IE901271L - A process for removing toxins from protein solutions - Google Patents
A process for removing toxins from protein solutionsInfo
- Publication number
- IE901271L IE901271L IE901271A IE127190A IE901271L IE 901271 L IE901271 L IE 901271L IE 901271 A IE901271 A IE 901271A IE 127190 A IE127190 A IE 127190A IE 901271 L IE901271 L IE 901271L
- Authority
- IE
- Ireland
- Prior art keywords
- protein
- acid
- detergent
- chelating agent
- proteins
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4721—Lipocortins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/18—Ion-exchange chromatography
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Analytical Chemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Toxicology (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
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
- 1A - 65260 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 5 from solutions of proteins, in particular from lipo-cortins 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 10 the amino acid sequences and belong to a family of proteins called lipocortins.
These proteins have antiinflammatory and anticoagulant effects. They have been detected in many organs and can be isolated from the latter.
Preparation from tissues, for example from human placenta, or of proteins prepared by gene manipulation, for example expressed in E. coli, such as rPP4 or rPP4-X, is associated with isolation, together with these proteins, of substances which are toxic for humans, such as bac-20 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 25 of body weight) to rabbits.
It was not possible to remove these evidently protein-associated contaminants either by chromatographic processes or filtration techniques such as sterile filtration or by the use of non-ionic detergents or of 30 chelating reagents, alone or in combination.
Hence the object of the invention was to develop processes for removing toxins deriving from organs, tissues and cell cultures in proteins of the lipocortin family which 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 in 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.
This process is especially applicable to lipocortins, which can be of natural or biotechnological origin, especially originating from gene manipulation.
Examples of chelating agents which can be used are EDTA, EGTA, a salt of citric acid or oxalic acid or a combina-20 tion of the latter.
Examples of ionic detergents which can be used are cholic acid, taurocholic acid, taurodehydrocholic acid, deoxy-cholic acid, taurodeoxycholic acid or taurochenodeoxy-cholic acid or a salt of the latter or a mixture of the 25 latter.
It is possible to use as ion exchanger an anion exchanger, preferably DEAE-aSepharose, -RSephacel, -HPractogel or Q-ESepharose, particularly preferably DEAE-^epharose.
The chelating agent and the detergent can be removed from 30 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, preferably pH 8.0-9.5, particularly preferably pH 8.0-9.0.
In one procedure, a solution of the protein which contains a buffer substance, such as tris, glycine, HEPES or 5 PBS, with a pH of 7.0-10.0, and at least 0.1 mmol/1 of a chelating reagent such as EDTA, E6TA, of a salt of citric acid or of oxalic acid or of a combination of these and at least 0.05 g/1 of an ionic detergent such as Na chol., Na Doc., Na Tdoc., Na Tchol., Tcheno-Doc or Tdcho. or of 10 a mixture of these, is brought into contact with an anion exchanger, the exchanger is washed with buffer solution, and the adsorbed protein is eluted with a salt gradient, for example using LiCl, KC1 or NaCl.
In a preferred procedure, a solution of the protein with 15 a concentration of 0.01-30 mg/ml, particularly preferably 0.2-5 mg/ml, which contains tris in a concentration of 2-80 mmol/1 and a pH of 7.0-9.5, particularly preferably 20 mmol/1 tris/HCl and a pH of 8.0-9.0, as well as 1-100 mmol/1 of a chelating reagent, particularly prefer-20 ably 5-20 mmol/1 EDTA, and 0.2-5 g/1, particularly preferably 0.8-1.5 g/1, Na chol. or Na Doc. or of a mixture, is brought into contact with DEAE-RSepharose, -RSephacel, -RFractogel, or Q-RSepharose, particularly preferably DEAE-RSepharose. After the exchanger has been 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, 30 HEPES, glycine or PBS, particularly preferably against a buffer solution of pH 8.0-9.0, or by 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 following abbreviations have been used for the descrip tion: DEAE: EDTA: 5 HEPES: Na Chol: Na Doc: Na Tchol: 10 Na Tdoc: PBS: rPP4s rPP4-X: PAP III: PAGE: Q: SDS: Tcheno-Docj Tdchol: tris: diethylaminoethyl ethylenediaminotetraacetic acid N-2-hydroxyethylpiperazine-N-2-ethane- sulfonic acid sodium cholate sodium deoxycholate sodium taurocholate sodium taurodeoxycholate sodium or potassium phosphate buffer PP4 prepared by genetically engineered expression in E. coli PP4-X prepared by genetically engineered expression in E. coli placental anticoagulant protein III polyacrylamide gel electrophoresis quaternary amine sodium dodecyl sulfate taurochenodeoxycholic acid taurodehydrocholic acid tris(hydroxymethyl)aminomethane The invention is illustrated by the examples which follow: The starting substances employed 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 30 protein content, in a buffer solution composed of 0.02 mol/1 tris/HCl, pH 8.5, with a protein concentration of 2.5 mg/ml. These preparations had an evident content of toxic substances (Table I) as was determined using the Limulus test (carried out in solutions at pH 7.2) and the 35 animal model.
