AU641916B2 - A process for removing toxins from protein solutions - Google Patents

Abstract

A process for separating toxins from solutions of proteins is described and entails an aqueous solution of a protein which contains a buffer substance, a chelating agent and a detergent being subjected to ion exchange chromatography.

Description

COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952-69 COMPL ETE SPECIFICAT CN (OR IGINA L) Class In Form it. Class Application Nu!Mber: Lodged: Complete Spefifilcation Lodged, Accepted Pul~lished: Priority .$eRelated Art Nam~?of Applicant: BEHRINGWERKE AKtIENG9SEI;LSCHAFT :Address of Applicant: D-3550 Marburg, Federal Republic of Germany e: acta Inetr JURGEN RCIAISCIH and NORBERT HEIMBURGER Address for Service: WATERMARK PATENT TRADEMARK ATTORNEYS.

LOCKED BAG NO. 5, HAWTHORN, VICTORIA 3122, AUSTRALIA Complete Specification for the invention entitled:' A PROCESS tIOR REM4OVING TIOXINS FROM4 PROTEIN SOLUTIONS The following statement is a full descrintfon of this -ivention, including the best method of performing it known to BEHRINGWERKE AKTIENGESELLSCHAFT HOE 89/B 012 Ma 745 Dr, Ha/Sd A process for removing toxins from protein solutions The invention relates to a process for removing of toxins from solutions of proteins, in particular from lipocortins PP4, PP4-X (PAP II), PAP III, p68 and lipocortins I and II.

Placental tissue protein PP4, proteins PP4-X, PAP III and p68, as well as lipocortins I and II, display homology in the amino acid sequences and belong to a family of proteins called lipocortins.

These proteins have antiinflampatory and anticoagulant effects. They have been detected in many organs and can o be isolated from the latter.

0 Preparation from tissues, for example from human placenta, or of proteins prepared by gene manipulation, for example expressed in E. co1-i such as rPP4 or rPP4-X, is associated with isolation, teter with the proteins, of substances which are toxi for humans, such as bac- 0 terial lipopolysaccharides. Despite high purity (greater than 95% based on the protein content), the isolated proteins showed heavy contamination with toxic substances in toxicity tests such as the Limulus test or after administration of therapeutic doses (1 mg of protein/kg of body weig) to rabbits.

It was not ossible to remove these evidently proteinassociated contaminants either by chromatographic processes or filtration techniques such as sterile filtration or by the use of non-ionic detergents or of chelating reagents, alone or in combination.

Hence the object of the invention was to develop processes f6r removing toxins deriving from organs, tissues and cell cultures in proteins of the lipocortin family which It -2 have been isolated as well as prepared by-gene manipulation, which do not impair the biological activity of the proteins and thus permit potential use as therapeutics for coagulation and/or inflammatory disorders.

It has been found, surprisingly, that toxic substances can be removed from these proteins by ion exchange chromatography iti the presence of chelating reagents in combination with ionic detergents,, without an adverse effect on the biological activity.

Thus the invention relates to a process for removing toxins from a protein solution, which comprises suBjecting a protein in an aqueous buffer solution in the presence of a chelating agent and of an ionic detergent to an ion exchange chromatography.

0 15 This process is especially applicable to lipocortins, which can be of natural or biotechnological origin, especially originating from gene manipulation.

seee Examples of chelating agents which can be used are EDTA, EGTA, a salt of citric acid or oxalic acid or a combination of the latter.

Go: Examples ofzionic detergents which can be used are cholic s. acid, taurocholic acid, taurodehydrocholic acid, deoxycholic acid, taurodeoxycholic acid or taurochenodeoxycholic acid or a salt of the latter or a mixture of the latter.

It is possible to use as ion exchanger an anion exchanger, preferably DEAE-RSepharose, -RSephacel, -RFractogel or Q- Sepharose, particularly preferably DEAE-_Sepharose.

