CA1046052A - Process for the purification of fr-1923 substance - Google Patents
Process for the purification of fr-1923 substanceInfo
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- CA1046052A CA1046052A CA222,363A CA222363A CA1046052A CA 1046052 A CA1046052 A CA 1046052A CA 222363 A CA222363 A CA 222363A CA 1046052 A CA1046052 A CA 1046052A
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- substance
- process according
- resin
- aqueous solution
- solution
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07G—COMPOUNDS OF UNKNOWN CONSTITUTION
- C07G11/00—Antibiotics
Abstract
ABSTRACT OF THE DISCLOSURE
A process is provided for the purification of an antibiotic substance FR-1923 which comprises contacting an aqueous solution containing the FR-1923 substance, together with impuri-ties, with a macroporous non-ionic adsorption resin.
A process is provided for the purification of an antibiotic substance FR-1923 which comprises contacting an aqueous solution containing the FR-1923 substance, together with impuri-ties, with a macroporous non-ionic adsorption resin.
Description
~04~;0SZ
The present invention relates to a new process for purification of FR-1923 substance. ~lore particularly, it relates to a new process for purification of FR-1923 sub-stanee, using a macroporous non-ionic adsorption resin.
FR-1923 substance is a known antibiotie isolated from the fermentation broth of a strain of genus Stre~tomvce~, as described, for example, in German Offenlegungsschrift 2242699 corresponding to Canadian Patent Application Ser.~o. 150,731 filed August 30,1972 Hatsuo Aoki et al.
In said prior literature, there are disclosed processes using aetivated ehareoal, anion exehange resin and DEAE-Sephadex (trade mark, made by Pharmacia Co.) for isolation and purification of FR-1923 substance from a fermentation broth.
However, the prior proeesses have not led to satisfae-tory results in an industrial manufacture of FR-1923 sub-stanee beeause they require relatively many steps for the isolation and purifieation of FR-1923 substanee, so that the overall recovery of the antibiotic is relatively poor.
As the results of the extensive studying, the present inventors have now newly found a new process for purification of FR-1923 substance as a step in the recovery thereof, by means of which FR-1923 substance can be obtained in higher yield than by the prior processes. Further, this new process is simpler and more convenient in practical operation and 1046()52 more economical on an industrial scale because the spent resin can be regenerated to be used again.
Accordingly, it is an object of the present invention to provide a new process for purification of FR-1923 substance usinq a macroporous non-ionic adsorption resin.
Another object of the present invention is to provide a new process for purification of FR-1923 substance, by means of which FR-1923 substance is obtained in higher yield than by the prior processes.
A further object of the present invention is to provide a simpler, more convenient and more economical process for puri-fication of FR-1923 substance.
According to the invention there is provided a process for the purification of FR-1923 substance which comprises:
contacting an aqueous solution containing FR-1923 substance, together with impurities, with a macroporous nonrionic adsorption resin, adsorbing one of said FR-1923 Aubstance and said impurities in ~aid solution on said re~in, and recovering said FR-1923 sub-stance.
In one aRpect of the invention the aqueous solution is contacted with the macroporous non-ionic adsorption resin to adsorb FR-1923 substance on the resin, the adsorbed FR-1923 substance i~ eluted from the re~in, and the FR-1923 substance is recovered from the eluate.
In another aspect of the invention the aqueous solution is contacted with a macroporous non-ionic adsorption resin saturated with FR-1923 substance, impurities are adsorbed on the saturated resin, and FR-1923 substance is recovered from the thus contacted solution.
As described above, the present invention provides a new process for purification of FR-1923 substance, using a macroporous non-ionic adsorption resin, which comprises alter-k ~ Jl ~ ~ 2 -r, ~, native methods in u~ing a macroporous non-ionic adsorption resin:
That is, one of them is a method that an aqueous solution containing FR-1923 substance, together with impurities, is contacted with a macroporous non-ionic adsorption resin, whereafter the adsorbed FR-1923 substance is eluted with, for example, a hydrophilic solvent system (hereinafter con-veniently referred to method A), The other is a method that an aqueous solution containing FR-1923 substance, together with impuritie~, is contacted with a macroporous non-ionic adsorption resin saturated by FR-1923 substance to adsorb the impurities, and FR-1923 substance is recovered from the B- 2a -passed solution (hereinafter conveniently referred to method B).
Alternative methods are explained in more detail hereinafter.
M~thod A
According to method A of the present invention, there is provided a process for purification of FR-1923 substance, wherein an aqueous solution containing FR-1923 substance, together with impurities, is contacted with a macroporous non-ionic adsorption resin, whereafter the adsorbed FR-1923 substance is eluted with, for example, a hydrophilic solvent system.
.
The macroporous non-ionic adsorption resins used according to the present invention may include a cross-linked . resin with an aromatic basic structure. The preferred resins include styrene polymers cross-linked with divinyl benzene. Examples of such resins include Amberlite XAD-l, XAD-2, XAD-4, XAD-7 and XAD-8 (trade mark, maker; Rohm &
Haas Co.), Diaion HP 10, HP 20, HP 30, HP 40 and HP 50 (trade mark, maker; Mitsubishi Kasei Co., Ltd.) and Hitachi-gel No. 3010 (trade mark, maker; Nissei Sangyo Co., Ltd.).
The process for purification of FR-1923 substance, using the macroporous non-ionic adsorption resin, in accord-ance with the method A of the present invention is applicable to any aqueous solution containing FR-1923 substance, together with impurities. Examples of such solutions include a filtrate of culture broth per se prepared by cultivating FR-1923 substance - producing microorganism in a nutrient medium, and a pre-extracted culture solution as well as an aqueous solution containing crude solid material containing F'R-1923 substance (e.g. crude powder or crude crystals containing FR-1923 substance) which first is isolated and purified to some degree from a filtrate of the culture broth by means of conventional manners, such as an optional treatment of the filtrate with an appropriate solvent for extraction and/or by adsorption on activated charcoal.
When using a culture filtrate, it is advantageous to use an optionally pre-extracted culture solution, this optionally pre-extracted culture solution being brought into contact with a macroporous non-ionic adsorption resin in the usual manner, preferably with use of a column containing a bed of resin.
Treatment of the aqueous solution containing FR-1923 substance in admixture with impurities with the non-ionic adsorption resin is advantageously carried out at pH value of from 2-7 and preferably of from 4 to 6. The desired acidic pE~ values can be achieved by using any desired acid, for example, an organic acid, such as oxalic acid, or preferably a mineral acid, such as hydrochloric, phosphoric or sulphuric acid.
Further, treatment of the aqueous solution containing FR-1923 substance in admixture with impurities with the non-ionic adsorption resin is advantageously carried out in case of the impurities being inorganic salt, such as sodium chloride, sodium biphosphate or disodium orthophosphate.
