CA1074789A - Process for purifying fortimicin a - Google Patents
Process for purifying fortimicin aInfo
- Publication number
- CA1074789A CA1074789A CA281,947A CA281947A CA1074789A CA 1074789 A CA1074789 A CA 1074789A CA 281947 A CA281947 A CA 281947A CA 1074789 A CA1074789 A CA 1074789A
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- Canada
- Prior art keywords
- fortimicin
- exchange resin
- solution
- acidic cation
- cation
- Prior art date
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/20—Carbocyclic rings
- C07H15/22—Cyclohexane rings, substituted by nitrogen atoms
- C07H15/222—Cyclohexane rings substituted by at least two nitrogen atoms
- C07H15/224—Cyclohexane rings substituted by at least two nitrogen atoms with only one saccharide radical directly attached to the cyclohexyl radical, e.g. destomycin, fortimicin, neamine
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Saccharide Compounds (AREA)
- Peptides Or Proteins (AREA)
Abstract
ABSTRACT OF THE DISCLOSURE
Fortimicin A is isolated and purified from a solution containing Fortimicin A and other co-produced antibiotic factors by passing the solution through a column of an acidic cation-exchange resin and thereafter eluting out Fortimicin A under controlled conditions.
Fortimicin A is isolated and purified from a solution containing Fortimicin A and other co-produced antibiotic factors by passing the solution through a column of an acidic cation-exchange resin and thereafter eluting out Fortimicin A under controlled conditions.
Description
~1~7~9 BACKGROUND OF THE ~NVENTION
_ _ .
The prPsent 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, Fortimicin C, etc. using a cationic ion-exchange resin.
Fortimicin ~ is a water-soluble, basic antibiotic produced by bacteria of the genus Micromonospora, e.g., Micromonospora olivoasterospora MK70 (ATCC 21819) Micromonaspora olivoasterospora ~ATCC 31009)) and Micromonospora .. .. .
olivoasterospora tATCC 31010), showing a strong antibacterial .. ...
activ~ity against various Gram-positive and Gram-negative strains.
Fortimicin A and the microbiological process ~or producing same utilizing the aforementioned microorganisms is described in United States Patent No. 3,~76,768 issued August 24, 1976.
The chemical structure of Fortimicin A is also set forth in such patent as follows:
~ O N ~
~ ~ ~ OCH3 O=C-CH2-NH
It has been found, and subsequently reported, that the culture liquor of known Fortimicin A-producing strains belonging to thè genus Micromanospora contains co-produced compounds analogous to Fortimicin A such as Fortimicin B
(U.S. Patent No. 3,g31,~00), and Fortimicin C (c~-pendi~g Canadian Patent Application No. 258,074 filed on July 29, 1976~, in addition to Fortimicin A. Due to the presence of these bm/p~
~7~7~
factors in the cultuxe li~uor, the separation o~ ~ortimicin A
therefrom has heretofore heen di~ficult.
SUMMARY OF THE INVENTION
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 For~imicins;
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, 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 adsorb Fortimicin A and other Fortimicins on the resin; and, thereafter conducting a chromatographic separation using a basic eluent, whereby :i Fortimicin A is eluted out in substantially puri~ied form.
DETAILED DESCRIPTION OF THE INVENTION
.. ..
In accordance with the present invention, Fortimicin A is 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 pa.ssing the solution or culture liquor through a column of an ion-exchange :
xesin such as a strongly acidic cation~exchange resin having a low cross-linking de~ree, a~macroreticular type strongly.
acidic cation-exchange resin or a weakly acidic cation-exchange resin, all of which`are - 4 ~ ...
bm~
.
78~
'.
¦¦to h~ve comparatively small-sized resin particles whexeby Fort~micin A and other Fortimicins are adsor~ed on ~he resin; an-l then conducting a chromatographic separation using an ~uent~
~ When the solution or culture liquor con~ains salts such as Na2S04~ (N~4)2S04, NaCl, NH4Cl, etc., the efficiency 6 of ~he chromatographic separation tends to be lowered as the 7 ¦concentration of the salts i~ the solution increases. Therefore, 8 llit is preferable ~o desalt the solution before the chromatography.
