CA2035927A1 - Process for purifying lipocortins - Google Patents

Abstract

Abstract of the disclosure:

A process for purifying lipocortins A process for purifying lipocortins, in particular the proteins rPP4, rPP4-X and rPP4-delta prepared by gene manipulation, by treatment of a solution of a lipocortin with an anion exchanger in the presence of a chelating agent and of a detergent is described.

Description

2n~27 BEHRINGWERRE ARTIENGESELLSC~AFT HOE 90/B 004 - Ma 777 Dr. Ha/Sd Description A process for purifying lipocortins The invention relates to a process for purifying lipo-cortins, especially the proteins rPP4, rPP4-X and rPP4-delta prepared by gene manipulation. These proteins have anticoagulant and antiinflammatory effects.

The natural protein PP4 is described in EP-A 0 123 307.

The preparation of PP4, of a protein PP4-X which also has anticoagulant activity, and of a deletion variant with increased activity (PP4-delta) by gene manipulation is described in DE-A 39 10 240.

These proteins can be regarded as belonging to a group of proteins which are called lipocortins (glucocorticoid-induced phospholipase-inhibiting proteins) or calpactins (calcium-dependent phospholipid- and actin-binding proteins).

The object of the invention is to provide a process for obtaining and purifying lipocortins, especially the proteins PP4, PP4-X and PP4-delta prepared by gene manipulation (rPP4, rPP4-X and rPP4-delta).

Although processes for purifying PP4 are known, they are unsatisfactory for obtaining lipocortins prepared by gene manipulation because the composition of the initial solution differs.

The invention relates to a process for purifying a lipocortin by treating a solution thereof with an anion exchanger, which comprises adding to this solution a chelating agent and a detergent and contacting this solution with the exchanger and separatin~ off the liquid.

It is then possible to wash the exchanger with a deter-gent-free buffer solution and elute the lipocortin.

However, the lipocortin is obtained from the liquid where appropriate.

The described process is particularly suitable for lipocortins prepared by gene manipulation. A lipocortin of this type is, in particular, rPP4, rPP4-X or rPP4-delta. If addition of a chelating agent to a lipocortin solution results in a precipitate, the latter is prefer-ably separated off before adding a detergent.

A lipocortin-containing solution is, in particular, an E.coli lysate as are produced in gene manipulation pro-cesses using E.coli. The chelating reagent is used in a concentration of 0.001-0.05 mol/l, preferably 0.002-0.02 mol/l. The pH is ad~usted to 6.5-9.5, preferably pH
6.8-7.5. Detergents which can be used are ionic or nonionic or zwitterionic, preferably derivatives of cholic acid and salts thereof and RTriton X-100 in a concentration of 0.1-2.0% (generally).

Ion exchangers which can be used are basic ion exchangers, preferably Q-, QAE- or DEAE-RSepharose, -cellulose, -RSephadex or -RFractogel (a copolymer of pentaerythritol, methacrylic acid derivatives and poly-ethylene glycol which carries amino groups and is cross-linked with divinylbenzene) or hydroxyapatite. It is possible to carry out adsorption from a buffer solution of 0.01-0.1 mol/l tris, HEPES or glycine at a pH of 6.5-9.5.

Whereas rPP4 and rPP4-delta are bound to the anion exchanger under the described conditions, rPP4-X is not 3 ~ ~

bound at pH 6.5-8Ø Xowever, enrichment of this protein is achieved because a large part of the contaminating proteins is adsorbed onto the exchanger.

It has also been found, surprisingly, that rPP4-X and rPP4-delta also, besides rPP4, bind to immobilized polysulfuric acid esters of saccharides or carrier-bound sulfated sugars, but only in the presence of calcium, at a conductivity of 0-10 mS and a pH of 6.5-9.5; the proteins can be eluted by increasing the ionic strength.
These proteins do not bind to these adsorbents in the absence of calcium or in the presence of chelating reagents.

rPP4, rPP4-X or rPP4-delta can be further purified by adsorption onto kaolin or RAerosil in the presence of calcium. Elution is effected by increasing the ionic strength and/or using chelating reagents.

It is also possible to use binding to calcium phosphate for further purification of these proteins. These pro-teins can be eluted by increasing the ionic strength or using chelating reagents such as EDTA or salts of citric acid or by a mixture containing these reagents.

The lipocortins can be subjected to affinity chromato-graphy for further purification. For this purpose, the lipocortin-containing solutions can be dialyzed, adsorbed in a buffer containing 0.01-0.05 mol/l, preferably 0.02 mol/l, tris-HCl, pH 8.5, after addition of calcium ions in a batch process onto carrier-bound heparin, the adsorbent can be washed, and the proteins can be eluted using a stepped gradient and subsequently dialyzed. The dialysates are mixed with 0.01-20 mmolJl, preferably 1 mmol/l, EDTA, preferably contacted once more with carrier-bound heparin, and the lipocortins dialyzed.