In this method, the aqueous solution containing FR-1923 1046Qsz substance in admixture with impurities is brought into contact with the macroporous non-ionic adsorption resin to adsorb FR-1923 substance by a conventional manner, for example batch operation or column operation, whereafter adsorbed FR-1923 substance is eluted from the resin with, for example, a hydrophilic solvent system.
The hydrophilic solvent systems used for eluting FR-1923 substance from the resin, include, for example, a lower dialkyl ketone (e.g. acetone and methyl ethyl ketone); and a lower alkanol (e.g. methanol, ethanol, n-propanol, iso-propanol, n-butanol and isobutanol). There may also be used mixtures of the above lower dialkylketone and lower alkanol.
Further, mixtures of watér or of a lower alkyl ester of a lower alkanoic acid, for example methyl acetate, ethyl acetate and butyl acetate, and the above lower alkanol or lower dialkyl ketone may also be used. Furthermore, a large volume of water and an alkaline aqueous solution Ipreferably pH 8 - 13) may be used.
The eluate thus obtained is treated by conventional means such as concentration, pH adjustment, lyophilization and recrystallization to give crystalline FR-1923 substance which frequently is sufficiently pure for use as a medicine.
Method B
According to method B of the present invention, there is provided a process for purification of FR-1923 substance wherein an aqueous solution containing FR-1923 substance, together with impurities, is contacted with a macroporous non-ionic adsorption resin saturated in advance by FR-1923 substance to adsorb impurities, and FR-1923 substance is recovered from the passed solution.
Thi~ method is based on the fact that adsorption capacity of a macroporous non-ionic adsorption resin to FR-1923 substance is smaller than that to specific impurities such as coloring materials stemming from fermentation broth and isomer of FR-1923 substance. By using the difference of capacity of macroporous non-ionic adsorption reqin between FR-1923 substance and the specific impurities, the specific impurities are selectively adsorbed to the resin saturated by FR-1923 substance from an aqueous solution containing FR-1923 substance, together with im-purities, and FR-1923 substance is recovered from the passed solution.
Ad~orption capacity of a macroporous non-ionic adRorption resin to ~R-1923 substance varies with condition of aqueous solution containing FR-1923 substance, together with impurities, and with the kind of the macroporous non-ionic adsorption reqin. Exampleq of adsorption capacity of a macroporous non-ionic adQorption reqin to FR-1923 sub-stance are as follows. The following data is given by contacting 0.2M phosphate buffer solution ~H4) containing FR-1923 substance with the resin.
Examples of adsorPtion caPacitY to FR-1923 substance Ca . lOg/l~ of Diaion HP 20*
Ca. 18g~1~ of Amberlite XAD-4*
*trademark t . - 6 -iO460S2 The macroporous non-ionic adsorption resin saturated by FR-1923 substance is prepared by contacting an aqueous solution containing FR-1923 substance with a macroporous non-ionic adsorption resin, wherein said solution is preferably neutral or slightly alkaline.
As examples of a macroporous non-ionic adsorption resin, the same examples as mentioned in the method A are exemplified.
The process for purification of FR-1923 substance using the macroporous non-ionic adsorption resin, in accordance with the method B of the present invention is applicable to any aqueous solution containing FR-1923 substance, together with impurities, as well as that of method A mentioned above, as examples of which the same examples as mentioned in the method A are exemplified.
Especially, when coloring materials or isomer of FR-1923 substance are contained as impurities in the aqueous solution,thls method B gives better results.
When an aqueous solution containing FR-1923 substance, together with impurities, is contacted with the macroporous non-ionic adsorption resin saturated by FR-1923 substance, FR-1923 substance can be adsorbed no more to it and, on the other hand, the impurities are selectively adsorbed to it.
Volume of the macroporous non-ionic adsorption resin to be used in this method varies with a content of FR-1923 substance in an aqueous solution containing FR-1923 substance, together with impurities, and with the kind of the resin to be used, and it, generally, may be lQ per 100 - 500 g of FR-1923 substance in the aqueous solution.
FR-1923 substance is recovered from the passed through solution, which is obtained by contacting an aqueous solution containing FR-1923 substance, together with impurities, with macroporous non-ionic adsorption resin saturated by FR-1923 substance. The recovery can be conducted by conventional manners such as pH adjustment, treatment with activated charcoal, concentration, lyophilization and recrystallization to give crystalline FR-1923 substance which frequently is sufficiently pure for use as a medicine.
Further, an aqueous solution containing FR-1923 substance, together with impurities, is contacted with a macroporous non-ionic adsorption resin saturated by FR-1923 substance to adsorb impurities, whereafter the column is washed with a small volume of water to obtain FR-1923 substance staying of the space among the resin in the column.
The spent resin can be regenerated by conventional manners, e.g. by washing the said resin with a relatively strong alkaline aqueous or alkaline aqueous-alcoholic solution.
The method B is superior to the method A and is more convenientin practiceand more economical on an industrial scale, in the following points. That is, the method B does not require a process for elution of FR-1923 substance from the resin, and volume of the resin used for purification in method B is less than 10% in comparison with that in method A, though recovery and purity of FR-1923 substance are almost 1046~SZ
same degree in both methods.
The following testsare given for the purpose of illustra-ting a comparison between method A and B as a process for ~urification of FR-1923 substance.
The comparison-tests are carried out by using the same crude crystals of FR-1923 substance and the same resin in ` the same volumein both methods, but volume of the crude crystals of FR-1923 substance in the method A is decided within the capacity of the resin to FR-1923 substance, and volume of the crude crystals of FR-1923 substance used in the method B is about 20 times that in method A.
Test A lI] Adsorption capacity of Diaion HP 20 to FR-1923 substance:
1.4 g of sodium salt of FR-1923 (purity : 95%) substance were dissolved in 1 liter of 0.2 M phosphate buffer solution (pH 4.0). The solution was adjusted to pH 4.0 with 5N
hydrochloric acid with stirring. The solution (concentration of FR-1923 substance : 1310 r/ml) was passed through a column packed with 100 ml of Diaion HP 20 (internal diameter of the column : 30 mm, packing height of the resin : 160 mm) at a rate of SV=l ("SV" means abbreviation of "Space Velocity").
The passed solution were collected lOOml by lOOml. Content of FR-1923 substance in the passed solution was determined by bioassay using Pseudomonas aeruginosa. Adsorption capacity of Diaion HP 20 to FR-1923 substance was calculated at the time when concentration of FR-1923 substance in the passed solution reaches the same concentration as that of the ~ ~r~o~ark g starting solution (1310 r/mQ~ to give 10.4 g / lite~.
