IE903230A1 - A process for the purification of plasminogen activator inhibitor 2 (PAI) 2 - Google Patents

Abstract

Purification of plasminogen activator inhibitor 2 (I) comprises (1) pre-incubating (I)-contg. soln. with an agent (A) for splitting disulphide bridges (2) treating with a water-soluble acridine or quinoline base (pref. 2-ethoxy-6,9-diaminoacridine lactate (II)) so that the ppte. formed contains little or no (I), (3) sepn. of this ppte. and (4) recovery of (I) from the supernatant and opt. further purification by known methods. (A) is pref. dithiothreitol (DTT) used at a concn. of 10-250 (pref. 100) mM, and preincubation is for 0.25-3 (esp. 1) hr. at 10-40 (pref. 37 deg.C). The starting soln. is pref. placental extract or contains (I) made by recombinant DNA methods, and after incubation with DTT is treated with (II) at 0.2-2g/g protein in soln. at pH 5.5-8.5. The resulting supernatant (which contains 85-90% of the (I) but only 5-8% of total proteins) can be treated with 5% NaCl to salt out (II), then subjected to pptn. with (NH4) S04 (a) at 30% satn. to remove more impurities and (b) at 80% satn. to recover (I). (I) is used therapeutically to regulate the fibrinolytic system. Compared with known methods for purifying (I) (which involve 8 stages) this process is simpler and is suitable for large scale operation. The base pptes. impurities while (I) remains in soln.

Description

A process for the purification of plasminogen activator 5 inhibitor 2 (PAI-2) The invention relates to a process for the purification of plasminogen activator inhibitor 2 (PAI-2), wherein impurities are precipitated with an acridine or quinoline base.

Plasminogen activators (PA) are serine proteases which activate the fibrinolytic system. They convert the inactive proenzyme plasminogen into the active enzyme plasmin which degrades fibrin and fibrinogen. In the body there are two PA, the tissue plasminogen activator (t-PA) and the PA which is identical with urokinase (u-PA) and which is produced by renal cells but also other normal or malignant cells. The t-PA possesses particular importance in the prevention of thromboses. u-PA prevents formation of fibrin clots in the urinary tract but also contributes to the healing of wounds and has been found where there is neoplastic growth.

The concentration and activity of the PA are regulated by their synthesis but also by inhibitors. Two specific PA inhibitors (PAI) which do not inhibit plasmin or other proteases are known. One of them is produced in endothelial cells (PAI-1), the other one found in placenta (PAI2) has also been called minactivin.

Starting from an extract from placenta a 120-fold concentration of PAI-2 can be achieved according to the state of the art by an ammonium sulfate fractionation, adsorption of impurities on CM-Sephadex C-50, a gel filtration and hydroxyapatite chromatography. This process results in two forms of PAI-2 with molecular weights of 70 and 43 kDa. Immunoaffinity chromatography and a combination - 2 of immunoaffinity chromatography and FPLC have also been used for purification. A combination of 8 process steps comprising a precipitation with neutral salts, chromatographies on CM- and DEAE- RSepharose and hydroxyapatite, and a preparative electrophoresis led, starting from placenta, to a pure PAI-2 with a molecular weight of 47 kDa. It has also been known that chromatographic purification steps can be influenced advantageously by the addition of reducing agents of the dithiothreitol type.

The object of the invention is to find a simpler process which can be used on the production scale for the isolation of PAI-2.

Surprisingly it has been that impurities can be precipit15 ated from a solution of PAI-2 using 2-ethoxy-6,9-diaminoacridine lactate while the PAI-2 itself remains in solution in the supernatant.

A preincubation with dithiothreitol (DTT) proved to be advantageous.

The invention relates to a process for the purification of plasminogen activator inhibitor 2 (PAI-2), which comprises a PAI-2-containing solution being preincubated with an agent cleaving disulfide linkages, preferably dithiothreitol (DTT), and being mixed with a suitable, water-soluble acridine or quinoline base, preferably 2ethoxy-6,9-diaminoacridine lactate, in such an amount that the precipitate formed does not contain more than a little PAI-2, this precipitate being separated off, and the PAI-2 being obtained from the supernatant and, where appropriate, further purified using known processes.

PAI-2 will also precipitate if small amounts of acridine or quinoline base in relation to the amount of proteins in solution are added. An appropriate concentration of a water-soluble salt of the base, preferably 2-ethoxy-6,9IE 903230 - 3 diaminoacridine lactate, is from 200 mg to 2 g/g of protein in solution (PAI-2 remains substantially in solution). The process is carried out at a pH of 5.5-8.5.

The PAI-2-containing solution may have been pasteurized 5 beforehand, where appropriate with the addition of stabilizers, preferably glycine and/or sucrose.

The PAI-2-containing solution is preferably an extract of placenta or a solution containing genetically engineered PAI-2.

The disulfide-cleaving agent is used in a concentration of from 10 mmol/1 to 250 mmol/1, preferably 100 mmol/1. Preincubation is carried out for from 15 min to 3 hours, preferably for 1 hour, at 10 to 40°C, preferably about 37°C. 85 - 90 % of the employed PAI-2 activity, but only 5 8 % of the employed amount of protein, are present in the supernatant when the process is carried out as described.