9 I~For example, the culture li~uor is treated with an acid in order ¦to dissol~e a small quanti~y of Fortimici~A adsorbed Il on th~ bacteria into a liquid phase. This acid treatment is ' -12 conducted by adjusting the pH of the culture liquor to 1-3 with l3 suluric aci~, hydrochloric acid~ phosphoric acid, etc. and stirring the liquor at room temperature for about 1 hour. Ater ~5 A stirring, a filtering aid such as Radiolite ~600 ~product of Showa ~6 1l Chemical Industry Co., Ltd.) is added to the liquor, and the 7 ¦¦liquor is then filtered using a filter press to obtain a ~iltrate.
9 ~¦The filtrate is adjusted to pH 7 with alkali (NaOH, NH40~I or KOH) 9 Ilalld passed ~hrough a column of a weakly acidic cation~exchange !~ ¦ resin such as Amberlite IRC-50 (NH4 type) (product of Rohm & Haas ~t ¦Co.) to adsorb the Forti~icins including Fortimicin A on the resin~
_ _ .
The prPsent 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, Fortimicin C, etc. using a cationic ion-exchange resin.
Fortimicin ~ is a water-soluble, basic antibiotic produced by bacteria of the genus Micromonospora, e.g., Micromonospora olivoasterospora MK70 (ATCC 21819) Micromonaspora olivoasterospora ~ATCC 31009)) and Micromonospora .. .. .
olivoasterospora tATCC 31010), showing a strong antibacterial .. ...
activ~ity against various Gram-positive and Gram-negative strains.
Fortimicin A and the microbiological process ~or producing same utilizing the aforementioned microorganisms is described in United States Patent No. 3,~76,768 issued August 24, 1976.
The chemical structure of Fortimicin A is also set forth in such patent as follows:
~ O N ~
~ ~ ~ OCH3 O=C-CH2-NH
It has been found, and subsequently reported, that the culture liquor of known Fortimicin A-producing strains belonging to thè genus Micromanospora contains co-produced compounds analogous to Fortimicin A such as Fortimicin B
(U.S. Patent No. 3,g31,~00), and Fortimicin C (c~-pendi~g Canadian Patent Application No. 258,074 filed on July 29, 1976~, in addition to Fortimicin A. Due to the presence of these bm/p~
~7~7~
factors in the cultuxe li~uor, the separation o~ ~ortimicin A
therefrom has heretofore heen di~ficult.
SUMMARY OF THE INVENTION
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 For~imicins;
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, 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 adsorb Fortimicin A and other Fortimicins on the resin; and, thereafter conducting a chromatographic separation using a basic eluent, whereby :i Fortimicin A is eluted out in substantially puri~ied form.
DETAILED DESCRIPTION OF THE INVENTION
.. ..
In accordance with the present invention, Fortimicin A is 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 pa.ssing the solution or culture liquor through a column of an ion-exchange :
xesin such as a strongly acidic cation~exchange resin having a low cross-linking de~ree, a~macroreticular type strongly.
acidic cation-exchange resin or a weakly acidic cation-exchange resin, all of which`are - 4 ~ ...
bm~
.
78~
'.
¦¦to h~ve comparatively small-sized resin particles whexeby Fort~micin A and other Fortimicins are adsor~ed on ~he resin; an-l then conducting a chromatographic separation using an ~uent~
~ When the solution or culture liquor con~ains salts such as Na2S04~ (N~4)2S04, NaCl, NH4Cl, etc., the efficiency 6 of ~he chromatographic separation tends to be lowered as the 7 ¦concentration of the salts i~ the solution increases. Therefore, 8 llit is preferable ~o desalt the solution before the chromatography.
9 I~For example, the culture li~uor is treated with an acid in order ¦to dissol~e a small quanti~y of Fortimici~A adsorbed Il on th~ bacteria into a liquid phase. This acid treatment is ' -12 conducted by adjusting the pH of the culture liquor to 1-3 with l3 suluric aci~, hydrochloric acid~ phosphoric acid, etc. and stirring the liquor at room temperature for about 1 hour. Ater ~5 A stirring, a filtering aid such as Radiolite ~600 ~product of Showa ~6 1l Chemical Industry Co., Ltd.) is added to the liquor, and the 7 ¦¦liquor is then filtered using a filter press to obtain a ~iltrate.
