CA1292963C - Process for the purification of plasminogen activators (pa) - Google Patents
Process for the purification of plasminogen activators (pa)Info
- Publication number
- CA1292963C CA1292963C CA000506372A CA506372A CA1292963C CA 1292963 C CA1292963 C CA 1292963C CA 000506372 A CA000506372 A CA 000506372A CA 506372 A CA506372 A CA 506372A CA 1292963 C CA1292963 C CA 1292963C
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- Prior art keywords
- plasminogen activator
- bound
- mol
- affinity material
- impurities
- Prior art date
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Abstract
BEHRINGWERKE AKTIENGESELLSCHAFT
Abstract of the disclosure A process is described for the purification of plas-minogen activator (PA), wherein a solution containing such a plasminogen activator is brought into con-tact with a carrier-bound polysulfate of a saccharide or sulfated sugar, the liquid is removed, and the PA bound by this material is eluted.
Abstract of the disclosure A process is described for the purification of plas-minogen activator (PA), wherein a solution containing such a plasminogen activator is brought into con-tact with a carrier-bound polysulfate of a saccharide or sulfated sugar, the liquid is removed, and the PA bound by this material is eluted.
Description
lZ'~Z~ 3 BEHRINGWERKE AKTIENGESELLSCHAFT 85/B 008 - Ma 514 Dr. Ha/Sd.
.
A process for the purification of plasminogen activators (PA) . ..
The ;nvent;on relates to a process for the pur;f;cation of plasminogen activators (PA). The term plasminogen activa-tor ;s ;ntended to mean proteins having urokinase and tissue plasminoaen activator tt-PA) activity as well as their ,derivatives obtained by synthetical or gentechnolo~ical processes.
.
Plasm;nogen ;s converted ;nto plasmin by plas,minogen activa-tors. Catalysts for this react;on include urokinase and t-PA. The therapeutic'use of these activators as fibrino-lytics ;s known. ~ecause of its high affinity for fibrin, t-PA is of particular significance for lysis therapy.
Methods for the ;solat;on and purif;cation of PA from cell culture supernatants and urine have already been descr;bed ~German Offenlegungsschrift 2,815,853, European Patent 0,041,766). These processes are complicated and are there-fore not suitable for industrial use.
A process for the isolation of t-PA is described ;n European Patent 0,041,766. Zinc chelate SepharoseR, concanavalin-A-AgaroseR and SephadexR G-150 (superfine) are used for this. Each of these purification steps is associated ~ith great disadvantages: zinc and concanavalin A can contami-nate the product, and SephadexR G-150 ~superfine)'cannot be used in an industrial process because of its capacity and ;ts flow propert;es. In European Patent 0,D23,869, the purification of t-PA with a carrier onto which soluble fragments of fibrin are immob;lized by covalent bonding ;s described. Th;s process is l;kewise unsuitable for industr;al ;solat;on processes.
The object of the present ;nvent;on ;s to develop a s;mple pur;fication process for PA.
.
A process for the purification of plasminogen activators (PA) . ..
The ;nvent;on relates to a process for the pur;f;cation of plasminogen activators (PA). The term plasminogen activa-tor ;s ;ntended to mean proteins having urokinase and tissue plasminoaen activator tt-PA) activity as well as their ,derivatives obtained by synthetical or gentechnolo~ical processes.
.
Plasm;nogen ;s converted ;nto plasmin by plas,minogen activa-tors. Catalysts for this react;on include urokinase and t-PA. The therapeutic'use of these activators as fibrino-lytics ;s known. ~ecause of its high affinity for fibrin, t-PA is of particular significance for lysis therapy.
Methods for the ;solat;on and purif;cation of PA from cell culture supernatants and urine have already been descr;bed ~German Offenlegungsschrift 2,815,853, European Patent 0,041,766). These processes are complicated and are there-fore not suitable for industrial use.
A process for the isolation of t-PA is described ;n European Patent 0,041,766. Zinc chelate SepharoseR, concanavalin-A-AgaroseR and SephadexR G-150 (superfine) are used for this. Each of these purification steps is associated ~ith great disadvantages: zinc and concanavalin A can contami-nate the product, and SephadexR G-150 ~superfine)'cannot be used in an industrial process because of its capacity and ;ts flow propert;es. In European Patent 0,D23,869, the purification of t-PA with a carrier onto which soluble fragments of fibrin are immob;lized by covalent bonding ;s described. Th;s process is l;kewise unsuitable for industr;al ;solat;on processes.