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 pyrogen-free tube, and the tube was incubated at 37 °C without shaking for 60 min. After the end of the incubation time, the tube was examined visually to find whether a solid gel had formed. 10 The pyrogenicity of the tested substance, expressed in EU (endotoxin units), was determined using a calibration plot constructed with the aid of a reference endotoxin (EC-5). 2. Pyrogen test on rabbits: The toxicity of the protein samples was determined by measuring the increase in the body temperatures (rectal) of rabbits compared with the body temperature determined in a 90-minute preliminary test. Protein samples were administered i.v. in a bolus (1 mg/kg of body weight) 20 into an ear vein of the rabbits, and the body temperature 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 of the temperature differences of 6 animals was less than or equal to 2.2°C.
Example 1 After addition of EDTA to a final concentration of 0.01 mol/1, while checking the pH, and 0.1% Na Doc, the PP4-, rPP4-, PP4-X-, rPP4-X-, PAP III-, p68- or lipo-cortin I- or II-containing solutions were brought into 30 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 proteins were eluted with an NaCl gradient increasing linearly.
The eluates were extensively dialyzed against a buffer solution composed of 0.02 mol/1 tris/HCl, pH 8.5, and 5 subsequently against a buffer solution composed of 0.02 mol/1 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 in 10 the pyrogen test or scarcely measurable endotoxin contents in the Limulus test (Table I), and it was possible to assess them as pyrogen-free.
Proteins PP4-X and rPP4-X were not adsorbed onto the gel material under the said conditions and were found in the 15 column flow-through, but they were likewise pyrogen-free after the 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 20 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 lipocor-25 tin I- or II-containing buffer solutions 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/1 EDTA, the latter were brought into contact with Q-RSepharose (from Pharmacia, Sweden) equilibrated with 0.02 mol/1 PBS, pH 8.5, 30 0.01 mol/1 EDTA, 0.05 g/1 Na Chol and 0.5 g/1 Na Tchol (column buffer) in a column, the gel material was washed, and adsorbed proteins were eluted with an NaCl 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.
Table I Protein Starting material After detoxification Toxicity test Toxicity test Coagulation Yield3 Limulus Rabbits1 Limulus Rabbits inhibition2 EU/ml t(°C) EU/ml t(°C) % % PP4 * 250 6.6 ** 0.25 0.8 100 70 rPP4 * 250 .8 2.0 1.4 100 65 PP4-X 50 4.8 ** 0.25 1.0 96 80 rPP4-X * 250 9.0 2.0 1.2 95 70 p68 250 6.0 0.5 0.8 97 65 Lipocortin I 50 .4 0.5 1.4 93 70 Lipocortin II 50 4.8 0.25 1.0 98 85 Factor XIII 50 3.6 50 3.4 * 80 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%) 3 based on the amount (= 100%) of extra pure proteins employed for the detoxification
Claims (9)
1. A process for removing toxins from protein solutions, which comprises subjecting a protein in an aqueous buffer solution in the presence of a 5 chelating agent and o£ an ionic detergent to an ion exchanger chromatography.
2. The process as claimed in claim 1, wherein the protein is a lipocortin.
3. The process as claimed in claim 1, wherein EDTA, 10 EGTA, a salt of citric acid or oxalic acid or a combination of the latter is used as chelating agent.
4. The process as claimed in claim 3, wherein the chelating agent is used in a concentration of 15 1-100 mmol/1.
5. The process as claimed in claim 1, wherein cholic acid, taurocholic acid, taurodehydrocholic acid, deoxycholic acid, taurodeoxycholic acid or tauro-chenodeoxycholic acid or a salt of the latter or a 20 mixture of the latter 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/1.
7. The process as claimed in claim 5, wherein DEAE-RSepharose, -RSephacel, -RPractogel or Q-RSepharose 25 is used as ion exchanger.
8. The process as claimed in claim 1, wherein DEAE-RSepharose is used as ion exchanger. 30
9. The process as claimed in claim 1, wherein the chelating agent and detergent are removed from the protein-containing solution by dialysis or - 10 - chromatography in a buffer solution of pH 7.5-9.5. A process according to claim 1 for removing toxins from protein solutions, substantially as hereinbefore described and exemplified. A protein solution whenever obtained by a process claimed in a preceding claim. F. R. KELLY & CO., AGENTS FOR THE APPLICANTS.
