monohydrate, 11.688g of sodium chloride and 50mg of sodium azide were dissolved in 1 liter of water. The flow rate was 0.5 mL/min.

Detection was done spectrophotometrically at 280 nm. Comparing the obtained chromatogram with a chromatogram of a reference solution. The chromatograms were integrated and peaks were identified according to the following protocol:

polymer (>1200kD), 10-13 min

High molecular weight protein (150-

Glu-plasminogen (92 kDa): 22-24 minutes

Albumin (66kD), 25-27 min

Protein fragment (<100kD), 26-40 min

Determination of proteolytic Activity

Proteolytic activity was assessed by mixing a chromogenic substrate (in particular a substrate sensitive to at least one serine protease) with a sample of the Glu-plasminogen formulation (usually diluted in a buffer to meet the linear range of the assay) at 37 ℃ and 70 ℃. Absorption kinetics were monitored using a spectrophotometer. The proteolytic activity of the sample is calculated from the initial absorption (extinction E) difference (Δ E/min) according to the formula C (U/L) ═ S x Δ E/min x F (C ═ proteolytic activity; S ═ conversion factor related to specific adsorption change of the chromogenic substrate; F ═ dilution factor). The substrate was used according to the instructions of the substrate supplier. In particular, proteolytic activity can be assessed by:

(a) 25mg of substrate S-2288(Chromogenix) was dissolved in 7.2mL of water for injection;

(b) samples of the Glu-plasminogen formulation were diluted into buffer (100mM Tris-HCl pH 8.4, 106mM NaCl) to bring the assay in the linear range and the temperature was adjusted to 37 ℃;

(c) mixing equal amounts (e.g. 100 μ l) of the diluted Glu-plasminogen formulation and dissolved substrate;

(d) absorption kinetics were measured at 405nm at 37 ℃ for 1 to 3 minutes using a spectrophotometer;

(e) the proteolytic activity of the sample was calculated from the initial absorption difference (Δ E/min) according to the formula C (U/L) ═ 313 × Δ E/min × F (C ═ proteolytic activity, F ═ dilution factor)

The limit of quantitation for this method was 8U/L, and proteolytic activity could not be detected with samples of Glu-plasminogen formulation. Thus, the level of proteolytic activity in the final product was demonstrated to be below 8U/L.