The object of the present ;nvent;on ;s to develop a s;mple pur;fication process for PA.
- 2 l~Z9~3 It was found, surpris;ngly, that the abovement;oned PA
exhibit a high affinity ~or polysulfates of saccharides or sulfated sugars and a purification of these PA is possible v;a th;s type of substances if the latter is bound to a carrier.
~he invention therefore relates to a process for the puri-f;cat;on of plasminocen activators , ~h;ch comprises br;nging a solution containing one of these act;vators into contact w;th a carrier-bound poly-sulfate of a saccharide or sulfated sugar ("aff;n;ty mater;al"), removal of the l;qu;d and elution of the act;vator boùnd by this material.
Plasminogen activator is to mean a protein having uroki-nase or tissue plasminogen activator (t-PA) activity as well as synthetically or gentechnologically prepared -derivatives.
The plasminogen activator is preferably of human origin.
Preferred i8 also an enbodiement of the invention in which the plasminogen activator i8 tissue plasminogen activator or a derivatlve thereof.
The plasminogen activator may preferably also be urokinase or a derivative thereof.
~ 3 ~ 129Z~3 Preferably, impurities bound by the affin;ty material are removed by washing before the elution of the act;vator.
Furthermore, a preferred process is one ;n ~h;ch the loaded aff;n;ty mater;al ;s freed from ;mpur;t;es us;ng a buffer conta;n;ng sod;um sulfate, ammonium sulfate, NaCl, LiCl or c;trate, and the activator is eluted uith a buffer solution contain;ng potassium nit~ate, ammonium chlor;de, barium chloride, potass;um bromide, calcium chloridej magnesium chloride, potassium thiocyanate, urea or a mixture of thesè -substances.
Insoluble ~garose, dextran, acrylamide or polyethylene gly-col glycidyl methacrylate polymers or a combination thereof may be used, for example, as carrier materials for a co-valent coupl;ng of polysulfates of a saccharide or sulfated sugars. Dextran or Agarose matr;ces are preterred.
~he coupling of polysulfates of sacchar;des or sulfated sugars takes place according to known methods such as, for example, by binding to carrier material preactivated with cyanogen bromide, or by bind;ng to amino-functionallzed resin by means of carbod;;m;de concdensation, but preferably by coupling to lysine-functionalized carr;er material by means of carbodi;mide condensation.
1292~3 Dextran sulfate, heparan sulfate, chondroitin sulfate, keratan sulfate, dermatan sulfate, pentosan sulfate or Arte-paronR, preferably heparin, may be used as polysulfates of a saccharide or sulfated sugars.
A further advantageous process compr;ses mixing of the solut;on conta;ning plasminogen activator with a carrier-bound polysulfate of a saccharide or sulfated sugar, preferably heparin-lysine-Sepharose, washing of the loaded affinity material with a buffer of pH 3 to 9 which, where appropriate, contains NaCl, elution of the impurities bound on the resin with a buffer of pH 3 to 9 containing sodium sulfate, ammonium sulfate, NaCl, LiCl or citrate, preferably with a 0.1 to 2 mol/l citrate solution of pH 3 to 9, preferably with 0.5 mol/l citrate pH 3.5 to 6, and elution of the plasm;nogen activator with a buffer of pH 3 to 9 containing KN03, KSCN, NH4Cl, CaCl2, MgCl2, K3r, BaCl2 or ured, preferably 1 to 2 mol/l KSCN, preferably pH S-8, containing, where appropr;ate, detergents, preferably 0.1 to 1 g/l TweenR 80 ~polyoxyethylene sorbi-tan monooleate).
In a particularly preferred embodiment, the process may be such that the solution containing PA, for example mela-noma cell culture supernatant or urine, demineral;zed where appropr;ate, is m;xed w;th heparin-, dextran sulfate-or pentosan sulfate-SepharoseR, preferably heparin-Sepha-roseR, hepar;n-lysine-SepharoseR being preferred, prefer-ably ;n a ratio of 1û l cell culture supernatant to 100 9 affinity material, the resin ;s freed from impurities using a 0.1 to 2 mol/l citrate solution, pH 3-9, preferably with 0.5 mol/l citrate, pH 3.5-6, and plasm;nogen act;vator ;s eluted with a buffer contain;ng 1-2 mol/l KSCN, pH 5-8, where appropriate containing 0.1 -1 g/l TweenR 80.