[II] Purification of FR-1923 substance by the method A:
9.5 g of crude crystals of FR-1923 substance (FR-1923 substance content : 7.9 g) were suspended in 2.5 liters of water. The suspension was adjusted to pH 7.5 with 6N sodium hydroxide aqueous solution to dissolve in. Further, the solution was adjusted to pH 6.0 with 6N hydrochloric acid, and to the solution there were added 150 g of sodium chloride and water to give 3 liters of the solution. The solution was passed through a column packed with 1 liter of Diaion HP 20 (internal diameter of the column: 77 mm, packing height of the resin: 230 mm) at a rate of SV=l. The adsorbed FR-1923 substance was eluted with 30% aqueous methanol after the column was washed with water. Active fraction (1950 ml) were collected and concentrated under reduced pressure. The concentrate was adjusted to pH 2.2 with stirring and allowed to stand overnight in the refrigerator to give crystals.
The crystals were collected by filtration and dried. The results are shown in the following table.
lIII] Purification of FR-1923 substance by the method B:
Two hundred gram of crude crystals of FR-1923 substance (FR-1923 substance content : 168 g) being the same crude crystals as that used in Test [II], were suspended in 2 liters of water. The suspension was adjusted to pH 7.2 to A~ dissolve in. The solution was passed through a column packed ;~ with 1 liter of Diaion HP 20 saturated by FR-1923 substance (internal diameter of the column : 77 mm, packing height of the resin : 230 mm) at a rate of SV=l. Four liters of water ~rao~ n a~k -- :LO --.. .. . .
10460S;~
were passed through the column, 4500 ml of active fractions were collected from the passed solution, and to the solution obtained there was added water to give 16 liters of the solution. The solution was àdjusted to pH 2.5 with stirring and then allowed to stand overnight in the refrigerator to give crystals. The crystals were collected by filtration and dried. The results are shown in the following table.
The method A The method B
Capacity of the resin for treating crude 9.5 g / lQ of 187 g / lQ of crystals of FR-1923 resin resin substance Recovery of crystals 1 7.2 g 160 g Purity of crystals 92.7 % 98.4 %
Purity-calcd,recovery 84.5 % ¦ 93.6 %
of FR-1923 substance The above results show that volume of FR-lg23 substance treated by method B is about 20 times that by method A, though purity and recovery of FR-1923 substance are almost the same degree in both methods, respectively.
The following examples are given for the purpose of illustrating the present invention.
Example 1 To a culture broth prepared by culturing Nocardia ~niformis var. tsuyamanensis ATCC 21806 at 30C for 120 hours in 150 liters of a nutrient medium containing by weight 3%
glycerol, 2% cottonseed meal, 2% dried yeast, 2.18% potassium dihydrogen phosphate, 1.43% disodium hydrogen phosphate dodecahydrate, 0.5% magnesium chloride heptahydrate, there was added 6% of Radiolite (trade mark, a filter aid material sold by Showa Kagaku Kogyo Co., Ltd.) and then the mixture was filtered. Three liters of the filtrate obtained (FR-1923 substance content: 3030 mg) were passed through a A column packed with a macroporous non-ionic adsorption resin, Diaion HP 20 (internal diameter of the column : 8 cm, packing . height of the resin : 26 cm) at a rate of SV=l. The column was washed with water, whereafter FR-1923 substance was eluted with 20% of aqueous methanol solution. Three liters of eluate obtained were concentrated under reduced pressure and then adjusted to pH 2.5. The solution was allowed to stand in refrigerator to give 2490 mg of colorless crystals of FR-1923 substance.
Example 2 A culture broth which was prepared by substantially the same manner as described in Example 1 was adjusted to pH 4.0 with diluted sulfuric acid, whereafter the culture broth was filtered. To the filtrate obtained was added 6% of Radiolite, whereafter the mixture was filtered. Nine hundred ml of the filtrate obtained (FR-1923 substance content : 774 mg) were passed through a column packed with a macroporous non-ionic adsorption resin, Amberlite XAD-4(internal diameter of the t ~a O~ ark 1046C)52 column: 3.6 cm, packing height of the resin~30 cm) at a rate of SV=l. After the column was washed with water, FR-1923 sub-stance was eluted with 20~ aqueous acetone. Six hundred ml of eluate obtained were concentrated under reduced pressure, adjusted to pH 2.0 with diluted hydrochloric acid, and then allowed to stand in a refrigerator to give 365 mg of colorless crystals of FR-1923 substance.
Example 3 Three hundred ml of a culture filtrate (pH`4.0), obtained by substantially the same manner as described in Example 1, were passed through a column of a macroporous non-A~ ionic adsorption resin, Diaion HP 20 (internal diameter of the column : 30 mm, packing height of the resin : 160 mm) at a rate of SV=l. The column was washed with water, whereafter FR-1923 substance was eluted with aqueous sodium hydroxide solution (pH 12.0). Six hundred ml of active fraction obtained were collected and concentrated to 15 ml under reduced pressure. The concentrate was adjusted to pH 2.5 with 6N hydrochloric acid while stirring, whereafter the solution was allowed to stand for 24 hours in the refrigerator to give 360 mg of colorless crystals of FR-1923 substance.
Example 4 Twenty g of crude powder (FR-1923 substance content :
10.2 g) of FR-1923 substance were suspended in 6.2 Q of water. The suspension was adjusted to pH 8.1 with 6N aqueous sodium hydroxide solution, whereafter 50 g of sodium chloride were added to the solution; The solution was passed through ~ ~ r6(a~ h?ark ~046~52 a column of a macroporousnon-ionic adsorption resin, Diaion HP 20 (internal diameter of the column : 87 mm, packing height of the resin : 520 mm) at a rate of SV=l. The column was washed with water, whereafter FR-1923 substance was eluted with 30% aqueous methanol solution. Active fractions (4.48 liters) obtained were collected and then concentrated to the volume of 400 ml under reduced pressure. The concentrate was adjusted to pH 2. 5 with 6N hydrochloric acid and then allowed to stand overnight in refrigerator to give 10.0 g of colorless crystals of FR-1923 substance.
Example 5 Eighteen gram of crude powder (FR-1923 substance content:
10.2 g) of FR-1923 substance were suspended in 360 ml of water, whereafter the suspension was adjusted to p~I 7.0 with 6N sodium hydroxide aqueous solution to dissolve in. The solution was passed through a column packed with a macroporous non-ionic adsorption resin, Amberlite XAD-8 (internal diameter of the column : 30 mm, packing helght of the resin :
160 mm) saturated by FR-1923 substance at a rate of SV=l, whereafter 400 ml of water were passed through the column.
700 ml of active fractions were collected from the passed solution, and then the solution was concentrated under reduced pressure to a volume of 200 ml. The concentrate obtained was adjusted to pH 2. 5 with 6N hydrochloric acid while stirring, and then allowed to stand in a refrigerator for 24 hours to give 10.13 g of colorless crystals of FR-1923 substance.
~ ~ æhn~ r ~
. .