After the precipitation the acridine base can be salted out of the supernatant, preferably using NaCl, preferably %, and the PAI-2 can be concentrated by an ammonium sulfate precipitation, for example by 80 % saturation of the solution with ammonium sulfate, which yields an additional purification in combination with a preliminary precipitation at 30 % saturation of the solution with ammonium sulfate. Since about 90 % of the employed protein are removed by the Rivanol and ammonium sulfate precipitation, the small residual amount can then be subjected to purification processes which are subject to volume limitation: for example a chromatography on DEAE30 RAffigel Blue, a chromatography gel with bifunctional affinity which is composed of diethylaminoethyl groups and RCibacron Blue F3GA dye covalently bonded to agarose, and on hydroxyapatite. Material of this degree of purity should already be suitable for therapeutic use. UltraIE 903230 - 4 purification can be carried out by hydrophobic chromatography on a phenylalanine column but is associated with substantial losses.

The PAI-2 which is obtained may additionally be pas5 teurized, if this has not been done beforehand.

The process according to the invention is described hereinafter: An amidolytic method was used in combination with the chromogenic substrate S-2444 (Glu-Gly-Arg-pNA) from Kabi for monitoring the isolation of PAI-2.

For the determination, 50 μΐ of urokinase (u-PA) (1000 U/ml) were incubated with 100 μΐ of PAI-2-containing sample for 4 min at room temperature, and 80 μΐ of this mixture were transferred into a plastic cuvette which had been prewarmed to 37’C. 50 μΐ of buffer A and 20 μΐ of S-2444 (6 mM) were then added. The increase in absorption at 4 05 nm was determined in a Cobas Bio centrifugal analyzer. The measurements were evaluated by comparison with a dilution plot which had been con20 structed via serial dilution of an in-house PAI-2 standard.

Materials which were used for the isolation of PAI-2: 2-Ethoxy-6,9-diaminoacridine lactate (6,9-diamino2-ethoxyacridine lactate): Sigma Chemie GmbH, D-6100 Darmstadt; a 2.5 % (w/v) strength solution in 12.5 mM tris, pH 6.8, was used for the fractional precipitation.

Dithiothreitol (DTT): Serva Feinbiochemika GmbH & Co., D-6900 Heidelberg - Hydroxyapatite: RBioRad, Richmond, CA, USA Phenylalanine-RSepharose and CNBr-activated - 5 RSepharose 4B: Deutsche Pharmacia GmbH, D-6900 Freiburg S-2444: Kabi Vitrum, S-11287 Stockholm, Sweden Tris (hydroxymethyl) aminomethane (tris): E. Merck, 5 D-6100 Darmstadt Urokinase (RActosolv): Behringwerke AG, D-3550 Marburg Buffer A: 50 mM tris, pH 8.4, 1 % polygeline, 100 mM NaCl, 0.01 % RTriton X 100 and 0.01 % NaN3 - Buffer B: 20 mM tris, pH 7.5, 20 mM DTT Buffer C: 20 mM Na2HPO4, pH 6.8, 20 mM DTT Buffer D: 20 mM tris, pH 7.5, 20 mM DTT, 30 % saturation with ammonium sulfate Buffer E: 20 mM tris, pH 7.5, 100 mM NaCl - Buffer F: 20 mM tris, pH 6.8 Example Frozen human placenta which had been washed free from blood was used as raw material for the preparation of PAI-2; the placenta had been chopped up in a cutter and then extracted with 0.5 % NaCl and 3 mM EDTA. The remnants of cells were removed by centrifugation and the supernatant was precipitated with 8 % Rivanol, and the precipitate was dissolved and subjected to a fractional precipitation with ammonium sulfate. The precipitate which contained the PAI-2 was dissolved in buffer F and dialyzed against it. The concentrated extract of placenta contained 4,615 U of PAI-2/ml with a specific activity of 120 U/mg. This material was used for the ultrapurification. - 6 - Rivanol and ammonium sulfate precipitation 500 ml of the starting material described above (Tab. 1) were incubated with 7.7 g of DTT (final concentration 100 mM) at 37°C for 1 hour. 1,520 ml of a 2.5 % strength Rivanol solution were added dropwise to this solution while continuously stirring cautiously, thus reaching a final concentration corresponding to 200 %. The precipitate resulting from the precipitation was removed by centrifugation, and excess Rivanol was removed from the supernatant by addition of 100 g of solid NaCl to final concentration of 5 %. 343 g of solid ammonium sulfate were then added to the supernatant until a final concentration corresponding to 30 % saturation was reached. The precipitate was removed by centrifugation (20 min, 3000 rpm), and 694 g of solid ammonium sulfate were then added to a final concentration of 80 % saturation. The precipitate resulting from this was dissolved buffer B in high concentration and dialyzed. This resulted in 80 ml of dissolved and dialyzed ammonium sulfate residue.