9 ~¦The filtrate is adjusted to pH 7 with alkali (NaOH, NH40~I or KOH) 9 Ilalld passed ~hrough a column of a weakly acidic cation~exchange !~ ¦ resin such as Amberlite IRC-50 (NH4 type) (product of Rohm & Haas ~t ¦Co.) to adsorb the Forti~icins including Fortimicin A on the resin~
2 Tll~ resin is then washed thoroughly with water and the Fortimicins 1, ~3 are subsequently eluted out with 005N - 2.0N aqueous ammonia.
~4 ~mmonia in the eluate is removed by concentrating under reduced s pxessure, and the pH of the concentrate is adjusted to 5 - 9 with ~6 sulfuric acid. ~he thus obtained desalted solution is xeady for 7 ¦ chromatographic separation.
l The cation-exchange resins suitable for the chromato-9 jigraphic separation of the solution containing Fortimicin A and ` jlother For~imicins are limited to three types. Moreover, resin particle si~e greatly influences the eficiency o~ chroma~o~raphic _ 7~
` separation. That is, the smaller the particle size, ~he better the separating efficiency. Cation-exchange resins having a particle size passing through 30 mesh (by Tyler standard sieve~, are preferred. Cation-exchange resins having a particle size of 30 mesh-400 mesh are especially preferred. As used herein, '30 mesh-400 mesh' means a particle size which will pass through 30 mesh sieve but not through 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 0/0 or less, especially 8 0/0-0.5 0/0, including Dowex 50W~ (X2, X4, X6; Dow Chemical Co.), Diaion SK~ (102, 101, 104, 106), Diaion PK~ (204, 206, 208, 212; Mitsubishi Kasei Kogyo, Co., Ltd., Japan), Duolite C-10~ (Chemical Process Co), etc. The second type is a macroreticular type strongly acidic cation-exchange resin having a cross-linking degree of 16% or mroe, especially 16 0/0-50 0/0 including Diaion HPK~ (16, 25, 30, 40), Amberlite 15~, Amberlite XN-1004~ (Rohm & Haas Co.), etc. 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 type9 NA type, K type, NH4 type, etc. are usable. Those of NH., type are preferably used. Exemplary is Amberlite CG-50~ (Rohm & ~laas Co~).
Elution of Fortimicin A and other Fortlmicins absorbed on one of the above-described three types of cation-exchange resins is conducted as follows. When elution is conducted with . .
n basic eluent having a suitable pH and concentration using the respective resins, Fortimicin B~ Fortimicin C, ~ -6-A
~L~)7~78~
etc. are eluted first, then Fortimicin A~ Thus, For-timicin A can be chromatographically separated from the other Fortimicins.
Suitable eluents are selected from sodium hydroxide, potassium -hydroxide, aqueous ammonia,ammonium phosphate, ammonium acetate, ammonium chloride, sodium chloride and the like.
- 6a -dc/.~ ~
~ 8~ . I
I ¦¦ The el~ent is removed from the Fortimicin A-containing 2 ~Ifraction by conc~ntration under reduced pressure or by ion- ¦
~4 ~mmonia in the eluate is removed by concentrating under reduced s pxessure, and the pH of the concentrate is adjusted to 5 - 9 with ~6 sulfuric acid. ~he thus obtained desalted solution is xeady for 7 ¦ chromatographic separation.