Example 6 Eighteen gram of crude powder (FR-1923 substance content:
10.2 g) of FR-1923 substance were suspended in 360 ml of water and adjusted to pH 7.0 with 6N sodium hydroxide aqueous solution to dissolve in. The solution was passed through a column packed with a macroporous non-ionic adsorption resin, Amberlite XAD-4 (internal diameter of the column : 30 mm, packing height of the resin : 160 mm) saturated by FR-1923 substance at a rate of SV=l, whereafter 540 ml of water were passed through the column. 900 ml of active fractions were collected from the passed solution, and then the solution was concentrated under reduced pressure to a volume of 350 ml. The concentrate obtained was adjusted to pH 2. 5 with 6N hydrochloric acid while stirring and then allowed to stand in a refrigerator for 24 hours to give 9. 41 g of colorless crystals of FR-1923 substance.
Example 7 Nineteen gram of crude power (FR-1923 substance content:
10. 7 g) of FR-1923 substance were suspended in 380 ml of water and then the suspension was adjusted to pH 7.0 with 6N sodium hydroxide aqueous solution to dissolve in. ~he solution was passed through a column packed with macroporous non-ionic adsorption resin, Amberlite XAD-7 (internal diameter of the column: 30 mm, packing height of the resin:
160 mm) saturated by FR-1923 substance at a rate of SV=l, whereafter 400 ml of water were passed through the column.
700 ml of active fractions were collected from the passed solution, whereafter the solution obtained was adjusted to pH 2. 5 with 6N hydrochloric acid while stirring and a~lowed ~ ~a6~QrnQrk to stand in a refrigerator to give 10.28 g of colorless crystals of FR-1923 substance.
Example 8 19.75 g of crude powder of FR-1923 substance (FR-1923 substance content: 17.16 g) were suspended in 395 ml of water and the suspension was adjusted to pH 7.5 with 5N
sodium hydroxide aqueous solution to dissolve in. The solution was passed through a column packed with a macroporous 1 A ~ non-ionic adsorption resin Diaion HP 20 (internal diameter of the column : 30 mm, packing height of the resin : 160 mm) saturated by FR-1923 substance at a rate of SV=l, whereafter 400 ml of water were passed through the column. 600 ml of active fractions were collected from the passed solution~
whereafter the solution obtained was adjusted to pH 2.5 with 6N hydrochloric acid while stirring and allowed to stand in a refrigerator to give 16.8 g of colorless crystals of FR-1923 substance.
Example 9 Fifteen liters of a culture filtrate (pH 4.0) (FR-1923 substance content: 14.25 g), obtained by substantially the same manner as described in Example l,were passed through a column packed with a macroporous non-ionic adsorption resin, Diaion HP20 (internal diameter of the coulum: 110 mm, packing height of the resin: 550 mm) at a rate of SV=l. The column was washed with 5 liters of water, whereafter FR-1923 substance was eluted with 30~
aqueous methanol solution. 7,940 ml of active fractions obtained were collected and then adjusted to pH 3.3 with * ~ 1ar ~
6N hydrochloric acid. The solution was passed through a column of alumina (acidic, internal diameter of the column: 46 mm, packing height of alumina: 300 mm).
The column was washed with 500 ml of water, whereafter FR-1923 substance was eluted with 0. 3 M pottassium biphosphate aqueous solution. 1800 ml of active fractions were collected and then concentrated under reduced pressure to the volume of 800 ml. The concentrate was adjusted to pH 2.2 with 6N hydrochloric acid while stirring, whereafter the solution was allowed to stand overnight in a refrigerator to give 11.94 g of colorless crystals of FR-1923 substance.
Example 10 Thirty liters of a culture filtrate (pH 4.0) (FR-1923 substance content: 32.4 g), obtained by substantially the same manner as described in Example l,were passed through a column packed with a macroporous non-ionic adsorption resin, Diaion HP20 (internal diameter of the column: 140 mm, packing height of the resin: 600 mm) at a rate of SV=l. The column was washed with 10 liters of water, whereafter FR-1923 substance was eluted with 0.01 N sodium hydroxide aqueous solution. 30.1 liters of active fractions were collected and then adjusted to pH 3.3 with 6 N hydrochloric acid. The solution was passed through a column of alumina (acidic, internal diameter of the column: 77 mm, packing height of alumina: 440 mm).
The column was washed with 2 liters of water, whereafter FR-1923 substance was eluted with 0.3M potassium biphosphate aqueous solution. 12. 3 liters of active fractions were collected k -17 -1046~5Z
and then concentrated under reduced pressure to the volume of 1,600 ml. The concentrate was adjusted to pH 2.2 with 6N hydrochloric acid while stirring, whereafter the solution was allowed to stand overnight in a refrigerator to. give 25.95 g of colorless crystals of FR-1923 substance.
.
The present invention relates to a new process for purification of FR-1923 substance. ~lore particularly, it relates to a new process for purification of FR-1923 sub-stanee, using a macroporous non-ionic adsorption resin.
FR-1923 substance is a known antibiotie isolated from the fermentation broth of a strain of genus Stre~tomvce~, as described, for example, in German Offenlegungsschrift 2242699 corresponding to Canadian Patent Application Ser.~o. 150,731 filed August 30,1972 Hatsuo Aoki et al.
In said prior literature, there are disclosed processes using aetivated ehareoal, anion exehange resin and DEAE-Sephadex (trade mark, made by Pharmacia Co.) for isolation and purification of FR-1923 substance from a fermentation broth.
However, the prior proeesses have not led to satisfae-tory results in an industrial manufacture of FR-1923 sub-stanee beeause they require relatively many steps for the isolation and purifieation of FR-1923 substanee, so that the overall recovery of the antibiotic is relatively poor.
As the results of the extensive studying, the present inventors have now newly found a new process for purification of FR-1923 substance as a step in the recovery thereof, by means of which FR-1923 substance can be obtained in higher yield than by the prior processes. Further, this new process is simpler and more convenient in practical operation and 1046()52 more economical on an industrial scale because the spent resin can be regenerated to be used again.
Accordingly, it is an object of the present invention to provide a new process for purification of FR-1923 substance usinq a macroporous non-ionic adsorption resin.
Another object of the present invention is to provide a new process for purification of FR-1923 substance, by means of which FR-1923 substance is obtained in higher yield than by the prior processes.
A further object of the present invention is to provide a simpler, more convenient and more economical process for puri-fication of FR-1923 substance.
According to the invention there is provided a process for the purification of FR-1923 substance which comprises:
contacting an aqueous solution containing FR-1923 substance, together with impurities, with a macroporous nonrionic adsorption resin, adsorbing one of said FR-1923 Aubstance and said impurities in ~aid solution on said re~in, and recovering said FR-1923 sub-stance.