- DEAE-RAf figel Blue chromatography ml of the ammonium sulfate precipitate were applied to a DEAE-Affigel Blue column (17.5 x 4.4 cm, 260 ml gel bed) which had been equilibrated with buffer B. The column was eluted using an NaCl gradient in buffer B (2 x 1000 ml) which covered a range from 0 to 200 mM NaCl at a flow rate of 90 ml/h. The protein concentration was continuously measured via the OD at 280 nm and the PAI-2 activity was determined as described.

- Hydroxyapatite chromatography 472 ml of the PAI-2-containing fractions from the previous step were collected, dialyzed against buffer C and applied to a hydroxyapatite column (15 x 3.2 cm, 56 ml gel bed) (Table 1) which had been equilibrated with the same buffer. The column was eluted at a flow rate of - 7 same buffer. The column was eluted at a flow rate of 50 ml/h and using a salt gradient of sodium phosphate (0.02 to 0.3 M; 2 x 250 ml) in buffer C. The protein concentration and PAI-2 activity of the eluate were determined continuously.

- Phenylalanine-RSepharose The PAI-2-containing fractions after hydroxyapatite chromatography were collected, and 13.2 g of solid ammonium sulfate were added to 120 ml of this material to reach 30 % saturation. This solution was applied to a phenylalanine-RSepharose column which had been equilibrated with buffer D. The resin was eluted at a flow rate of 20 ml/h and using a gradient formed from buffer D and B (2 x 100 ml). The PAI-2-containing fractions were col15 lected and characterized. The purest material had a specific activity of 60,000 U (= units)/mg and migrated in SDS polyacrylamide gel electrophoresis as one band which had a molecular weight of 43 kDa and could be demonstrated in an immunoblot with a specific monoclonal antibody. rt 3 a a σ 3 3 GJ CO so 3 ω cn ET Ό 3· 1—1 w Φ H· O 3 H· 3 r+ Φ Φ 3 a 3 > CO 3 1 Ό < rt 3 3 rt 3 Φ w 3· 3 00 Φ 3 Φ 3 i-f H· 0 1 0. o 3 3 3 rt H ft X 7 3 0) dP 3 0 H· H· h 3 Φ !> Φ 3 3 1—* 3 3 0 I—* 1 1—· Q) rt 1—1 tO cn 0) l-h l-h a 3 Φ 3 H· 3 § 3 H· (P rt 0 rt 3 Φ Φ Φ1 1 I-* NJ (-* 00 Ul NJ 00 Ul o O O NJ o o o NJ • GJ σι • CJ σι Ui 00 o o UJ h-» Ι-» kO w o o o 's tQ O o 3 O 1-» Ul NJ 3 • • . • • G O NJ σ» to (-* 00 NJ —- rt O o o Ul o O H· X X X X X X H· 1—* I—1 I-* 1—· 1-· I—· o o o o o o ut ut on m a> Ul NJ 3 NJ 00 to I—4 G n <» •x ft GJ I—* I—* GJ 3* 3 3· O o Ol -J Η* iQ < o o GJ o GJ Ul 3· rt 3! Ul ui M tfc -J ffc Ό Ul GJ tO f+ H0 l-h n rt O NJ Ul I-* Ul NJ Ul >J NJ O NJ O Table 1 Purification of plasminogen activator inhibitor 2 (PAI-2) Volume Protein PAI-2 Spec. Purifica- Yield

Claims (10)

1. Patent Claims: HOE 89/B 038

1. A process for the purification of plasminogen activator inhibitor 2 (PAI-2), which comprises a PAI-2-containing solution being preincubated with an 5 agent cleaving disulfide linkages, and being mixed with a suitable water-soluble acridine or quinoline base, preferably 2-ethoxy-6,9-diaminoacridine lactate, in such an amount that the precipitate formed does not contain more than a little PAI-2, 10 this precipitate being separated off and the PAI-2 being obtained from the supernatant and, where appropriate, further purified using known processes.

2. The process as claimed in claim 1, wherein the PAI2-containing solution is an extract of placenta or 15 a solution of genetically engineered PAI-2.

3. The process as claimed in claim 1, wherein the disulfide-cleaving agent is dithiothreitol.

4. The process as claimed in claim 1, wherein the disulfide-cleaving agent is used in a concentration 20 of from 10 mmol/1 to 250 mmol/1, preferably 100 mmol/1.

5. The process as claimed in claim 1, wherein preincubation is carried out for 15 min to 3 hours, preferably for 1 hour. 25

6. The process as claimed in claim 1, wherein preincubation is carried out at 10 to 40 e C, preferably about 3 7 0 C.

7. The process as claimed in claim 1, wherein the base is used in dissolved form at a pH of 5.5 -

8.5. 30 8. The process as claimed in claim 1, wherein a PAI-2containing solution is pasteurized. - 10

9. A process according to claim 1 for the purification of plasminogen activator inhibitor 2 (PAI 2), substantially as hereinbefore described and exemplified.

10. Plasminogen activator inhibitor 2 (PAI 2), whenever obtained by a process claimed in a preceding claim.