In another, particularly preferred, embod;ment the process may be such that PA is eluted with a buffer solution, conta;n;ng 1 to 2 mol/l CaCl2 with a pH of 4 to 9, prefer-12~29~3 ably ~ith a 2 mol/l CaCl2 solut;on of pH 5 to 8, contain-ing, where appropriate, D.1 to 1 g/l TweenR 80.
Th;s process ;s dist;ngu;shed by the fact that, ~ith one pur;f;cation step, PA can be obtained in a degree of pur;ty and specific activity ~hich are obtained accor-d;ng to convent;onal processes only after several puri-fication steps.
A further embodiment of the invention is a tissue plasminogen activator or a derivative thereof ob-tained ~y the disclosed process.
Still another embodiment of the invention is urokinase or a derivative thereof obtained by the disclosed process.
lZ9Z~3 , _ ~he_;nvention will_be lllustrated by_the following-ex~amp~s.
Example 1 10 l of cell culture supe;natant of melanoma cells produc-ing t-PA were mixed, with stirring, with 100 9 of a heparin-SepharoseR at room temperature. After removal of the protein liquid, the affinity material was washed w;th 0.1 mol/l tris.HCl, 0.1 mol/l NaCl, pH 7.5 containing 0.1%
TweenR 80 and subsequently freed from impur;ties w;th 0.5 mol/l citrate pH 5Ø The PA was eluted w;th a buffer conta;n;ng 0.1 mol/l tr;s.HCl, 2 mol/l KSCN and 0.û1%
TweenR 80, pH 7.5. The eluate was dialyzed, concentrated and tested for t-PA activity. Of the t-PA activity present ;n the starting solution, 99X were bound to heparin-Sepha-rose. After elut;on, approximately 90% of the t-PA activity were recovered.
. .
A polyacrylamide gel electrophoresis showed tvo prote;n ~ .
~ bands of different molecular weight. One band could be ~ -, ~ ~ immunologically assigned to t-PA.
~, Example 2 10 l of cell culture supernatant were treated in the same manner as indicated in Example 1, and subsequently eluted with a 2 mol/l CaCl2 solut;on containing 0.1 mol/l tris, pH 8.0, and 0.1X TweenR 80. 7he results were comparable ` with those ;ndicated ;n Example 1.
~, :
Example 3 %~?~3 Salts were removed from 10 l of ur;ne by d;alys;s, and subsequently, w;th st;rring; 100 9 of a heparin-Sephar-oseR were added, at room temperature. After removal of the protein l;quid, the affinity material was washed with 0.1 mol/l tris.HCl pH 7.5 and then eluted with a buffer solution conta;ning 2 mol/l KSCN, 0.1 mol/l tris.HCl, pH
7.5. 80X of the starting act;vity was bound to heparin-Sepharose. After elut;on approximately 80~ of the uro-k;nase activity were recovered.
Example 4 10 l of a cell culture supernatant of melanoma cells produc-ing t-PA were m;xed w;th 500 9 pentosan sulfate-Sepharose and processed as in Example 3. Approx;mately 80X of the activ;ty were bound on the resin.
exhibit a high affinity ~or polysulfates of saccharides or sulfated sugars and a purification of these PA is possible v;a th;s type of substances if the latter is bound to a carrier.
~he invention therefore relates to a process for the puri-f;cat;on of plasminocen activators , ~h;ch comprises br;nging a solution containing one of these act;vators into contact w;th a carrier-bound poly-sulfate of a saccharide or sulfated sugar ("aff;n;ty mater;al"), removal of the l;qu;d and elution of the act;vator boùnd by this material.
Plasminogen activator is to mean a protein having uroki-nase or tissue plasminogen activator (t-PA) activity as well as synthetically or gentechnologically prepared -derivatives.
The plasminogen activator is preferably of human origin.
Preferred i8 also an enbodiement of the invention in which the plasminogen activator i8 tissue plasminogen activator or a derivatlve thereof.
The plasminogen activator may preferably also be urokinase or a derivative thereof.
~ 3 ~ 129Z~3 Preferably, impurities bound by the affin;ty material are removed by washing before the elution of the act;vator.
Furthermore, a preferred process is one ;n ~h;ch the loaded aff;n;ty mater;al ;s freed from ;mpur;t;es us;ng a buffer conta;n;ng sod;um sulfate, ammonium sulfate, NaCl, LiCl or c;trate, and the activator is eluted uith a buffer solution contain;ng potassium nit~ate, ammonium chlor;de, barium chloride, potass;um bromide, calcium chloridej magnesium chloride, potassium thiocyanate, urea or a mixture of thesè -substances.
