GB1585725A - Protein adsorbent and process for the production of urokinase - Google Patents

Description

(54) PROTEIN ADSORBENT AND PROCESS FOR' PRODUCTION OF UROKINASE (71) We, NIPPON SODA CO., LTD., of No. 2-1, Ohtemachi 2-chome, Chiyoda-ku, Tokyo, Japan, a company organised under the laws of Japan, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:- This invention relates to a protein adsorbent and to the production of highly pure urokinase from human urine by absorbing impurities on the adsorbent.

Urokinase is a plasminogen-activating enzyme found in trace amount in human urine and is used as an effective thrombolytic agent and a drug used together with anticancer. High purity urokinase is required because these drugs are used by an intravenous injection.

Recently, affinity chromatography has been used as a method for the purification of human urokinase. For example, the method using basic amino acids, i.e. lysine, arginine, as a ligand (Japanese Patent Publication 441953/1976, Japanese Patent Publication 20596/1976, Japanese Patent Publication (Kokai Koho ) 95183/1976, Japanese Patent Publication (Kokai Koho) 35481--35483/1976), and the method using an urokinase inhibitor contained in the tissue of the placenta as a ligand (Japan ese Patent Publication 205977/1976) are known. It is, however, difficult to obtain a large amount of those materials used as a ligand in those methods.

According to one aspect of the present in vention there is provided a protein adsorbent comprising a water insoluble carrier to which a compound of the general formula

wherein X is hydrogen, alkyl, halogen or alkoxycarbonyl, is united by a spacer of a compound of the general formula NH2 (CH2)1NHCOCH2CH2COOH, or NH2 ( CH2 )1COOH wherein 1 is an integer of from 3 to 10.

According to a second aspect of the invention there is provided a process for the purification of impure urokinase, comprising the step of adsorbing impurities by contacting an impure aqueous solution of urokinase with the above protein adsorbent.

Human urine is usually pre-treated before it is applied to the column. For example, the foam made by stirring human urine is collected and a defoaming agent is added thereto and the solution is adjusted to pH 8.5-9 to form precipitates. The resulting supernatant solution may be applied to the column. The resulting supernatant solution may be treated, namely, a weakly acidified supernatant solution is contacted with diatomaceous earth such as CELITE (Registered Trade Mark) to recover the crude urokinase in the usual way. The crude urokinase is dissolved in water and the resulting solution may be applied to the column.

A solution containing crude urokinase which is prepared as mentioned above may be purified by hydrophobic chromatography on a protein adsorbent and the partially purified urokinase solution then applied to an affinity column.

Recently, hydrophobic chromatography wherein the interaction between the hydrophobic region of a protein and the hydrophobic group such as a long chain including methylene groups and aromatic rings has been utilized as a means for the purification of enzyme by fractionation.

For protein adsorbent for the hydrophobic chromatography is the material which comprises a water insoluble carrier such as agarose and the compounds indicated by the formula (I)

wherein X is hydrogen, a halogen, an alkyl group, or an alkoxycarbonyl group.

The compound of the formula (I) is coupled to the carrier through spacers. The spacer is first coupled to the carrier, namely, the group of the formula (II) or (III) --NH( CH2)lNHCOCH2CH2COOH (11) -NH(CH2)1COOH (III) in which 1 is an integer of from 3 to 10, is bound to the carrier. The compound of the formula (I) is coupled to the modified carrier.

These protein adsorbents are constructed in a usual method similarly to the adsorbent for urokinase. As the water insoluble carrier, any material having the functional group to which the terminal amino group of the spacer can be coupled may be used. Suitable carriers are polysaccharides such as agarose, crosslinked dextran, celluloses and agar-agars, polymer such as acrylamide and glass powder. Agarose is the most preferable carrier. Agarose or modified agarose, for example agarose to which hexamethylenediamine is coupled, is sold on the market as a carrier for affinity chromatography.