l The cation-exchange resins suitable for the chromato-9 jigraphic separation of the solution containing Fortimicin A and ` jlother For~imicins are limited to three types. Moreover, resin particle si~e greatly influences the eficiency o~ chroma~o~raphic _ 7~
` separation. That is, the smaller the particle size, ~he better the separating efficiency. Cation-exchange resins having a particle size passing through 30 mesh (by Tyler standard sieve~, are preferred. Cation-exchange resins having a particle size of 30 mesh-400 mesh are especially preferred. As used herein, '30 mesh-400 mesh' means a particle size which will pass through 30 mesh sieve but not through 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 0/0 or less, especially 8 0/0-0.5 0/0, including Dowex 50W~ (X2, X4, X6; Dow Chemical Co.), Diaion SK~ (102, 101, 104, 106), Diaion PK~ (204, 206, 208, 212; Mitsubishi Kasei Kogyo, Co., Ltd., Japan), Duolite C-10~ (Chemical Process Co), etc. The second type is a macroreticular type strongly acidic cation-exchange resin having a cross-linking degree of 16% or mroe, especially 16 0/0-50 0/0 including Diaion HPK~ (16, 25, 30, 40), Amberlite 15~, Amberlite XN-1004~ (Rohm & Haas Co.), etc. 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 type9 NA type, K type, NH4 type, etc. are usable. Those of NH., type are preferably used. Exemplary is Amberlite CG-50~ (Rohm & ~laas Co~).
Elution of Fortimicin A and other Fortlmicins absorbed on one of the above-described three types of cation-exchange resins is conducted as follows. When elution is conducted with . .
n basic eluent having a suitable pH and concentration using the respective resins, Fortimicin B~ Fortimicin C, ~ -6-A
~L~)7~78~
etc. are eluted first, then Fortimicin A~ Thus, For-timicin A can be chromatographically separated from the other Fortimicins.
Suitable eluents are selected from sodium hydroxide, potassium -hydroxide, aqueous ammonia,ammonium phosphate, ammonium acetate, ammonium chloride, sodium chloride and the like.
- 6a -dc/.~ ~
~ 8~ . I
I ¦¦ The el~ent is removed from the Fortimicin A-containing 2 ~Ifraction by conc~ntration under reduced pressure or by ion- ¦
3 jlexchange separation The ~g o the Fortimicin ~-containing .
~ ¦solu~ion is then adjus~ed to 2.0 - 4.5 with sulfuric acid, S and an or~anic solvent such as ethanol is aaded there~o to 6 !precipitate the sulfate form o Fortimicin A. The precipitate ? ~is s~parated by filtration and dried to obtain a powdery product~
8 j Certain specific embodiments of the present invention 9 ,are illustrated by the following representative examples.
, E~Yample 1 ' I
Il ~ In thîs example, Micromonospora olivoasterospora 12 ~ 70 (ATCC 21819) ~Ferm~P No. 1560) is seed cultured at 30C for ~3` days in a m~dium containing 2 g/dl of~ glucose, 0.5 g/dl o peptone,
~ ¦solu~ion is then adjus~ed to 2.0 - 4.5 with sulfuric acid, S and an or~anic solvent such as ethanol is aaded there~o to 6 !precipitate the sulfate form o Fortimicin A. The precipitate ? ~is s~parated by filtration and dried to obtain a powdery product~
8 j Certain specific embodiments of the present invention 9 ,are illustrated by the following representative examples.
, E~Yample 1 ' I
Il ~ In thîs example, Micromonospora olivoasterospora 12 ~ 70 (ATCC 21819) ~Ferm~P No. 1560) is seed cultured at 30C for ~3` days in a m~dium containing 2 g/dl of~ glucose, 0.5 g/dl o peptone,
4 1l 0 . 5 g/dl of yeast extract and 0.1 gjdl o calcium carbonate ~ (pH: 7.5 beore sterilization)O The thus l6 1 obtained seed culture is then cul~ured at 37C for 4 days in a l7 1 culture medium containing 4 g/dl o soluble starch, 2 g/dl of l8 l soybean meal, 1 g/dl o coin steep liquor, 0.05 g/dl of K2HPO4, -S g/dl o MgSO~.7H2O, 0.03 g/dl of KCi and 0.01 g/dl o~ CaCO3.
~0 1 In thè culture liquor 45 y/ml of Fortimicin A is acc~ulated I ` `~1 l as well as 20 y/ml of Fortimicin B and 15 y/ml o l2 Foxtimicin C.