The chelating agent and the detergent can be removed from the protein-containing solution after the treatment according to the invention by dialysis or by chromatography in a buffer solution of pH 7.4-9.5, 1 -3preferably pH 8.0-9.5, particularly preferably pH In one pcocedure, a solution of the protein which contains a buffer substance, such as tris, glycine, HEPES or PBS, with a pH of 7.0-10.0, and at least 0.1 mmol/l of a chelating reagent such as EDTA, EGTA, of a salt of citric acid or of oxalic acid or of a combination of these and w at least 0.05 g/l of an ionic detergent such as Na cho7., Na Doc., Na Tdoc., Na Tchol., Tcheno-Doc or Tdcho. Pr of a mixture of these, is brought into contact with an anion exchanger, the exchanger is washed with buffer solution, S. and the adsorbed protein is eluted with a salt gradient, for example using LiCI, KCI or NaCl.

t G. In a preferred procedure, a solution of the protein with a concentration of 0.01-30 mg/ml, particularly preferably 0.2-5 mg/ml, which contains tris in a concentration of 2mmol/l and a pH of 7.0-9.5, particularly preferably mmol/1 tris/HCl and a pH of 8.0-9.0, as well as 1- 100 mmol/l of a chelating reagent, particularly preferably 5-20 mmol/l EDTA, and 44-/ g/1, particularly preferably 0.8-1.5 g/l, Na chol. or Na Doc. or of a mixture, is brought into contact with DEAE-RSepharose, -'Sephacel, -RFractogel, or Q-RSepharose, particularly preferably DEAE-RSepharose. After the exchanger has been 0.•25 washed with buffer solution, the adsorbed protein is **eluted with a linear increasing NaCl gradient.

Chelating reagents and detergents can be removed from the protein-containing column flow-throughs or eluates by dialysis against a buffer solution composed of tris, HEPES, glycine or PBS, particularly preferably against a buffar solution of pH 8.0-9.0, orby a further chromatographic step such as gel permeation chromatography with AcA 202 or AcA 54.

It is possible, where appropriate, for the preparations treated in this way to be further purified. The A*STa V following abbreviations have been used for the description: DEAE: diethylaminoethyl EDTA: ethylenediaminotetraacetic acid EGTA: ethyleneglycol-(-aminoethylether)-N-N'-tetraacetic acid HEPES: N-2-hydroxyethylpiperazine-N-2-ethane-sulfonic acid Na Chol: sodium cholate Na Doc: sodium deoxycholate Na Tchol: sodium taurocholate Na Tdoc: sodium taurodeoxycholate PBS: sodium or potassium phosphate buffer ZrPP4: PP4 prepared by genetically engineered expression in E. coli rPP4-X: PP4-X prepared by genetically engineered expression in E. coli PAP m: placental anticoagulant protein III PAGE: polyacrylamide gel electrophoresis Q: quaternary amine SDS: sodium dodecyl sulfate Tcheno-Doc: taurochenodeoxycholic acid Tdchol: taurodehydrocholic acid Tris: tris (hydroxymethyl) aminomethane 20 The invention is illustrated by the examples which follow: The starting substances eimployed for the detoxification were preparations of the proteins PP4, PP4-X, PAP III, p68, lipocortins I and II from human placenta and of proteins rPP4 and rPP4-X from transformed E. coli cultures with a purity of greater than 95% based on the protein content, in a 25 buffer solution composed of 0.02 moill triS/HCI, pH 8.5, with a protein concentration of 2.5 mg/mi. These preparations had an evident content of toxic substances (Table I) as was determined using the Limulus test (carried out in solution at pH 7.2) and the animal model.

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5 Toxicity tests 1. Limulus test This test was carried out as described by Concept GmbH (Heidelberg, Germany): 0.1 ml of the protein-containing solution to be tested was gently mixed with 0.1 ml of Limulus amebocyte lysate in a frogen-free tube, and the /jiabe was incubated at 37"C without shaking for 60 min.

After the end of the incubation time, the tube was excamined visually to find whether a solid gel had formed.

The pyrogenicity of the tested substance, expressed in EU *I (endotoxin units), was determined using a calibration plot constructed with the aid of a reference endotoxin k 2. Pyrogen test on rabbits: The toxicity of the protein samples was determined by measuring the increase in the body temperatures (rectal) see of rabbits compared with the body temperature determined 6 in a 90-minute preliminary test. Protein samples were administered i.v. in a bolus (1 mg/kg of body weight) into an ear vein of the rabbits, and the body temperature 00"" was recorded for a period of 180 min. The highest value was used as basis for the evaluation. Samples were assessed as pyrogen-free if the total f the temperature differences of 6 animals was less than equl to 2.2"C.

215 Example 1 A ter addition of EDTA to a final concentration of 0. 4 mol/l, while chedking the pH, and 0.1% Na Doc, the PP4-, rPP4-, PP4-X-, rPP4-X-, PAP III-, p68- or lipocortin I- or II-containing solutions were brought into contact with DEAE-RSepharose (from Pharmacia, Sweden) equilibrated with 0.02 M tris/HCl, pH 8.5, 0.01 M EDTA and 0.1% Na Doc. (column buffer) in a column, the gel material was washed with column buffer, and adsorbed 6 proteins were eluted with an NaCI gradient increasing linearly.