When carrying out the hydrophobic chromatography, the solution containing the crude urokinase which is prepared from human urine in a usual simple method as mentioned above, is adjusted to a salt concentration of 0.2 to 2 M preferably 0.2 to 1 M. The solution is then made to flow through the hydrophobic column.

Proteins having higher affinity to the protein adsorbent than urokinase is removed by the adsorption. The effluent is then applied to the affinity column. The pH is not critical when carrying out the hydrophobic chromatography, however, the pH is usually adjusted to pH 5.510, preferably 6-8.5 at which pH urokinase is stable against inactivation.

The above method combining the affinity chromatography with hydrophobic chromatography is very advantageous for producing highly pure urokinase. In this combination the order of sequence may be reversed, it is however, preferable in the industrious process to apply the effluent of hydrophobic chromatography to the affinity chromatography.

To further illustrate this invention, and not by way of limitation, the following examples are given: Pre-treatment of human urine Fresh human urine was stirred and the resulting foam was separated. A defoaming agent was added to the foam and adjusted to pH 8.7 by adding aqueous NaOH. The resulting precipitate was removed to obtain the supernatant solution containing crude urokinase. The supernatant solution is hereinafter called "crude urokinase solution A ". After adjusting the crude urokinase solution A to pH 5.5 by adding aqueous HC1, it was contacted with diatomaceous earth to adsorb urokinase. The adsorbed urokinase was eluted with 3% aqueous solution of NH, after washing the diatomaceous earth with water to remove impurities. The eluate was ultrafiltrated by follow-fibre and lyophilized to obtain a powder of crude urokinase. The powder of the crude urokinase is hereinafter called "crude urokinase B".

Production of adsorbent for urokinase To a suspension of 20 ml AH-Sepharose (Registered Trade Mark) (product by Pharmacia Fine Chemicals) in 20 ml of water were added 2 g of succinic anhydride and the reaction was allowed to proceed at 40 C, maintaining the pH at 6 by adding aqueous NaOH.

After the change of the pH disappeared, the reaction was further continued for 5 hours. The reaction mixture was filtered and washed with 11 of water to obtain the modified Sepharose, succinyl AH Sepharose. To a suspension of the resulting succinyl AH-Sepharose in 50 ml of 40% aqueous solution of dimethyl formamide were added 470 mg of p-aminobenzamidine HCI and the pH was adjusted to 4.8. To the mixture were added 1.23 g of 1 - ethyl - 3 - (3 - dimethyl - aminopropyl)carbodiimide dissolved in 2 ml of water and the reaction was allowed to proceed at room temperature for 18 hours. During the first one hour of the reaction the pH was maintained at 4.8 by adding 1 N HCI. The reaction mixture was filtered and washed with 500 ml of 0.5 M NaCI and then 500 ml of distilled water to obtain the adsorbent for urokinase.

Example 1.

To a suspension of 100 ml of Sepharose 4B (Registered Trade Mark) (product by Pharmacia Fine Chemicals) in 100 ml of water were added 200 ml of 5% aqueous solution of CNBr with stirring. The reaction was allowed to proceed for 10 minutes at 200 C while adding NaOH aqueous solution to keep the reaction mixture at pH 11. The reaction mixture was then filtered and the activated Sepharose was washed with 1.5 1 of 0.1 M NaHCO,. The activated Sepharose was rapidly suspended in a solution of s-aminocaproic acid which was prepared by dissolving 1.31 g of e-aminocaproic acid in 100 ml of 0.1 M NaHCO3 and then adjusting to pH 9.5 and was reacted for 16 hours at 40 C. The reaction product was washed with 1 1 of 0.5 M NaCI and then 1 1 of distilled water to obtain the modified Sepharose, Sepharose.

e-aminocaproic acid. To this modified Sepharose were added an aniline solution which was prepared by dissolving 400 mg of aniline in 250 ml of 40% dimethyl formamide and adjusting to pH 4.7. To the mixture were added 3 g of 1 - ethyl - 3 (3 - dimethylamino propyl)carbodiimide HC1 dissolved in 5 ml of water and the reaction was allowed to proceed for 18 hours at room temperature.

