GB1587932A - Process for purifying fortimicin a - Google Patents

Description

(54) PROCESS FOR PURIFYING FORTIMICIN A (71) We, ABBOT LABORATORIES, a Corporation organized and existing under the Laws of the State of Illinois, United States of America, of 14th Street and Sheridan Road, North Chicago, County of Lake, State of Illinois, United States of America, 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 process for chromatographically isolating Fortimicin A from a solution containing Fortimicin A and co-produced metabolic compounds analogous to Fortimicin A such as Fortimicin B and Forticimin C using a cationic ion-exchange resin.

Fortimicin A is a water-soluble, basic antibiotic produced by bacteria of the genus Micromonospora, e.g. Micromonospora olivoasterospora MK70 (ATCC 21819) Micromonospora olivoasterospora (ATCC 31009), and Micromonospora olivoasterospora (ATCC 31010), showing a strong antibacterial activity against various Gram-positive and Gram-negative strains. Fortimicin A and the microbiological process for producing same utilizing the aforementioned micro-organisms is described in British Patent Specification No. 1 473 356. Forticimin A is characterised by a molecular weight of 405, a molecular formula of C17 H35 N5 06, an optical rotation [a225 of +26 (C = 0.2, H20) and ultraviolet and infrared absorption spectra essentially as shown in the Drawings accompanying Specification No. 1 473 356. The chemical structure of Fortimicin A is also set forth in that Patent as follows:

It has been found, and subsequently reported, that the culture liquor of known Fortimicin A-producing strains belonging to the genus Micromonospora contains coproduced compounds analogous to Fortimicin A such as Fortimicin B and Fortimicin C, in addition to Fortimicin A. Due to the presence of these factors in the culture liquor, the separation of Fortimicin A therefrom has heretofore been difficult.

As a result of various investigations on processes for separating Fortimicin A from other Fortimicins, it has been discovered that Fortimicin A can be more readily isolated and recovered in good yield by desalting a filtrate of the culture liquor containing Fortimicin A and other Fortimicins. passing the resultant desalted solution through a column of an ion-exchange resin such as a strongly acidic cation-exchange resin having a low cross-linking degree, as herein defined. a macroreticular type strongly acidic cation-exchange resin or a weakly acidic cation-exchange resin, all of which are to have comparatively small-sized resin particles, to absorb Fortimicin A and other Fortimicins on the resin and thereafter conducting a chromatographic separation using a basic eluent, whereby Fortimicin A is eluted out in substantially purified form.

Therefore according to the invention there is provided a process for separating Fortimicin A substantially free of co-produced substances from a solution containing the Fortimicin A and co-produced substances, which comprises passing the solution through an acidic cation-exchange resin which is a strongly acidic cation-exchange resin having a cross-linking degree, as herein defined of 8 percent or less, a macroreticular type strongly acidic cation-exchange resin having a cross-linking degree, as herein defined, of 16 percent or more or a weakly acidic cation-exchange resin which weakly acidic cation exchange resin passes through a 100 mesh (Tyler Standard Sieve) and passing a basic eluent through the cation-exchange resin to elute out the Fortimicin A in eluate fractions containing Fortimicin A.

As used herein the term "cross-linking degree" expressed as a percentage is the percentage by weight of the crosslinking monomer to the remaining monomer.

In one embodiment of the present invention, the eluate fractions containing Fortimicin A are combined and the Fortimicin A is separated from them.

In another embodiment of the present invention, the Fortimicin A is separated from the eluate fractions by precipitation with an organic solvent. Fortimicin A is preferably isolated and recovered from a solution or culture liquor containing Fortimicin A and other co-produced metabolic products such as Fortimicin B and Fortimicin C by passing the solution or culture liquor through a column of an ion-exchange resin such as a strongly acidic cation-exchange resin having a low cross-linking degree, a macroreticular type strongly acidic cation-exchange resin or a weakly acidic cation-exchange resin, all of which are to have comparatively small-sized resin particles whereby Fortimicin A and other Fortimicins are adsorbed on the resin; and then conducting a chromatographic separation using an eluent.