Insoluble ~garose, dextran, acrylamide or polyethylene gly-col glycidyl methacrylate polymers or a combination thereof may be used, for example, as carrier materials for a co-valent coupl;ng of polysulfates of a saccharide or sulfated sugars. Dextran or Agarose matr;ces are preterred.
~he coupling of polysulfates of sacchar;des or sulfated sugars takes place according to known methods such as, for example, by binding to carrier material preactivated with cyanogen bromide, or by bind;ng to amino-functionallzed resin by means of carbod;;m;de concdensation, but preferably by coupling to lysine-functionalized carr;er material by means of carbodi;mide condensation.
1292~3 Dextran sulfate, heparan sulfate, chondroitin sulfate, keratan sulfate, dermatan sulfate, pentosan sulfate or Arte-paronR, preferably heparin, may be used as polysulfates of a saccharide or sulfated sugars.
A further advantageous process compr;ses mixing of the solut;on conta;ning plasminogen activator with a carrier-bound polysulfate of a saccharide or sulfated sugar, preferably heparin-lysine-Sepharose, washing of the loaded affinity material with a buffer of pH 3 to 9 which, where appropriate, contains NaCl, elution of the impurities bound on the resin with a buffer of pH 3 to 9 containing sodium sulfate, ammonium sulfate, NaCl, LiCl or citrate, preferably with a 0.1 to 2 mol/l citrate solution of pH 3 to 9, preferably with 0.5 mol/l citrate pH 3.5 to 6, and elution of the plasm;nogen activator with a buffer of pH 3 to 9 containing KN03, KSCN, NH4Cl, CaCl2, MgCl2, K3r, BaCl2 or ured, preferably 1 to 2 mol/l KSCN, preferably pH S-8, containing, where appropr;ate, detergents, preferably 0.1 to 1 g/l TweenR 80 ~polyoxyethylene sorbi-tan monooleate).
In a particularly preferred embodiment, the process may be such that the solution containing PA, for example mela-noma cell culture supernatant or urine, demineral;zed where appropr;ate, is m;xed w;th heparin-, dextran sulfate-or pentosan sulfate-SepharoseR, preferably heparin-Sepha-roseR, hepar;n-lysine-SepharoseR being preferred, prefer-ably ;n a ratio of 1û l cell culture supernatant to 100 9 affinity material, the resin ;s freed from impurities using a 0.1 to 2 mol/l citrate solution, pH 3-9, preferably with 0.5 mol/l citrate, pH 3.5-6, and plasm;nogen act;vator ;s eluted with a buffer contain;ng 1-2 mol/l KSCN, pH 5-8, where appropriate containing 0.1 -1 g/l TweenR 80.
In another, particularly preferred, embod;ment the process may be such that PA is eluted with a buffer solution, conta;n;ng 1 to 2 mol/l CaCl2 with a pH of 4 to 9, prefer-12~29~3 ably ~ith a 2 mol/l CaCl2 solut;on of pH 5 to 8, contain-ing, where appropriate, D.1 to 1 g/l TweenR 80.
Th;s process ;s dist;ngu;shed by the fact that, ~ith one pur;f;cation step, PA can be obtained in a degree of pur;ty and specific activity ~hich are obtained accor-d;ng to convent;onal processes only after several puri-fication steps.
A further embodiment of the invention is a tissue plasminogen activator or a derivative thereof ob-tained ~y the disclosed process.
Still another embodiment of the invention is urokinase or a derivative thereof obtained by the disclosed process.
lZ9Z~3 , _ ~he_;nvention will_be lllustrated by_the following-ex~amp~s.
Example 1 10 l of cell culture supe;natant of melanoma cells produc-ing t-PA were mixed, with stirring, with 100 9 of a heparin-SepharoseR at room temperature. After removal of the protein liquid, the affinity material was washed w;th 0.1 mol/l tris.HCl, 0.1 mol/l NaCl, pH 7.5 containing 0.1%
TweenR 80 and subsequently freed from impur;ties w;th 0.5 mol/l citrate pH 5Ø The PA was eluted w;th a buffer conta;n;ng 0.1 mol/l tr;s.HCl, 2 mol/l KSCN and 0.û1%
TweenR 80, pH 7.5. The eluate was dialyzed, concentrated and tested for t-PA activity. Of the t-PA activity present ;n the starting solution, 99X were bound to heparin-Sepha-rose. After elut;on, approximately 90% of the t-PA activity were recovered.
. .
A polyacrylamide gel electrophoresis showed tvo prote;n ~ .