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE3911629A DE3911629A1 (en) | 1989-04-10 | 1989-04-10 | METHOD FOR SEPARATING TOXINES FROM PROTEIN SOLUTIONS |
Publications (2)
Publication Number | Publication Date |
---|---|
IE901271L true IE901271L (en) | 1990-10-10 |
IE65260B1 IE65260B1 (en) | 1995-10-18 |
Family
ID=6378296
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
IE127190A IE65260B1 (en) | 1989-04-10 | 1990-04-09 | A process for removing toxins from protein solutions |
Country Status (12)
Country | Link |
---|---|
EP (1) | EP0395896B1 (en) |
JP (1) | JP2833824B2 (en) |
KR (1) | KR900015783A (en) |
AT (1) | ATE118014T1 (en) |
AU (1) | AU641916B2 (en) |
CA (1) | CA2014222C (en) |
DE (2) | DE3911629A1 (en) |
DK (1) | DK0395896T3 (en) |
ES (1) | ES2068270T3 (en) |
GR (1) | GR3015579T3 (en) |
IE (1) | IE65260B1 (en) |
PT (1) | PT93704B (en) |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB9019919D0 (en) * | 1990-09-12 | 1990-10-24 | Delta Biotechnology Ltd | Purification of proteins |
DE4003773A1 (en) * | 1990-02-08 | 1991-08-14 | Behringwerke Ag | METHOD FOR PURIFYING LIPOCORTINES |
US5063912A (en) * | 1990-07-16 | 1991-11-12 | Hughes John S | Sleep inducing device |
AUPM388494A0 (en) * | 1994-02-16 | 1994-03-10 | Csl Limited | Process for removing endotoxins |
DE19847074C1 (en) * | 1998-10-06 | 2000-07-13 | Chiron Behring Gmbh & Co | Process for the removal of lipopolysaccharides from aqueous, protein-containing solutions |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0286830A3 (en) * | 1987-03-13 | 1990-01-24 | BEHRINGWERKE Aktiengesellschaft | Process for the extraction of protein pp4 from tissues, and the use of citric acid therefor |
DE3724726A1 (en) * | 1987-07-25 | 1989-02-02 | Behringwerke Ag | METHOD FOR PURIFYING THE PLACENTARY TISSUE PROTEIN PP4 |
US4981952A (en) * | 1988-10-04 | 1991-01-01 | Eli Lilly And Company | Method for the purification of vitamin K-dependent proteins |
DE4003773A1 (en) * | 1990-02-08 | 1991-08-14 | Behringwerke Ag | METHOD FOR PURIFYING LIPOCORTINES |
-
1989
- 1989-04-10 DE DE3911629A patent/DE3911629A1/en not_active Withdrawn
-
1990
- 1990-04-03 ES ES90106334T patent/ES2068270T3/en not_active Expired - Lifetime
- 1990-04-03 EP EP90106334A patent/EP0395896B1/en not_active Expired - Lifetime
- 1990-04-03 AT AT90106334T patent/ATE118014T1/en not_active IP Right Cessation
- 1990-04-03 DK DK90106334.7T patent/DK0395896T3/en active
- 1990-04-03 DE DE59008392T patent/DE59008392D1/en not_active Expired - Fee Related
- 1990-04-09 JP JP2092355A patent/JP2833824B2/en not_active Expired - Fee Related
- 1990-04-09 IE IE127190A patent/IE65260B1/en not_active IP Right Cessation
- 1990-04-09 KR KR1019900004811A patent/KR900015783A/en not_active Application Discontinuation
- 1990-04-09 AU AU52992/90A patent/AU641916B2/en not_active Ceased
- 1990-04-09 PT PT93704A patent/PT93704B/en not_active IP Right Cessation
- 1990-04-09 CA CA002014222A patent/CA2014222C/en not_active Expired - Fee Related
-
1995
- 1995-03-30 GR GR950400756T patent/GR3015579T3/en unknown
Also Published As
Publication number | Publication date |
---|---|
ES2068270T3 (en) | 1995-04-16 |
DK0395896T3 (en) | 1995-06-26 |
DE59008392D1 (en) | 1995-03-16 |
PT93704A (en) | 1990-11-20 |
EP0395896B1 (en) | 1995-02-01 |
PT93704B (en) | 1997-12-31 |
DE3911629A1 (en) | 1990-10-11 |
EP0395896A2 (en) | 1990-11-07 |
AU641916B2 (en) | 1993-10-07 |
GR3015579T3 (en) | 1995-06-30 |
KR900015783A (en) | 1990-11-10 |
IE65260B1 (en) | 1995-10-18 |
AU5299290A (en) | 1990-10-11 |
JP2833824B2 (en) | 1998-12-09 |
CA2014222C (en) | 2000-05-09 |
EP0395896A3 (en) | 1991-07-17 |
ATE118014T1 (en) | 1995-02-15 |
JPH02290899A (en) | 1990-11-30 |
CA2014222A1 (en) | 1990-10-10 |
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MM4A | Patent lapsed |