When the solution or culture liquor contains salts such as Na2SO4, (NH4)S04, NaCI and NH4Cl, the efficiency of the chromatographic separation tends to be lowered as the concentration of the salts in the solution increases. Therefore, it is preferable to desalt the solution before the chromatography. For example, the culture liquor is treated with an acid in order to dissolve a small quantity of Fortimicin A adsorbed on the bacteria into the liquid phase. This acid treatment is conducted by adjusting the pH of the culture liquor to 1-3 with sulfuric acid, hydrochloric acid or phosphoric acid, and stirring the liquor at room temperature for about 1 hour. After stirring, a filtering aid such as Radiolite #600 (product of Showa Chemical Industry Co., Ltd.) is added to the liquor, and the liquor is then filtered using a filter press to obtain a filtrate. The filtrate is adjusted to pH 7 with alkali (NaOH, NH40H or KOH) and passed through a column of a weakly acidic cation-exchange resin such as Amberlite IRC-50 (NH+ type) (product of Rohm & Haas Co. The word "Amberlite" is a Registered Trade Mark) to adsorb the Fortimicins including Fortimicin A on the resin. The resin is then washed thoroughly with water and the Fortimicins are subsequently eluted out with O.5N - 2.()N aqueous ammonia. Ammonia in the eluate is removed by concentrating under reduced pressure, and the pH of the concentrate is adjusted to 5 - 9 with sulfuric acid. The thus obtained desalted solution is ready for chromatographic separation.

The cation-exchange resins suitable for the chromatographic separation of the solution containing Fortimicin A and other Fortimicins are limited to three types. Moreover, resin particle size greatly influences the efficiency of chromatographic separation. That is, the smaller the particle size. the better the separating efficiency. Cation-exchange resins having a particle size passing through 3() mesh (by Tyler standard sieve) are preferred.

Cation-exchange resins having a particle size of 30 mesh to 400 mesh are especially preferred. As used herein. 30 mesh to 400 mesh means a particle size which will pass through a 30 mesh sieve but not through a 400 mesh sieve.

Specific preferable examples of the three types of cation-exchange resins suitable for the present invention are as follows. The first type is a strongly acidic cation-exchange resin having a cross-linking degree of 8% or less. especially 8 to 0.5%, including Dowex 50W (X2. X4, X6) (trade name of the Dow Chemical Co. - The word "Dowex" is a Registered Trade Mark). Diaion SK (1()2. 101. 104, 106), Diaion PK (2()4, 206. 2()8. 2l2) (trade name of Mitsubishi Kasci Kogyo, Co.. Ltd.. Japan) and Duolite C-l0 (trade name of Chemical Process Co. - The word "Duolite" is a Registered Trade Mark). The second type is a macroreticular type strongly acidic cation-exchange resin having a cross-linking degree of 16% or more, especially 16 to 50C/c. including Diaion HPK (16. 25. 30, 40), Amberlyst 15 and Amberlyst XN-10()4 (trade names of Rohm & Haas Co. - The word "Amberlyst" is a Registered Trade Mark. The third type is a weakly acidic cation-exchange resin having a small resin particle size (100 mesh - 400 mesh). As for these three types of cation-exchange resins, those which are H+ type, Na+ type, K+ type or NH4+ type, are useable. Those of NH4+ type are preferably used. Preferred is Amberlite CG 50 (trade name of Rohm & Haas Co.).

Elution of Fortimicin A and other Fortimicins adsorbed on one of the above-described three types of cation-exchange resins is conducted as follows. When elution is conducted with a basic eluent having a suitable pH and concentration using the respective resins, Fortimicin B, Fortimicin C, etc. are eluted first, then Fortimicin A. Thus Fortimicin A can be chromatographically separated from the other Fortimicins. Suitable eluents may be selected from sodium hydroxide, potassium hydroxide, aqueous ammonia, ammonium phosphate, ammonium acetate, ammonium chloride and sodium chloride.

The eluent may be removed from the Fortimicin A-containing fraction by concentration under reduced pressure or by ion-exchange separation. The pH of the Fortimicin A-containing solution may then be adjusted to 2.0 - 4.5 with sulfuric acid, and an organic solvent such as ethanol may be added thereto to precipitate the sulfate form of Fortimicin A. The precipitate is separated by filtration and dried to obtain a powdery product.