~3 The culture liquor (300 ~.), is adjusted to p~ 1.5 t~ wi.th sulfuric acid, and s~irred for lIi~ur ~ dissolve a ~ma~ ~ti~ of the IS Fo~timicins which are aasorbed on the ~acteria. After ~ s~irring, the liquox is fil~exed using a filter press. The 17 . filtrate is then adjusted to pH 7 wi~h aqueous ammonia and ~8 A l!~passed through a column of S L. of Amberlite IRC-50 (NH4 type)O
~9 , After washing the resin with water, elution with lN aqueous lo ammonia is carried out. The eluate is then concentrated under 3~ ~,reduced pressure to remove the ammonia, and the concen~rate is l _ 7 _ .. i~ , , ~
, adjusted to pll 7 with s~uric ~cid.
Tllis pH-adjusted solution is ~hen passed through a column of 5 L. of 100-150 mesh strong y acidic cation-A exchange resin, Diaion SK 102 (NH4 typei, having a cross-linking degree of 2 percent, 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 subs~ances such as Fortimicin B, 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 Amberlite IRC-50 to the solution obtained by combining Fraction Nos. 11 - 20 o~ 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 l I.. 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 lN 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 10.3g of the sulfate of Fortimicin A
(molar ratio of sulfuric acid to Fortimicin A is 2. This is the same in the subsequent examples.) in powder form with a purity of 95%
~xample 2 In this example, 500 Lo of a culture liquor obtained in the same manner as in Example 1 containing 38 r/ml of Fortimicin A, and 7 r/ml of Fortimicin B and 15 y/ml of Fortimicin C. The liquor is suhjected to the same desalting .
dc/~
'78~
procedure as in Example l; and thereaf~er, 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 A~ cation-exchange resin, Amberlite CG-5 ~ (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 1 L. portions. Other Fortimicins such as Fortimicin B, 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 ~ante manner as in Example 1 to obtain 15.5g of sulfate of Fortimicin A with a purity of 93%.
Example 3 In this example, 300 L. of a culture liquor o~tained in the same manner as in Example 1 containing 23 r/ml of Fortimicin A, and 10 ~/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 Ispassed 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 percent, to adsorb all the Fortimicins on the resin. Then, 0.7 M
an~onium phosphate (pH: 9.0) is passed through the column at S.~. 2/hour and the eluate is taken in 4 L. portionsO Othex Fortimicins, e.g., Fortimicin B and Foxtimicin C are contained in the solution obtained by combining Fraction Nos. 1 - 15.
- 30 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 5.lg of the sulfat~ of _ g _ dc/~
7~ I
1 I1Fortimicin A with a purity o~ ~2%u 2 !!Example 4 I
3 1 In this example, 30~ L. of a culture liquor obtained-~ 1 in the same manner as in Example l containing 23 y/ml of Forti-5micin A, and l0 y~ml of Fortimicin B and 4 y/ml of Forti-6 !¦micin C, is subiected to the desalting procedure of Example l.
Th~ pEI o~ the desalte~ solution is adjusted to 6.5 with sulfuric ~ '¦acid, and the resultant solution is passed through a column o 9 'j4 L. of 40-60 mesh cation~exchange resin having a low cross-10 ~ ~llinking deg.~e, ~owex 50W X2~(NH4 t~pe), hAYing a cros~:l..ink~g degree of ~1 ' 2 percent, to a~orb ~l the Fortimicins.
121 Then 0.6 M ammonium phosphate ~pH: 9.5) is passed 13 1 th~ough the column at S.V. 2/hour and the eluate is taken -~in 4 L. portions. Other Fortimicins, e.g., Fortimicin B and IS I Fortimicin C, are contained in the solution obtained by combining t6 ~ Fraction Nos. l - 20. Fortimicin A is contained in the solution 17~ obtained by combining Fraction Nos~ ?1 - 40 in about 55~ yield.
l9 j Fraction Nos. 21 - 40 are then processed in the ts ¦ same manner as in Example l to obtain 4.8g of sulfate o~
10Fortimicin A with a purity o 94%.
2t lt 1~
~ , ' ', :" .
2'.
1 .