In one aRpect of the invention the aqueous solution is contacted with the macroporous non-ionic adsorption resin to adsorb FR-1923 substance on the resin, the adsorbed FR-1923 substance i~ eluted from the re~in, and the FR-1923 substance is recovered from the eluate.
In another aspect of the invention the aqueous solution is contacted with a macroporous non-ionic adsorption resin saturated with FR-1923 substance, impurities are adsorbed on the saturated resin, and FR-1923 substance is recovered from the thus contacted solution.
As described above, the present invention provides a new process for purification of FR-1923 substance, using a macroporous non-ionic adsorption resin, which comprises alter-k ~ Jl ~ ~ 2 -r, ~, native methods in u~ing a macroporous non-ionic adsorption resin:
That is, one of them is a method that an aqueous solution containing FR-1923 substance, together with impurities, is contacted with a macroporous non-ionic adsorption resin, whereafter the adsorbed FR-1923 substance is eluted with, for example, a hydrophilic solvent system (hereinafter con-veniently referred to method A), The other is a method that an aqueous solution containing FR-1923 substance, together with impuritie~, is contacted with a macroporous non-ionic adsorption resin saturated by FR-1923 substance to adsorb the impurities, and FR-1923 substance is recovered from the B- 2a -passed solution (hereinafter conveniently referred to method B).
Alternative methods are explained in more detail hereinafter.
M~thod A
According to method A of the present invention, there is provided a process for purification of FR-1923 substance, wherein an aqueous solution containing FR-1923 substance, together with impurities, is contacted with a macroporous non-ionic adsorption resin, whereafter the adsorbed FR-1923 substance is eluted with, for example, a hydrophilic solvent system.
.
The macroporous non-ionic adsorption resins used according to the present invention may include a cross-linked . resin with an aromatic basic structure. The preferred resins include styrene polymers cross-linked with divinyl benzene. Examples of such resins include Amberlite XAD-l, XAD-2, XAD-4, XAD-7 and XAD-8 (trade mark, maker; Rohm &
Haas Co.), Diaion HP 10, HP 20, HP 30, HP 40 and HP 50 (trade mark, maker; Mitsubishi Kasei Co., Ltd.) and Hitachi-gel No. 3010 (trade mark, maker; Nissei Sangyo Co., Ltd.).
The process for purification of FR-1923 substance, using the macroporous non-ionic adsorption resin, in accord-ance with the method A of the present invention is applicable to any aqueous solution containing FR-1923 substance, together with impurities. Examples of such solutions include a filtrate of culture broth per se prepared by cultivating FR-1923 substance - producing microorganism in a nutrient medium, and a pre-extracted culture solution as well as an aqueous solution containing crude solid material containing F'R-1923 substance (e.g. crude powder or crude crystals containing FR-1923 substance) which first is isolated and purified to some degree from a filtrate of the culture broth by means of conventional manners, such as an optional treatment of the filtrate with an appropriate solvent for extraction and/or by adsorption on activated charcoal.
When using a culture filtrate, it is advantageous to use an optionally pre-extracted culture solution, this optionally pre-extracted culture solution being brought into contact with a macroporous non-ionic adsorption resin in the usual manner, preferably with use of a column containing a bed of resin.
Treatment of the aqueous solution containing FR-1923 substance in admixture with impurities with the non-ionic adsorption resin is advantageously carried out at pH value of from 2-7 and preferably of from 4 to 6. The desired acidic pE~ values can be achieved by using any desired acid, for example, an organic acid, such as oxalic acid, or preferably a mineral acid, such as hydrochloric, phosphoric or sulphuric acid.
Further, treatment of the aqueous solution containing FR-1923 substance in admixture with impurities with the non-ionic adsorption resin is advantageously carried out in case of the impurities being inorganic salt, such as sodium chloride, sodium biphosphate or disodium orthophosphate.
In this method, the aqueous solution containing FR-1923 1046Qsz substance in admixture with impurities is brought into contact with the macroporous non-ionic adsorption resin to adsorb FR-1923 substance by a conventional manner, for example batch operation or column operation, whereafter adsorbed FR-1923 substance is eluted from the resin with, for example, a hydrophilic solvent system.
The hydrophilic solvent systems used for eluting FR-1923 substance from the resin, include, for example, a lower dialkyl ketone (e.g. acetone and methyl ethyl ketone); and a lower alkanol (e.g. methanol, ethanol, n-propanol, iso-propanol, n-butanol and isobutanol). There may also be used mixtures of the above lower dialkylketone and lower alkanol.
Further, mixtures of watér or of a lower alkyl ester of a lower alkanoic acid, for example methyl acetate, ethyl acetate and butyl acetate, and the above lower alkanol or lower dialkyl ketone may also be used. Furthermore, a large volume of water and an alkaline aqueous solution Ipreferably pH 8 - 13) may be used.
The eluate thus obtained is treated by conventional means such as concentration, pH adjustment, lyophilization and recrystallization to give crystalline FR-1923 substance which frequently is sufficiently pure for use as a medicine.
Method B
According to method B of the present invention, there is provided a process for purification of FR-1923 substance wherein an aqueous solution containing FR-1923 substance, together with impurities, is contacted with a macroporous non-ionic adsorption resin saturated in advance by FR-1923 substance to adsorb impurities, and FR-1923 substance is recovered from the passed solution.
Thi~ method is based on the fact that adsorption capacity of a macroporous non-ionic adsorption resin to FR-1923 substance is smaller than that to specific impurities such as coloring materials stemming from fermentation broth and isomer of FR-1923 substance. By using the difference of capacity of macroporous non-ionic adsorption reqin between FR-1923 substance and the specific impurities, the specific impurities are selectively adsorbed to the resin saturated by FR-1923 substance from an aqueous solution containing FR-1923 substance, together with im-purities, and FR-1923 substance is recovered from the passed solution.
Ad~orption capacity of a macroporous non-ionic adRorption resin to ~R-1923 substance varies with condition of aqueous solution containing FR-1923 substance, together with impurities, and with the kind of the macroporous non-ionic adsorption reqin. Exampleq of adsorption capacity of a macroporous non-ionic adQorption reqin to FR-1923 sub-stance are as follows. The following data is given by contacting 0.2M phosphate buffer solution ~H4) containing FR-1923 substance with the resin.
Examples of adsorPtion caPacitY to FR-1923 substance Ca . lOg/l~ of Diaion HP 20*
Ca. 18g~1~ of Amberlite XAD-4*
*trademark t . - 6 -iO460S2 The macroporous non-ionic adsorption resin saturated by FR-1923 substance is prepared by contacting an aqueous solution containing FR-1923 substance with a macroporous non-ionic adsorption resin, wherein said solution is preferably neutral or slightly alkaline.
As examples of a macroporous non-ionic adsorption resin, the same examples as mentioned in the method A are exemplified.