Certain specific embodiments of the present invention are illustrated by the following representative examples.

Example I In this example, Micromonospora olivoasterospora MK 70 (ATCC 21819) (Ferm-P No.

1560) is seed cultured at 300C for 3 days in a medium containing 2 g/dl of glucose, 0.5 g/dl of peptone 0.5 g/dl of yeast extract and 0.1 g/dl of calcium carbonate (pH: 7.5 before sterilization). The thus obtained seed culture is then cultured at 370C for 4 days in a culture medium containing 4 g/dl of soluble starch, 2 g/dl of soybean meal, 1 g/dl of corn steep liquor, 0.05 g/dl of K2HPO4, 0.05 g/dl of MgSO4.7H2O, 0.03 g/dl of KCI and 0.01 g/dl of CaCO3. In the culture liquor 45 y/ml of Fortimicin A is accumulated as well as 20 y/ml of Fortimicin B and 15 y/ml of Fortimicin C.

The culture liquor (300 1), is adjusted to pH 1.5 with sulfuric acid, and stirred for 1 hour to dissolve a small quantity of the Fortimicins which are adsorbed on the bacteria. After stirring, the liquor is filtered using a filter press. The filtrate is then adjusted to pH 7 with aqueous ammonia and passed through a column of 5 L. of Amberlite IRC-50 (NH4 type).

After washing the resin with water, elution with 1N aqueous ammonia is carried out. The eluate is then concentrated under reduced pressure to remove the ammonia, and the concentrate is adjusted to pH 7 with sulfuric acid.

This pH-adjusted solution is then passed through a column of 5 L. of 100-150 mesh strongly acidic cation-exchange resin, Diaion SK 102 (NR+4 type), having a cross-linking degree of 2%, to adsorb all the Fortimicins on the resin.

Thereafter, 0.3 N aqueous ammonia is passed through the column at S.V. 2/hour to conduct an elution and the eluate is taken in 5 L. portions. Other active substances such as Fortimicin B and Fortimicin C are contained in the solution obtained by combining Fraction Nos. 1 - 10. Fortimicin A is contained in the solution obtained by combining Fraction Nos.

11 - 20 in about 50% yield. (This yield is that of the process from the eluate of the treatment with Aniberlite IRC-50 to the solution obtained by combining Fraction Nos. 11 - 20 of the treatment with Diaion SK 102.) The solution containing Fortimicin A is adjusted to pH 7 with sulfuric acid and passed through a column of 1 L. of Amberlite IRC 50 (NH4 type) to adsorb Fortimicin A on the resin. The resin is then washed with water and elution is conducted with IN aqueous ammonia. The thus obtained eluate is concentrated under reduced pressure to remove ammonia. The resultant concentrate is adjusted to pH 4.5 with sulfuric acid, and added to a 15- fold amount of ethyl alcohol to obtain a precipitate. This precipitate is separated and dried to obtain 10.3 g of the sulfate of Fortimicin A (molar ratio of sulphuric acid to Fortimicin A is 2; this is the same in the subsequent Examples) in powder form with a purity of 95%.

Example 2 In this example, 500 L. of a culture liquor is obtained in the same manner as in Example 1 containing 38 y/ml of Fortimicin A, and 7 y/ml of Fortimicin B and 15 y/ml of Fortimicin C.

The liquor is subjected to the same desalting procedure as in Example l: and thereafter, the pH of the desalted solution is adjusted to 7. The solution is passed through a column of 3 L.

of 200 - 400 mesh weakly acidic cation-exchange resin, Amberlite CG-50 (NH4 type) to adsorb all the Fortimicins thereon.

Then. 0.15 N aqueous ammonia was passed through the column at S.V. 2/hour to conduct an elution, and the eluate is taken in l L. portions. Other Fortimicins such as Fortimicin B and Fortimicin C are contained in the solution obtained by combining Fraction Nos. 1 - 10.

Fortimicin A is contained in the solution obtained by combining Fraction Nos. 11 - 20 in about 60% yield.