This affinity chromatography can generally ~e carried out on carrier-bound polysulfuric acid esters of saccharides ?f~?~27 or sulfated sugars, for example carrier-bound heparin, dextran sulfate, heparan sulfate, chondroitin sulfate, keratan sulfate or dermatan sulfate, preferably heparin-RSepharose or -RFractogel or dextran sulfate-R~epharose or -RFractogel, from a buffer solution which contains 0.01-O.1 mol/l tris, HEPES or glycine, at a pH of 6.5-9.5, preferably pH 6.8-8.5, which preferably contains 5 mmol/l calcium, and by elution of adsorbed proteins by increas-ing the ionic strength as for the ion exchanger chromato-graphy. In the absence of calcium or presence of chelat-ing reagents, rPP4, rPP4-X and rPP4-delta do not bind to these adsorbents.

Further purification can be carried out by adsorption onto RAerosil or kaolin, especially onto RAerosil 200 (from Serva) or kaolin (from Ser~a) and from E.coli fermentation media (see above) or from a buffer solution with a pH of 6.5-9.5 which contains 0.01-0.1 mol/l tris, HEPES or glycine and 1-20 mmol/l calcium. Adsorbed proteins can be eluted by increasing the ionic strength as for the ion exchanger chromatography and/or using chelating reagents, preferably 1-20 mmol/l EDTA or 0.01-0.2 mol/l of a salt of citric acid or a mixture.

In a preferred procedure, lipocortin-containing solutions from E.coli fermentation mixtures or buffer solutions composed of 0.01-0.1 mol/l tris/HCl, HEPES or glycine and a pH of 6.5-9.5 are mixed with calcium to a final concen-tration of 1-10 mmol/l, contacted with kaolin, RAerosil 200 or calcium phosphate while shaking, the liquids are separated off, the adsorbent is washed and adsorbed proteins are eluted using a chelating r~agent, preferably EDTA or salts of citric acid or a mixture. After dialysis of the eluates, further purification of the proteins can be continued by adsorption onto a heparin-resin as described above.

Adsorption and elution of the proteins on calcium phos-phate is carried out as described for adsorption onto RAerosil and kaolin, but the presence of calcium is not necessary for binding.

A particularly preferred procedure is the combination (EDTA/RTriton) DEAE exchanger, then heparin chromatography with Ca2t and then heparin chromatography with EDTA as described in Example 1.

All the process steps can be carried out at room tempera-ture. Exceptions are indicated.

The following abbreviations are used for the description:

RAerosil 200: silicon dioxide t DEAE: diethylaminoethyl EDTA: ethylenediaminetetraacetic acid HEPES: N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid Kaolin: hydrated aluminum silicate PAGE: polyacrylamide gel electrophoresis PP4: placental protein 4 Q: quaternary amino RRivanol: 6,9-diamino-2-ethoxyacridine lactate SDS: sodium dodecyl sulfate Tris: tris(hydroxymethyl)aminomethane VAC: vascular anticoagulant The in~ention is illustrated by the examples which follow:

~ample 1 a) Solubilization of phospholipid-associated proteins 1.0 1 of centrifuged E.coli lysates which contained 1.0 mg/ml rPP4 or rPP4-delta in 0.05 mol/l tris/HCl, pH
7.5, 5 mmol/l EDTA were mixed with RTriton X-100 to a final concentration of 1.0~, incubated at room tempera-ture while shaking for 10 min and then centrifuged.

- 6 ~ 3 2 7 b) Ion exchanger chromatography The supernatant after c~ntrifugation was contacted with 300 ml of DEAE-~Sepharose ("resin material~') equilibrated with 0.02 mol/l tris/HCl, pH 7.5 (column buffer 1), while stirring for one hour, the liquid was removed by filtra-tion, the adsorbent was washed with column buffer l, and the resin material was packed into a column. Adsorbed proteins were eluted with column buffer 1 which contained 0.25 mol/l NaCl.

c) Adsorption onto carrier-bound heparin (+calcium) After dialysis, the ion exchanger eluate (300 ml) was, if necessary, adjusted to a protein concentration of 0.2-3.0 mg/ml of rPP4 or rPP4-delta after addition of CaCl2 to a final concentration of 5 mmol/l, contacted with 70 ml of heparin-RFractogel equilibrated with 0.02 mol/l tris/HCl, pH 8.5, 5 mmol~l CaCl2 (column buffer 2), while stirring for one hour, the supernatant liquid was fil-tered off, the resin was washed with column buffer 2, and adsorbed proteins were eluted by increasing the ionic strength using a 0.3 molar NaCl buffer solution.

d) Heparin chromatography (+EDTA) After dialysis, the heparin eluate (100 ml) was mixed with EDTA to a final concentration of 1 mmol/l, contacted with 50 ml of heparin-resin, equilibrated with 0.02 M
tris/HCl, pH 8.5, 1 mM EDTA (column buffer 3) in a column, and the flow-through containing rPP4 or rPP4-delta was dialyzed against a solution of 0.02 M tris/HCl, pH 6.8-8.5, and then lyophilized. After the lyophilisates had been dissolved in appropriate volumes of distilled water or buffer solutions, the recovered biological activities were 95-100% of those of the materials before freeze-drying.