The process for purification of FR-1923 substance using the macroporous non-ionic adsorption resin, in accordance with the method B of the present invention is applicable to any aqueous solution containing FR-1923 substance, together with impurities, as well as that of method A mentioned above, as examples of which the same examples as mentioned in the method A are exemplified.
Especially, when coloring materials or isomer of FR-1923 substance are contained as impurities in the aqueous solution,thls method B gives better results.
When an aqueous solution containing FR-1923 substance, together with impurities, is contacted with the macroporous non-ionic adsorption resin saturated by FR-1923 substance, FR-1923 substance can be adsorbed no more to it and, on the other hand, the impurities are selectively adsorbed to it.
Volume of the macroporous non-ionic adsorption resin to be used in this method varies with a content of FR-1923 substance in an aqueous solution containing FR-1923 substance, together with impurities, and with the kind of the resin to be used, and it, generally, may be lQ per 100 - 500 g of FR-1923 substance in the aqueous solution.
FR-1923 substance is recovered from the passed through solution, which is obtained by contacting an aqueous solution containing FR-1923 substance, together with impurities, with macroporous non-ionic adsorption resin saturated by FR-1923 substance. The recovery can be conducted by conventional manners such as pH adjustment, treatment with activated charcoal, concentration, lyophilization and recrystallization to give crystalline FR-1923 substance which frequently is sufficiently pure for use as a medicine.
Further, an aqueous solution containing FR-1923 substance, together with impurities, is contacted with a macroporous non-ionic adsorption resin saturated by FR-1923 substance to adsorb impurities, whereafter the column is washed with a small volume of water to obtain FR-1923 substance staying of the space among the resin in the column.
The spent resin can be regenerated by conventional manners, e.g. by washing the said resin with a relatively strong alkaline aqueous or alkaline aqueous-alcoholic solution.
The method B is superior to the method A and is more convenientin practiceand more economical on an industrial scale, in the following points. That is, the method B does not require a process for elution of FR-1923 substance from the resin, and volume of the resin used for purification in method B is less than 10% in comparison with that in method A, though recovery and purity of FR-1923 substance are almost 1046~SZ
same degree in both methods.
The following testsare given for the purpose of illustra-ting a comparison between method A and B as a process for ~urification of FR-1923 substance.
The comparison-tests are carried out by using the same crude crystals of FR-1923 substance and the same resin in ` the same volumein both methods, but volume of the crude crystals of FR-1923 substance in the method A is decided within the capacity of the resin to FR-1923 substance, and volume of the crude crystals of FR-1923 substance used in the method B is about 20 times that in method A.
Test A lI] Adsorption capacity of Diaion HP 20 to FR-1923 substance:
1.4 g of sodium salt of FR-1923 (purity : 95%) substance were dissolved in 1 liter of 0.2 M phosphate buffer solution (pH 4.0). The solution was adjusted to pH 4.0 with 5N
hydrochloric acid with stirring. The solution (concentration of FR-1923 substance : 1310 r/ml) was passed through a column packed with 100 ml of Diaion HP 20 (internal diameter of the column : 30 mm, packing height of the resin : 160 mm) at a rate of SV=l ("SV" means abbreviation of "Space Velocity").
The passed solution were collected lOOml by lOOml. Content of FR-1923 substance in the passed solution was determined by bioassay using Pseudomonas aeruginosa. Adsorption capacity of Diaion HP 20 to FR-1923 substance was calculated at the time when concentration of FR-1923 substance in the passed solution reaches the same concentration as that of the ~ ~r~o~ark g starting solution (1310 r/mQ~ to give 10.4 g / lite~.
[II] Purification of FR-1923 substance by the method A:
9.5 g of crude crystals of FR-1923 substance (FR-1923 substance content : 7.9 g) were suspended in 2.5 liters of water. The suspension was adjusted to pH 7.5 with 6N sodium hydroxide aqueous solution to dissolve in. Further, the solution was adjusted to pH 6.0 with 6N hydrochloric acid, and to the solution there were added 150 g of sodium chloride and water to give 3 liters of the solution. The solution was passed through a column packed with 1 liter of Diaion HP 20 (internal diameter of the column: 77 mm, packing height of the resin: 230 mm) at a rate of SV=l. The adsorbed FR-1923 substance was eluted with 30% aqueous methanol after the column was washed with water. Active fraction (1950 ml) were collected and concentrated under reduced pressure. The concentrate was adjusted to pH 2.2 with stirring and allowed to stand overnight in the refrigerator to give crystals.
The crystals were collected by filtration and dried. The results are shown in the following table.
lIII] Purification of FR-1923 substance by the method B:
Two hundred gram of crude crystals of FR-1923 substance (FR-1923 substance content : 168 g) being the same crude crystals as that used in Test [II], were suspended in 2 liters of water. The suspension was adjusted to pH 7.2 to A~ dissolve in. The solution was passed through a column packed ;~ with 1 liter of Diaion HP 20 saturated by FR-1923 substance (internal diameter of the column : 77 mm, packing height of the resin : 230 mm) at a rate of SV=l. Four liters of water ~rao~ n a~k -- :LO --.. .. . .
10460S;~
were passed through the column, 4500 ml of active fractions were collected from the passed solution, and to the solution obtained there was added water to give 16 liters of the solution. The solution was àdjusted to pH 2.5 with stirring and then allowed to stand overnight in the refrigerator to give crystals. The crystals were collected by filtration and dried. The results are shown in the following table.
The method A The method B
Capacity of the resin for treating crude 9.5 g / lQ of 187 g / lQ of crystals of FR-1923 resin resin substance Recovery of crystals 1 7.2 g 160 g Purity of crystals 92.7 % 98.4 %
Purity-calcd,recovery 84.5 % ¦ 93.6 %
of FR-1923 substance The above results show that volume of FR-lg23 substance treated by method B is about 20 times that by method A, though purity and recovery of FR-1923 substance are almost the same degree in both methods, respectively.
The following examples are given for the purpose of illustrating the present invention.
Example 1 To a culture broth prepared by culturing Nocardia ~niformis var. tsuyamanensis ATCC 21806 at 30C for 120 hours in 150 liters of a nutrient medium containing by weight 3%
glycerol, 2% cottonseed meal, 2% dried yeast, 2.18% potassium dihydrogen phosphate, 1.43% disodium hydrogen phosphate dodecahydrate, 0.5% magnesium chloride heptahydrate, there was added 6% of Radiolite (trade mark, a filter aid material sold by Showa Kagaku Kogyo Co., Ltd.) and then the mixture was filtered. Three liters of the filtrate obtained (FR-1923 substance content: 3030 mg) were passed through a A column packed with a macroporous non-ionic adsorption resin, Diaion HP 20 (internal diameter of the column : 8 cm, packing . height of the resin : 26 cm) at a rate of SV=l. The column was washed with water, whereafter FR-1923 substance was eluted with 20% of aqueous methanol solution. Three liters of eluate obtained were concentrated under reduced pressure and then adjusted to pH 2.5. The solution was allowed to stand in refrigerator to give 2490 mg of colorless crystals of FR-1923 substance.