~7 1 . ~`
' i . : :
30 .' ., ~ ~ ~ , , ', /.:
: ','' . ' '' '- . : ' ' : , - . : , . : ~ ':
~0 1 In thè culture liquor 45 y/ml of Fortimicin A is acc~ulated I ` `~1 l as well as 20 y/ml of Fortimicin B and 15 y/ml o l2 Foxtimicin C.
~3 The culture liquor (300 ~.), is adjusted to p~ 1.5 t~ wi.th sulfuric acid, and s~irred for lIi~ur ~ dissolve a ~ma~ ~ti~ of the IS Fo~timicins which are aasorbed on the ~acteria. After ~ s~irring, the liquox is fil~exed using a filter press. The 17 . filtrate is then adjusted to pH 7 wi~h aqueous ammonia and ~8 A l!~passed through a column of S L. of Amberlite IRC-50 (NH4 type)O
~9 , After washing the resin with water, elution with lN aqueous lo ammonia is carried out. The eluate is then concentrated under 3~ ~,reduced pressure to remove the ammonia, and the concen~rate is l _ 7 _ .. i~ , , ~
, adjusted to pll 7 with s~uric ~cid.
Tllis pH-adjusted solution is ~hen passed through a column of 5 L. of 100-150 mesh strong y acidic cation-A exchange resin, Diaion SK 102 (NH4 typei, having a cross-linking degree of 2 percent, 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 subs~ances such as Fortimicin B, 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 Amberlite IRC-50 to the solution obtained by combining Fraction Nos. 11 - 20 o~ 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 l I.. 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 lN 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 10.3g of the sulfate of Fortimicin A
(molar ratio of sulfuric acid to Fortimicin A is 2. This is the same in the subsequent examples.) in powder form with a purity of 95%
~xample 2 In this example, 500 Lo of a culture liquor obtained in the same manner as in Example 1 containing 38 r/ml of Fortimicin A, and 7 r/ml of Fortimicin B and 15 y/ml of Fortimicin C. The liquor is suhjected to the same desalting .
dc/~
'78~
procedure as in Example l; and thereaf~er, 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 A~ cation-exchange resin, Amberlite CG-5 ~ (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 1 L. portions. Other Fortimicins such as Fortimicin B, 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 ~ante manner as in Example 1 to obtain 15.5g of sulfate of Fortimicin A with a purity of 93%.
Example 3 In this example, 300 L. of a culture liquor o~tained in the same manner as in Example 1 containing 23 r/ml of Fortimicin A, and 10 ~/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 Ispassed 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 percent, to adsorb all the Fortimicins on the resin. Then, 0.7 M
an~onium phosphate (pH: 9.0) is passed through the column at S.~. 2/hour and the eluate is taken in 4 L. portionsO Othex Fortimicins, e.g., Fortimicin B and Foxtimicin C are contained in the solution obtained by combining Fraction Nos. 1 - 15.
- 30 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 5.lg of the sulfat~ of _ g _ dc/~
7~ I
1 I1Fortimicin A with a purity o~ ~2%u 2 !!Example 4 I
3 1 In this example, 30~ L. of a culture liquor obtained-~ 1 in the same manner as in Example l containing 23 y/ml of Forti-5micin A, and l0 y~ml of Fortimicin B and 4 y/ml of Forti-6 !¦micin C, is subiected to the desalting procedure of Example l.
Th~ pEI o~ the desalte~ solution is adjusted to 6.5 with sulfuric ~ '¦acid, and the resultant solution is passed through a column o 9 'j4 L. of 40-60 mesh cation~exchange resin having a low cross-10 ~ ~llinking deg.~e, ~owex 50W X2~(NH4 t~pe), hAYing a cros~:l..ink~g degree of ~1 ' 2 percent, to a~orb ~l the Fortimicins.
121 Then 0.6 M ammonium phosphate ~pH: 9.5) is passed 13 1 th~ough the column at S.V. 2/hour and the eluate is taken -~in 4 L. portions. Other Fortimicins, e.g., Fortimicin B and IS I Fortimicin C, are contained in the solution obtained by combining t6 ~ Fraction Nos. l - 20. Fortimicin A is contained in the solution 17~ obtained by combining Fraction Nos~ ?1 - 40 in about 55~ yield.
l9 j Fraction Nos. 21 - 40 are then processed in the ts ¦ same manner as in Example l to obtain 4.8g of sulfate o~
10Fortimicin A with a purity o 94%.