~ bands of different molecular weight. One band could be ~ -, ~ ~ immunologically assigned to t-PA.
~, Example 2 10 l of cell culture supernatant were treated in the same manner as indicated in Example 1, and subsequently eluted with a 2 mol/l CaCl2 solut;on containing 0.1 mol/l tris, pH 8.0, and 0.1X TweenR 80. 7he results were comparable ` with those ;ndicated ;n Example 1.
~, :
Example 3 %~?~3 Salts were removed from 10 l of ur;ne by d;alys;s, and subsequently, w;th st;rring; 100 9 of a heparin-Sephar-oseR were added, at room temperature. After removal of the protein l;quid, the affinity material was washed with 0.1 mol/l tris.HCl pH 7.5 and then eluted with a buffer solution conta;ning 2 mol/l KSCN, 0.1 mol/l tris.HCl, pH
7.5. 80X of the starting act;vity was bound to heparin-Sepharose. After elut;on approximately 80~ of the uro-k;nase activity were recovered.
Example 4 10 l of a cell culture supernatant of melanoma cells produc-ing t-PA were m;xed w;th 500 9 pentosan sulfate-Sepharose and processed as in Example 3. Approx;mately 80X of the activ;ty were bound on the resin.
Claims (13)
1. A process for the purification of a plasminogen activator (PA), which comprises bringing a solution containing urokinase, tissue plasminogen activator (t-PA) or derivatives thereof into contact with a carrier-bound polysulfate of a saccharide or sulfated sugar (affinity material), removal of the liquid, freeing the affinity material from impurities, and eluting the PA bound by this material.
2. The process as claimed in claim 1, wherein the polysulfate of a saccharide or sulfated sugar is bound to a carrier via lysine.
3. The process as claimed in claim 1, wherein the polysulfate of a saccharide is heparin.
4. The process as claimed in claim 1, wherein the carrier is agarose.
5. The process as claimed in claim 1, wherein before elution of the plasminogen activator the bound impurities are removed by washing the affinity material with a buffer solution containing NaCl, sodium sulfate, ammonium sulfate, LiCl or alkali metal citrate.
6. The process as claimed in claim 1, wherein before the elution of the plasminogen activator the bound impurities are removed by washing the affinity material with a 0.1 to 2 mol/l citrate solution, pH 3-9.
7. The process as claimed in claim 1, wherein before elution of the plasminogen activator the bound impurities are removed by washing the affinity material with a 0.4 to 0.6 mol/l citrate solution, pH 3.5-6.
8. The process as claimed in claim 1, wherein the loaded affinity material is freed from impurities with a buffer, and the plasminogen activator is eluted with a solution of potasium nitrate, ammonium chloride, calcium chloride, magnesium chloride, potassium bromide, barium chloride, urea or potassium thiocyanate or a mixture of these substances.
9. The process as claimed in claim 1, wherein the loaded affinity material is freed from impurities with a buffer, and the plasminogen activator is eluted with a buffer solution containing 0.5 to 2 mol/l KSCN, pH 4-9.
10. The process as claimed in claim 1, wherein the loaded affinity material is washed with a buffer of pH 4 to 9, which, where appropriate, contains NaCl, the bound impurities are removed by washing the affinity material with a 0.4 to 0.6 mol/l alkali metsl citrate solution, pH 3.5 - 6, and the plasminogen activator is eluted with a buffer solution containing 0.5 to 2 mol/l KSCN, pH 4-9.
11 The process as claimed in claim 5, wherein the buffer solution contains 0.1 to 1 g/l polyoxyethylene sorbitan monooleate.
12. The process as claimed in claim 6, 7 or 10 wherein the citrate solution contains 0.1 to 1 g/l polyoxyethylene sorbitan monooleate.
13. The process as claimed in claim 8, 9 or 10 wherein the solution used to elute the plasminogen activator contains 0.1 to 1 g/l polyoxyethylene sorbitan monooleate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000506372A CA1292963C (en) | 1986-04-10 | 1986-04-10 | Process for the purification of plasminogen activators (pa) |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000506372A CA1292963C (en) | 1986-04-10 | 1986-04-10 | Process for the purification of plasminogen activators (pa) |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1292963C true CA1292963C (en) | 1991-12-10 |
Family
ID=4132855
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000506372A Expired - Lifetime CA1292963C (en) | 1986-04-10 | 1986-04-10 | Process for the purification of plasminogen activators (pa) |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1292963C (en) |
-
1986
- 1986-04-10 CA CA000506372A patent/CA1292963C/en not_active Expired - Lifetime
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