Example 2 A culture broth which was prepared by substantially the same manner as described in Example 1 was adjusted to pH 4.0 with diluted sulfuric acid, whereafter the culture broth was filtered. To the filtrate obtained was added 6% of Radiolite, whereafter the mixture was filtered. Nine hundred ml of the filtrate obtained (FR-1923 substance content : 774 mg) were passed through a column packed with a macroporous non-ionic adsorption resin, Amberlite XAD-4(internal diameter of the t ~a O~ ark 1046C)52 column: 3.6 cm, packing height of the resin~30 cm) at a rate of SV=l. After the column was washed with water, FR-1923 sub-stance was eluted with 20~ aqueous acetone. Six hundred ml of eluate obtained were concentrated under reduced pressure, adjusted to pH 2.0 with diluted hydrochloric acid, and then allowed to stand in a refrigerator to give 365 mg of colorless crystals of FR-1923 substance.
Example 3 Three hundred ml of a culture filtrate (pH`4.0), obtained by substantially the same manner as described in Example 1, were passed through a column of a macroporous non-A~ ionic adsorption resin, Diaion HP 20 (internal diameter of the column : 30 mm, packing height of the resin : 160 mm) at a rate of SV=l. The column was washed with water, whereafter FR-1923 substance was eluted with aqueous sodium hydroxide solution (pH 12.0). Six hundred ml of active fraction obtained were collected and concentrated to 15 ml under reduced pressure. The concentrate was adjusted to pH 2.5 with 6N hydrochloric acid while stirring, whereafter the solution was allowed to stand for 24 hours in the refrigerator to give 360 mg of colorless crystals of FR-1923 substance.
Example 4 Twenty g of crude powder (FR-1923 substance content :
10.2 g) of FR-1923 substance were suspended in 6.2 Q of water. The suspension was adjusted to pH 8.1 with 6N aqueous sodium hydroxide solution, whereafter 50 g of sodium chloride were added to the solution; The solution was passed through ~ ~ r6(a~ h?ark ~046~52 a column of a macroporousnon-ionic adsorption resin, Diaion HP 20 (internal diameter of the column : 87 mm, packing height of the resin : 520 mm) at a rate of SV=l. The column was washed with water, whereafter FR-1923 substance was eluted with 30% aqueous methanol solution. Active fractions (4.48 liters) obtained were collected and then concentrated to the volume of 400 ml under reduced pressure. The concentrate was adjusted to pH 2. 5 with 6N hydrochloric acid and then allowed to stand overnight in refrigerator to give 10.0 g of colorless crystals of FR-1923 substance.
Example 5 Eighteen gram of crude powder (FR-1923 substance content:
10.2 g) of FR-1923 substance were suspended in 360 ml of water, whereafter the suspension was adjusted to p~I 7.0 with 6N sodium hydroxide aqueous solution to dissolve in. The solution was passed through a column packed with a macroporous non-ionic adsorption resin, Amberlite XAD-8 (internal diameter of the column : 30 mm, packing helght of the resin :
160 mm) saturated by FR-1923 substance at a rate of SV=l, whereafter 400 ml of water were passed through the column.
700 ml of active fractions were collected from the passed solution, and then the solution was concentrated under reduced pressure to a volume of 200 ml. The concentrate obtained was adjusted to pH 2. 5 with 6N hydrochloric acid while stirring, and then allowed to stand in a refrigerator for 24 hours to give 10.13 g of colorless crystals of FR-1923 substance.
~ ~ æhn~ r ~
. .
Example 6 Eighteen gram of crude powder (FR-1923 substance content:
10.2 g) of FR-1923 substance were suspended in 360 ml of water and adjusted to pH 7.0 with 6N sodium hydroxide aqueous solution to dissolve in. The solution was passed through a column packed with a macroporous non-ionic adsorption resin, Amberlite XAD-4 (internal diameter of the column : 30 mm, packing height of the resin : 160 mm) saturated by FR-1923 substance at a rate of SV=l, whereafter 540 ml of water were passed through the column. 900 ml of active fractions were collected from the passed solution, and then the solution was concentrated under reduced pressure to a volume of 350 ml. The concentrate obtained was adjusted to pH 2. 5 with 6N hydrochloric acid while stirring and then allowed to stand in a refrigerator for 24 hours to give 9. 41 g of colorless crystals of FR-1923 substance.
Example 7 Nineteen gram of crude power (FR-1923 substance content:
10. 7 g) of FR-1923 substance were suspended in 380 ml of water and then the suspension was adjusted to pH 7.0 with 6N sodium hydroxide aqueous solution to dissolve in. ~he solution was passed through a column packed with macroporous non-ionic adsorption resin, Amberlite XAD-7 (internal diameter of the column: 30 mm, packing height of the resin:
160 mm) saturated by FR-1923 substance at a rate of SV=l, whereafter 400 ml of water were passed through the column.
700 ml of active fractions were collected from the passed solution, whereafter the solution obtained was adjusted to pH 2. 5 with 6N hydrochloric acid while stirring and a~lowed ~ ~a6~QrnQrk to stand in a refrigerator to give 10.28 g of colorless crystals of FR-1923 substance.
Example 8 19.75 g of crude powder of FR-1923 substance (FR-1923 substance content: 17.16 g) were suspended in 395 ml of water and the suspension was adjusted to pH 7.5 with 5N
sodium hydroxide aqueous solution to dissolve in. The solution was passed through a column packed with a macroporous 1 A ~ non-ionic adsorption resin Diaion HP 20 (internal diameter of the column : 30 mm, packing height of the resin : 160 mm) saturated by FR-1923 substance at a rate of SV=l, whereafter 400 ml of water were passed through the column. 600 ml of active fractions were collected from the passed solution~
whereafter the solution obtained was adjusted to pH 2.5 with 6N hydrochloric acid while stirring and allowed to stand in a refrigerator to give 16.8 g of colorless crystals of FR-1923 substance.
Example 9 Fifteen liters of a culture filtrate (pH 4.0) (FR-1923 substance content: 14.25 g), obtained by substantially the same manner as described in Example l,were passed through a column packed with a macroporous non-ionic adsorption resin, Diaion HP20 (internal diameter of the coulum: 110 mm, packing height of the resin: 550 mm) at a rate of SV=l. The column was washed with 5 liters of water, whereafter FR-1923 substance was eluted with 30~
aqueous methanol solution. 7,940 ml of active fractions obtained were collected and then adjusted to pH 3.3 with * ~ 1ar ~
6N hydrochloric acid. The solution was passed through a column of alumina (acidic, internal diameter of the column: 46 mm, packing height of alumina: 300 mm).