During the first one hour of the reaction the pH was maintained at pH 4.8 by adding 1 N HCI. The reaction mixture was filtered and washed with 500 ml of 0.5 M NaCl and then 500 ml of distilled water to obtain protein adsorbent for hydrophobic chromatography.

A column of 2.5 cm diameter packed with this protein adsorbent was connected with another column of 2.5 cm diameter packed with 100 ml of the above mentioned adsorbent for urokinase. Through the hydrophobic column was passed a crude urokinase solution containing 10 million international units of urokinase which was prepared by dissolving the crude urokinase B in 500 ml of 0.2 M NaCI in 0.1 M phosphate buffer (pH 7.4). The effluent from the hydrophobic column was successively passed through the affinity column.

Then the hydrophobic column was removed and the urokinase adsorbed by the affinity column was eluted with 0.1 M acetate buffer (pH 5.0).

The fraction containing urokinase was ultrafiltrated, dialyzed and filtrated to obtain 8.56 million international units of urokinase with a specific activity of 82,700 international units/ mg protein.

Example 2.

A protein adsorbent for hydrophobic chromatography was constructed in the same way as in Example 1, using 500 mg of nbutyl-p-aminobenzoate instead of 400 mg of anline.

A crude urokinase solution containing 10 million international units of urokinase, which was prepared by dissolving the crude urokinase B in 500 ml of 0.4 MNaCl in 0.1 M phosphate buffer (pH 7.0), was treated in the same way as in Example 1 except that the eluting solution was 0.1 M acetate buffer (pH 4.5) instead of 0.1 M acetate buffer (pH 5.0). Thus, 7.8 million international units of purified urokinase with a specific activity of 95,200 international units/mg protein was obtained.

Pyrogen tests were applied to the urokfnase obtained in Examples 1 and 2 according to the Japanese pharmacopoeia. The results were negative at a dosage of 8,000 international units/p-kg.

WHAT WE CLAIM IS: 1. A protein adsorbent comprising a water insoluble carrier to which a compound of the general formula

wherein X is hydrogen, alkyl, halogen or alkoxycarbonyl, is united by a spacer of a compound of the general formula NH, (CH, )1NHCOCH,CH,COOH, or NH2(CH2),COOH wherein l is an integer of from 3 to 10.

2. A protein adsorbent as claimed in claim 1, wherein the spacer is a compound of the general formula, NH2(CH,)COOH, and X is hydrogen or alkoxycarbonyl containing 1 to 4 carbon atoms.

3. A protein absorbent as claimed in claim 2, wherein 1 is 5 and X is butoxycarbonyl.

4. A protein adsorbent substantially as described in claim 1 and as hereinbefore described.

5. A process for the purification of impure urokinase, comprising the step of adsorbing impurities by contacting an impure aqueous solution of urokinase with a protein adsorbent as claimed in any one of the preceding claims.

6. A process for the purification of impure urokinase, substantially as hereinbefore described with reference to either Example 1 or Example 2.

7. Pure urokinase produced by the process as claimed in either claim 5 or claim 6.

**WARNING** end of DESC field may overlap start of CLMS **.

Claims (7)