2t lt 1~
~ , ' ', :" .
2'.
1 .
~7 1 . ~`
' i . : :
30 .' ., ~ ~ ~ , , ', /.:
: ','' . ' '' '- . : ' ' : , - . : , . : ~ ':
Claims (6)
1. A process for separating Fortimicin A substan-tially free of co-produced substances from a solution containing said Fortimicin A and co-produced substances which comprises passing said solution through an acidic cation-exchange resin selected from the group consisting of strongly acidic cation-exchange resin having a cross-linking degree of 8% or less, macroreticular type strongly acidic cation-exchange resin having a cross-linking degree of 16% or more, and weakly acidic cation-exchange resin having at least a 100 mesh particle size; passing a basic eluent through said cation-exchange resin to elute out said Fortimicin A in eluate fractions; combining the fractions containing Fortimicin A and separating said Fortimicin A therefrom.
2. A process according to claim 1 wherein said solution is a culture liquor filtrate.
3. A process according to claim 1 including the step of desalting said solution prior to passing said solution through said cation-exchange resin.
4. A process according to claim 1 wherein said basic eluent is selected from the group consisting of sodium hyroxide, potassium hydroxide, aqueous ammonia, ammonium phosphate, ammonium acetate, ammonium chloride and sodium chloride.
5. A process according to claim 1 wherein said Fortimicin A is separated from said eluate fractions by precipi-tation utilizing an organic solvent.
6. A process according to claim 1 wherein said strongly acidic cation-exchange resins have an at least 30 mesh particle size.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP7885076A JPS597318B2 (en) | 1976-07-05 | 1976-07-05 | Purification method for hortimycin A |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1074789A true CA1074789A (en) | 1980-04-01 |
Family
ID=13673290
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA281,947A Expired CA1074789A (en) | 1976-07-05 | 1977-07-04 | Process for purifying fortimicin a |
Country Status (10)
| Country | Link |
|---|---|
| JP (1) | JPS597318B2 (en) |
| AR (1) | AR213118A1 (en) |
| AU (1) | AU511015B2 (en) |
| BE (1) | BE856509A (en) |
| CA (1) | CA1074789A (en) |
| GB (1) | GB1587932A (en) |
| GR (1) | GR63243B (en) |
| MX (1) | MX4552E (en) |
| NL (1) | NL7707436A (en) |
| PH (1) | PH13988A (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5721400A (en) * | 1980-07-12 | 1982-02-04 | Kyowa Hakko Kogyo Co Ltd | Novel fortimicin derivative |
-
1976
- 1976-07-05 JP JP7885076A patent/JPS597318B2/en not_active Expired
-
1977
- 1977-06-22 MX MX10071977U patent/MX4552E/en unknown
- 1977-07-04 GB GB2781177A patent/GB1587932A/en not_active Expired
- 1977-07-04 CA CA281,947A patent/CA1074789A/en not_active Expired
- 1977-07-05 AU AU26772/77A patent/AU511015B2/en not_active Expired
- 1977-07-05 BE BE179111A patent/BE856509A/en not_active IP Right Cessation
- 1977-07-05 AR AR26831777A patent/AR213118A1/en active
- 1977-07-05 GR GR53885A patent/GR63243B/en unknown
- 1977-07-05 PH PH19948A patent/PH13988A/en unknown
- 1977-07-05 NL NL7707436A patent/NL7707436A/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| GR63243B (en) | 1979-10-12 |
| JPS536487A (en) | 1978-01-20 |
| AR213118A1 (en) | 1978-12-15 |
| PH13988A (en) | 1980-11-28 |
| GB1587932A (en) | 1981-04-15 |
| AU2677277A (en) | 1979-01-11 |
| MX4552E (en) | 1982-06-10 |
| JPS597318B2 (en) | 1984-02-17 |
| NL7707436A (en) | 1978-01-09 |
| BE856509A (en) | 1978-01-05 |
| AU511015B2 (en) | 1980-07-24 |
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