The column was washed with 500 ml of water, whereafter FR-1923 substance was eluted with 0. 3 M pottassium biphosphate aqueous solution. 1800 ml of active fractions were collected and then concentrated under reduced pressure to the volume of 800 ml. The concentrate was adjusted to pH 2.2 with 6N hydrochloric acid while stirring, whereafter the solution was allowed to stand overnight in a refrigerator to give 11.94 g of colorless crystals of FR-1923 substance.
Example 10 Thirty liters of a culture filtrate (pH 4.0) (FR-1923 substance content: 32.4 g), obtained by substantially the same manner as described in Example l,were passed through a column packed with a macroporous non-ionic adsorption resin, Diaion HP20 (internal diameter of the column: 140 mm, packing height of the resin: 600 mm) at a rate of SV=l. The column was washed with 10 liters of water, whereafter FR-1923 substance was eluted with 0.01 N sodium hydroxide aqueous solution. 30.1 liters of active fractions were collected and then adjusted to pH 3.3 with 6 N hydrochloric acid. The solution was passed through a column of alumina (acidic, internal diameter of the column: 77 mm, packing height of alumina: 440 mm).
The column was washed with 2 liters of water, whereafter FR-1923 substance was eluted with 0.3M potassium biphosphate aqueous solution. 12. 3 liters of active fractions were collected k -17 -1046~5Z
and then concentrated under reduced pressure to the volume of 1,600 ml. The concentrate was adjusted to pH 2.2 with 6N hydrochloric acid while stirring, whereafter the solution was allowed to stand overnight in a refrigerator to. give 25.95 g of colorless crystals of FR-1923 substance.
.
Claims (21)
1. A process for the purification of FR-1923 substance which comprises:
contacting an aqueous solution containing FR-1923 substance, together with impurities, with a macroporous non-ionic adsorption resin, adsorbing one of said FR-1923 substance and said impurities in said solution on said resin, and recovering said FR-1923 substance.
contacting an aqueous solution containing FR-1923 substance, together with impurities, with a macroporous non-ionic adsorption resin, adsorbing one of said FR-1923 substance and said impurities in said solution on said resin, and recovering said FR-1923 substance.
2. A process according to claim 1, which comprises contacting said aqueous solution with said macroporous non-ionic adsorption resin to adsorb FR-1923 substance on said resin, eluting the adsorbed FR-1923 substance from the resin, and recovering FR-1923 substance from the eluate.
3, A process according to claim 2, wherein said adsorbed FR-1923 substance is eluted from the resin with a hydrophilic solvent system.
4, A process according to claim 2, wherein said adsorbed FR-1923 substance is eluted from the resin with an aqueous alkaline solution.
5. A process according to claim 1, which comprises con-tacting said aqueous solution with a macroporous non-ionic adsorption resin saturated with FR-1923 substance, adsorbing said impurities on said saturated resin and recovering FR-1923 substance from the thus contacted solution.
6. A process according to claim 2 or 5, wherein said aqueous solution is a filtrate of a culture broth containing FR-1923 substance.
7. A process according to claim 2 or 5, wherein said aqueous solution is a pre-extracted culture solution containing the FR-1923 substance.
8. A process according to claim 2 or 5, wherein said aqueous solution is in an aqueous solution of crude solid material containing FR-1923 substance.
9, A process according to claim 5, wherein said aqueous solution is in an aqueous solution containing FR-1923 substance together with coloring materials.
10. A process according to claim 5, wherein the aqueous solution containing the FR-1923 substance is in an aqueous solution containing FR-1923 substance together with isomer of FR-1923 substance.
11. A process according to claim 2 or 5, wherein the adsorption resin is a macroporous crosslinked resin of aromatic basic structure.
12. A process according to claim 2 or 5, wherein the resin is a macroporous styrene polymer crosslinked with di-vinylbenzene.
13. A process according to claim 3, wherein the hydro-philic solvent system is a lower dialkyl ketone, a lower alkanol, a mixture of a lower dialkyl ketone and a lower alkanol, or an aqueous solution of a lower dialkyl ketone, lower alkanol or mixture thereof.
14. A process according to claim 4, wherein the elution is carried out with said alkaline solution at a pH of from 8 to 13.
15. A process according to claim 13, wherein the hydro-philic solvent system is a mixture of water and a lower alkanol.
16. A process according to claim 13, wherein the hydro-philic solvent system is a mixture of water and a lower di-alkylketone.
17. A process according to claim 15, wherein the lower alkanol is methanol.
18. A process according to claim 15, wherein the lower alkanol is ethanol.
19. A process according to claim 15, wherein the lower alkanol is propanol.
20. A process according to claim 16, wherein the lower dialkylketone is acetone.
21, A process according to claim 1 or 2, wherein the contacting is carried out at a pH of from 4 to 6.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3607974A JPS57795B2 (en) | 1974-03-28 | 1974-03-28 | |
| JP2214075A JPS5737319B2 (en) | 1975-02-21 | 1975-02-21 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1046052A true CA1046052A (en) | 1979-01-09 |
Family
ID=26359317
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA222,363A Expired CA1046052A (en) | 1974-03-28 | 1975-03-18 | Process for the purification of fr-1923 substance |
Country Status (7)
| Country | Link |
|---|---|
| AU (1) | AU500312B2 (en) |
| CA (1) | CA1046052A (en) |
| DE (1) | DE2512396A1 (en) |
| ES (1) | ES436092A1 (en) |
| FR (1) | FR2265400B1 (en) |
| GB (1) | GB1441897A (en) |
| NL (1) | NL7503718A (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB8324152D0 (en) * | 1983-09-09 | 1983-10-12 | Glaxo Group Ltd | Chemical process |
-
1975
- 1975-03-18 CA CA222,363A patent/CA1046052A/en not_active Expired
- 1975-03-20 GB GB1165375A patent/GB1441897A/en not_active Expired
- 1975-03-21 DE DE19752512396 patent/DE2512396A1/en not_active Withdrawn
- 1975-03-26 ES ES436092A patent/ES436092A1/en not_active Expired
- 1975-03-27 AU AU79582/75A patent/AU500312B2/en not_active Expired
- 1975-03-27 FR FR7509678A patent/FR2265400B1/fr not_active Expired
- 1975-03-27 NL NL7503718A patent/NL7503718A/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| ES436092A1 (en) | 1977-06-16 |
| GB1441897A (en) | 1976-07-07 |
| DE2512396A1 (en) | 1975-10-02 |
| FR2265400B1 (en) | 1980-02-08 |
| FR2265400A1 (en) | 1975-10-24 |
| NL7503718A (en) | 1975-09-30 |
| AU500312B2 (en) | 1979-05-17 |
| AU7958275A (en) | 1976-09-30 |
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