**WARNING** start of CLMS field may overlap end of DESC **. Example 1. To a suspension of 100 ml of Sepharose 4B (Registered Trade Mark) (product by Pharmacia Fine Chemicals) in 100 ml of water were added 200 ml of 5% aqueous solution of CNBr with stirring. The reaction was allowed to proceed for 10 minutes at 200 C while adding NaOH aqueous solution to keep the reaction mixture at pH 11. The reaction mixture was then filtered and the activated Sepharose was washed with 1.5 1 of 0.1 M NaHCO,. The activated Sepharose was rapidly suspended in a solution of s-aminocaproic acid which was prepared by dissolving 1.31 g of e-aminocaproic acid in 100 ml of 0.1 M NaHCO3 and then adjusting to pH 9.5 and was reacted for 16 hours at 40 C. The reaction product was washed with 1 1 of 0.5 M NaCI and then 1 1 of distilled water to obtain the modified Sepharose, Sepharose. e-aminocaproic acid. To this modified Sepharose were added an aniline solution which was prepared by dissolving 400 mg of aniline in 250 ml of 40% dimethyl formamide and adjusting to pH 4.7. To the mixture were added 3 g of 1 - ethyl - 3 (3 - dimethylamino propyl)carbodiimide HC1 dissolved in 5 ml of water and the reaction was allowed to proceed for 18 hours at room temperature. During the first one hour of the reaction the pH was maintained at pH 4.8 by adding 1 N HCI. The reaction mixture was filtered and washed with 500 ml of 0.5 M NaCl and then 500 ml of distilled water to obtain protein adsorbent for hydrophobic chromatography. A column of 2.5 cm diameter packed with this protein adsorbent was connected with another column of 2.5 cm diameter packed with 100 ml of the above mentioned adsorbent for urokinase. Through the hydrophobic column was passed a crude urokinase solution containing 10 million international units of urokinase which was prepared by dissolving the crude urokinase B in 500 ml of 0.2 M NaCI in 0.1 M phosphate buffer (pH 7.4). The effluent from the hydrophobic column was successively passed through the affinity column. Then the hydrophobic column was removed and the urokinase adsorbed by the affinity column was eluted with 0.1 M acetate buffer (pH 5.0). The fraction containing urokinase was ultrafiltrated, dialyzed and filtrated to obtain 8.56 million international units of urokinase with a specific activity of 82,700 international units/ mg protein. Example 2. A protein adsorbent for hydrophobic chromatography was constructed in the same way as in Example 1, using 500 mg of nbutyl-p-aminobenzoate instead of 400 mg of anline. A crude urokinase solution containing 10 million international units of urokinase, which was prepared by dissolving the crude urokinase B in 500 ml of 0.4 MNaCl in 0.1 M phosphate buffer (pH 7.0), was treated in the same way as in Example 1 except that the eluting solution was 0.1 M acetate buffer (pH 4.5) instead of 0.1 M acetate buffer (pH 5.0). Thus, 7.8 million international units of purified urokinase with a specific activity of 95,200 international units/mg protein was obtained. Pyrogen tests were applied to the urokfnase obtained in Examples 1 and 2 according to the Japanese pharmacopoeia. The results were negative at a dosage of 8,000 international units/p-kg. WHAT WE CLAIM IS:

1. A protein adsorbent comprising a water insoluble carrier to which a compound of the general formula

wherein X is hydrogen, alkyl, halogen or alkoxycarbonyl, is united by a spacer of a compound of the general formula NH, (CH, )1NHCOCH,CH,COOH, or NH2(CH2),COOH wherein l is an integer of from 3 to 10.

2. A protein adsorbent as claimed in claim 1, wherein the spacer is a compound of the general formula, NH2(CH,)COOH, and X is hydrogen or alkoxycarbonyl containing 1 to 4 carbon atoms.

3. A protein absorbent as claimed in claim 2, wherein 1 is 5 and X is butoxycarbonyl.

4. A protein adsorbent substantially as described in claim 1 and as hereinbefore described.

5. A process for the purification of impure urokinase, comprising the step of adsorbing impurities by contacting an impure aqueous solution of urokinase with a protein adsorbent as claimed in any one of the preceding claims.

6. A process for the purification of impure urokinase, substantially as hereinbefore described with reference to either Example 1 or Example 2.

7. Pure urokinase produced by the process as claimed in either claim 5 or claim 6.