Fraction Nos. 11 - 20 are then processed in the same manner as in Example 1 to obtain 15.5 g of sulfate of Fortimicin A with a purity of 93%.

Example 3 In this example, 300 L. of a culture liquor obtained in the same manner as in Example 1 containing 23 y/ml of Fortimicin A, and 10 y/ml of Fortimicin B and 4 y/ml of Fortimicin C is desalted as in Example 1. The pH of the desalted solution is adjusted to 7, and the resultant solution is passed through a column of 4 L. of 80 - 120 mesh macroreticular type strongly acidic cation-exchange resin, Diaion HPK 25 (NH4 type), having a cross-linking degree of 25% to adsorb all the Fortimicins on the resin. Then, 0.7 M ammonium phosphate (pH: 9.0) is passed through the column at S.V. 2/hour and the eluate is taken in 4 L. portions.

Other Fortimicins, e.g. Fortimicin B and Fortimicin C, are contained in the solution obtained by combining Fraction Nos. 1 - 15. Fortimicin A is contained in the solution obtained by combining Fraction Nos. 16 - 30 in about 55% yield.

Fraction Nos. 16 - 30 are then processed in the same manner as in Example 1 to obtain S.1g of the sulfate of Fortimicin A with a purity of 92%.

Example 4 In this example, 300 L. of a culture liquor obtained in the same manner as in Example 1 containing 23 y/ml of Fortimicin A, and 10 y/ml of Fortimicin B and 4 y/ml of Fortimicin C, is subjected to the desalting procedure of Example 1. The pH of the desalted solution is adjusted to 6.5 with sulfuric acid, and the resultant solution is passed through a column of 4 L. of 40-60 mesh cation-exchange resin having a low cross-linking degree, Dowex 50W X2 (NH4 type) having a cross-linking degree of 2%, to adsorb all the Fortimicins.

Then 0.6 M ammonium phosphate (pH: 9.5) is passed through the column at S.V. 2/hour and the eluate is taken in 4 L. portions. Other Fortimicins, e.g. Fortimicin B and Fortimicin C, are contained in the solution obtained by combining Fraction Nos. 1 - 20. Fortimicin A is contained in the solution obtained by combining Fraction Nos. 21 - 40 in about 55% yield.

Fraction Nos. 21 - 40 are then processed in the same manner as in Example 1 to obtain 4.8g of sulfate of Fortimicin A with a purity of 94%.

WHAT WE CLAIM IS: 1. A process for separating Fortimicin A substantially free of co-produced substances from a solution containing the Fortimicin A and co-product substances, which comprises passing the solution through an acidic cation-exchange resin which is a strongly acidic cation-exchange resin having a cross-linking degree (as herein defined) of 8% or less, a macroreticular type strongly acidic cation-exchange resin having a cross-linking degree (as herein defined) of 16% or more or a weakly acidic cation-exchange resin which weakly acidic cation - exchange resin passes through a 100 mesh (Tyler Standard Sieve), and passing a basic eluent through the cation-exchange resin to elute out the Fortimicin A in eluate fractions containing Fortimicin A.

2. A process as claimed in Claim 1 in which the eluate fractions containing Fortimicin A are combined and the Fortimicin A is separated from them.

3. A process as claimed in Claim 2 in which the Fortimicin A is separated from the eluate fractions by precipitation with an organic solvent.

4. A process as claimed in any of Claims 1 to 3 in which the solution is a culture liquor filtrate.

5. A process as claimed in any preceding claim in which the solution is desalted prior to passing the solution through the cation-exchange resin.

6. A process os claimed in any preceding claim in which the basic eluent is sodium hydroxide. potassium hydroxide, aqueous ammonium. ammonium phosphate, ammonium acetate, ammonium chloride or sodium chloride.

7. A process as claimed in any preceding claim in which the strongly acidic cation-exchange resins pass through a 30 mesh (Tyler Standard Sieve).

8. A process for separating Fortimicin A substantially free of co-produced substances from a solution containing the Fortimicin A and the co-produced substances, substantially as herein described with reference to any Example.