The purity o the rPP4-heparin flow-throughs (d) was - 7 - ~ 7 >95%. The yield was about 55% based on the starting materials and about 40% for rPP4-delta.

~xample 2 a) Solubilization of phospholipid-associated proteins 1.0 1 of centrifuged E.coli lysates in 0.02 M tris/HCl, pH 6.8, which contained 0.8 mg/l rPP4-X at a total protein concentration of about 20 mg~ml was mixed with Triton X-100 to a final concentration of 1.0~, incubated with shaking for 10 min and then centrifuged.

b) Ion exchanger chromatography The supernatant after centrifugation was contacted with 300 ml of DEAE-Sepharose, equilibrated with 0.02 M
tris/HCl, pH 6.8, with stirring, the liquid was removed by filtration, the adsorbent was washed and the resin material was packed into a column. Adsorbed proteins were eluted with a buffer solution which contained 1.5 M NaCl.

The solution containing rPP4-X which was filtered off after the incubation with the ion exchanger resin was adjusted to a pH of 8.5, mixed with 5 mmol/l CaCl2, incubated with carrier-bound heparin, the adsorbent was washed with a solution of 0.02 M tris/HCl, pH 8.5, adsorbed proteins were eluted, the eluate was dialyzed and, after addition of 1 mmol/l EDTA, again incubated with heparin-resin as described in Example 1, c. and d.

2~ It was surprisingly found that rPP4-X did not bind to the anion exchanger under these conditions. Nevertheless, a considerable purifying effect was achieved thereby because part of the E.coli proteins were adsorbed onto the resin. The yield after the heparin chromatographies was about 60% based on the starting material, i.e. about 480 mg of rPP4-X with a purity of >95~.

- 8 - ~ 7 Example 3 Adsorption onto kaolin, RAerosil 200 or calcium phosphate Centrifuged E.coli lysate solutions from 0.05 N tris~HCl, pH 7.5, which contained 1.0 mg/ml rPP4, rPP4-X or rPP4-delta, with a total protein content of about 20 mg/ml,were mixed with 5 mM CaCl2 and, in separate mixtures, incubated in each case with 25 mg of kaolin or RAerosil 200 per ml of protein solution while stirring for an hour, and the adsorbent was washed with a solution of 0.02 mol/l tris/HCl, pH 8Ø Adsorption onto calcium phosphate took place under the same conditions but the presence of calcium was unnecessary for binding. Adsorbed proteins were eluted with a solution of 0.02 M tris/HCl, pH 8.0, which contained 0.02 mol/l EDTA or a mixture of 0.01 M EDTA and 0.1 M sodium citrate.

After the eluates had been dialyzed against a buffer solution of 0.02 M tris/HCl, pH 8.5, the purification of the proteins was continued by adsorption onto carrier-bound heparin as described in Example 1, c. and d. The yields of the >95% pure proteins rPP4 and rPP4-X after the heparin chromatographies were between 50 and 60%
based on the starting materials.

The proteins rPP4 and rPP4-X purified as described in the Examples were tested for their physicochemical and biological properties. All their properties were identi-cal to those of the corresponding proteins isolated from placenta.

Claims (10)

1. A process for purifying a lipocortin by treating a solution thereof with an anion exchanger, which comprises adding to this solution a chelating agent and a detergent and contacting this solution with the exchanger and separating off the liquid.

2. The process as claimed in claim 1, wherein the exchanger is washed with a detergent-free buffer solution and the lipocortin is eluted.

3. The process as claimed in claim 1, wherein the lipo-cortin is obtained from the liquid.

4. The process as claimed in claim 1, wherein the lipo-cortin is a lipocortin prepared by gene manipulation.

5. The process as claimed in claim 1, wherein the lipo-cortin is rPP4, rPP4-X or rPP4-delta.

6. The process as claimed in claim 1, wherein the lipo-cortin-containing solution derives from an E.coli, yeast or animal cell culture.

7. The process as claimed in claim 1, wherein the lipo-cortin is further purified by chromatography on an immobilized polysulfuric acid ester of a saccharide or on a carrier-bound sulfated sugar.

8. The process as claimed in claim 1, wherein the lipo-cortin is further purified by chromatography on an immobilized polysulfuric acid ester of a saccharide or on a carrier-bound sulfated sugar in the presence of calcium ions and subsequently in the presence of EDTA.

9. The process as claimed in claim 1, wherein the lipo-cortin is further purified by adsorption onto kaolin, RAerosil or calcium phosphate.

10. The process as claimed in claim 1 and substantially as described herein.