The eluates were extensively dialyzed against a buffer solution composed of 0,2 mol/1 tris/HCl, pH 8.5, and subsequently against ,a buffer solution composed of 0.02 mol/l tris/HCl, pH 7.2, and the dialyzates were examined for toxicity both in the Limulus test and in the pyrogen test on rabbits. The proteins treated in this way caused only very low or no increases in temperature i n the pyrogen test or scarcely measurable endotoxin contents in the Limulus test (Table and it was possible to assess them as pytpgen-free.

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06 0005 Proteins PP4-X and rPP4-X were not adsorbed onto the gel material under the said conditions and were found in the column flow-through, but they were likewise pyrogen-free after th1"stated process had been carried out (Table I).

The biological activity, examined using the modified.

prothrombin time, based on the protein concentration, was fully retained by comparison with the starting materials through this process step. The yields of the proteins were between 64 and 85% based on the toxic starting materials.

Example 2 PP4-, rPP4-, PP4-X-, rPP4-X-, PAP III-, p68- or lipocortin I- or II-containing bufferesolutions were mixed with Na Chol or Na Tchol to a final concentration of 0.5 g/1 in each case, as well as 0.01 mol/l EDTA, the latter were brought into contact with Q-tSepharose (from Pharmacia, SSweden) equilibrated with 0.02 mol/l PBS, pH 0.01 iol/l EDTA, 0.05 g/l Na Chol and 0.5 g/l Na Tchol (colunbuffer) in a column, the gel material was washed, and adsorbed proteins were eluted with an NaCI gradient increasingly linearly.

The procedures for further treatment of the protein solutions and examination thereof for toxicity were as described in Example 1. The results of these investigations corresponded to those for Example 1 and are listed in Table I.

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C *C C C*C *C *.C ~&2~k C C S S Y 5 6 Sb enS S S C Ce St C S C S S S *S CC 555 Table I Protein Starting material Toxicity test, Limualus Rabbits' EU/mi t( 0

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After detoxification Toxicity test Coagulation Limualus Rabbits inhibition 2 EU/mi t(-C) Yield' PP4 rPP4 PP4-X rPP4-X p68 Lipocortin I Lipocortin II Factor XIII 250 250 50 250 250 50 50 50 6.6 10.8 4.8 9.0 6.0 5.4 4.8 3.6 **0.25 2.0 **0.25 2.0 0.5 0.5 0.25 50 0.8 1.4 1.0 1.2 0.8 1.4 1.0 3.4 100 100 96 97 93 98 greater than less than 1 temperature difference (see text); total for 6 rabbits 2 based on the coagulation inhibition (modified thromboplastin test),by the starting materials 100%) 3based on the amount extra pure proteins employed for the detoxification

Claims (9)

1. A process for removing toxins from lipocortin solutions, which comprises subjecting a lipocortin in an aqueous buffer solution in the presence of a chelating agent and of an ionic detergent to an ion exchange chromatography.

2. A process as claimed in claim 1, wherein the lipocortin is PP4, PP4-X, PAP II, p68, lipocortins I and I, rPP4 or rPP4-X.

3. The process as claimed in claim 1, wherein EDTA, EGTA, a salt of citric acid or oxalic acid or a combination thereof is used as chelating agent.

4. The process as claimed in claim 3, wherein the chelating agent is used in a concentration of 1-100 mmol/l.

The process as claimed in claim 1, wherein cholic acid, taurocholic acid, taurodehydrocholic acid, deoxycholic acid, taurodeoxycholic acid or taurochenodeoxy- cholic acid or a salt of the latter or a mixture of thereof is used as detergent.

6. The process as claimed in claim 5, wherein the detergent is used in a concentration of 0.02-5 g/l.

7. The process as claimed in claim 5, wherein DEAE-RSepharose, "Sephacel, -RFractogel or Q-RSepharose is used as ion exchanger.

8. The process as claimed in claim 1, wherein DEAE-RSepharose is used as an ion exchanger. i

9. The process as claimed in claim 1, wherein the chelating agent and detergent are removed from the protein-containing solution by dialysis or S chromatography in a buffer solution of pH 7.5-9.5. DATED this 21st day of July, 1993. BEHRINGWERKE AKTIENGESELLSCHAFT WATERMARK PATENT TRADEMARK ATTORNEYS THE ATRIUM 290 BURWOOD ROAD HAWTHORN VICTORIA 3122 AUSTRALIA VAX doc 037 AU5299290.WPC 9