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

Claims (9)

**WARNING** start of CLMS field may overlap end of DESC **. Fortimicin A is contained in the solution obtained by combining Fraction Nos. 11 - 20 in about 60% yield. Fraction Nos. 11 - 20 are then processed in the same manner as in Example 1 to obtain 15.5 g of sulfate of Fortimicin A with a purity of 93%. Example 3 In this example, 300 L. of a culture liquor obtained in the same manner as in Example 1 containing 23 y/ml of Fortimicin A, and 10 y/ml of Fortimicin B and 4 y/ml of Fortimicin C is desalted as in Example 1. The pH of the desalted solution is adjusted to 7, and the resultant solution is passed through a column of 4 L. of 80 - 120 mesh macroreticular type strongly acidic cation-exchange resin, Diaion HPK 25 (NH4 type), having a cross-linking degree of 25% to adsorb all the Fortimicins on the resin. Then, 0.7 M ammonium phosphate (pH: 9.0) is passed through the column at S.V. 2/hour and the eluate is taken in 4 L. portions. Other Fortimicins, e.g. Fortimicin B and Fortimicin C, are contained in the solution obtained by combining Fraction Nos. 1 - 15. Fortimicin A is contained in the solution obtained by combining Fraction Nos. 16 - 30 in about 55% yield. Fraction Nos. 16 - 30 are then processed in the same manner as in Example 1 to obtain S.1g of the sulfate of Fortimicin A with a purity of 92%. Example 4 In this example, 300 L. of a culture liquor obtained in the same manner as in Example 1 containing 23 y/ml of Fortimicin A, and 10 y/ml of Fortimicin B and 4 y/ml of Fortimicin C, is subjected to the desalting procedure of Example 1. The pH of the desalted solution is adjusted to 6.5 with sulfuric acid, and the resultant solution is passed through a column of 4 L. of 40-60 mesh cation-exchange resin having a low cross-linking degree, Dowex 50W X2 (NH4 type) having a cross-linking degree of 2%, to adsorb all the Fortimicins. Then 0.6 M ammonium phosphate (pH: 9.5) is passed through the column at S.V. 2/hour and the eluate is taken in 4 L. portions. Other Fortimicins, e.g. Fortimicin B and Fortimicin C, are contained in the solution obtained by combining Fraction Nos. 1 - 20. Fortimicin A is contained in the solution obtained by combining Fraction Nos. 21 - 40 in about 55% yield. Fraction Nos. 21 - 40 are then processed in the same manner as in Example 1 to obtain 4.8g of sulfate of Fortimicin A with a purity of 94%. WHAT WE CLAIM IS:

1. A process for separating Fortimicin A substantially free of co-produced substances from a solution containing the Fortimicin A and co-product substances, which comprises passing the solution through an acidic cation-exchange resin which is a strongly acidic cation-exchange resin having a cross-linking degree (as herein defined) of 8% or less, a macroreticular type strongly acidic cation-exchange resin having a cross-linking degree (as herein defined) of 16% or more or a weakly acidic cation-exchange resin which weakly acidic cation - exchange resin passes through a 100 mesh (Tyler Standard Sieve), and passing a basic eluent through the cation-exchange resin to elute out the Fortimicin A in eluate fractions containing Fortimicin A.

2. A process as claimed in Claim 1 in which the eluate fractions containing Fortimicin A are combined and the Fortimicin A is separated from them.

3. A process as claimed in Claim 2 in which the Fortimicin A is separated from the eluate fractions by precipitation with an organic solvent.

4. A process as claimed in any of Claims 1 to 3 in which the solution is a culture liquor filtrate.

5. A process as claimed in any preceding claim in which the solution is desalted prior to passing the solution through the cation-exchange resin.

6. A process os claimed in any preceding claim in which the basic eluent is sodium hydroxide. potassium hydroxide, aqueous ammonium. ammonium phosphate, ammonium acetate, ammonium chloride or sodium chloride.

7. A process as claimed in any preceding claim in which the strongly acidic cation-exchange resins pass through a 30 mesh (Tyler Standard Sieve).

8. A process for separating Fortimicin A substantially free of co-produced substances from a solution containing the Fortimicin A and the co-produced substances, substantially as herein described with reference to any Example.

9. Fortimicin A when separated by a process as claimed in any preceding claim.