HU199563B - Process for production of antibiotics of nebramicin and neomicin type - Google Patents

Abstract

The present invention relates to a process for preparing nebramycin and neomycin-type antibiotics from a fermentation broth or from an aqueous solution containing antibiotics of any other origin using a carboxyl-type cation exchange resin and / or an adsorption resin on one or more occasions, the cation exchange resin being ammonium hydroxide. by eluting an aqueous solution of the eluate and the adsorbent effluent from the adsorption resin in a manner known per se, such that at least once prior to administration to the resin, the pH of the aqueous solution containing the antibiotic is 9.5-12.0, preferably 10.2-11. , Set to 0. Scope of the description: 12 pages, without figure EN 199563 A -1-

Description

The present invention relates to a process for the preparation of nebramycin and neomycin antibiotics by recovery and / or purification from fermentation broth or aqueous solution of any other origin containing the antibiotics using one or more carboxylic ion exchange resins and / or adsorbent resins prior to application to the resin. at least once, the pH of the fermentation broth or aqueous solution containing the antibiotic is adjusted from 9.5 to 12.0, preferably from 10.2 to 11.0.

The essence of the invention is in fact a high-purification step which can be implemented in two ways:

a) An aqueous solution of the antibiotic base (e.g. neomycin, tobramycin, kanamycin, apramycin) adjusted to an alkaline pH range is passed through a weakly acidic cationic resin. The valuable active ingredient binds to the resin, while in the effluent brine and in the washing water, very large amounts of impurities are removed. The resin-bound and impurity-free antibiotic base is eluted in a known manner with an aqueous solution of ammonium hydroxide.

b) passing an aqueous solution of an antibiotic base (e.g. apramycin) adjusted to an alkaline pH range through an adsorption resin. The resin binds a very large amount of contaminants, leaving the purified antibiotic in the effluent and in the wash water.

The resulting purified antibiotic-containing solutions can be prepared in substantially higher yields than the prior art using a highly purified antibiotic base or a pharmaceutically acceptable salt thereof.

In practice, the production of aminoglycoside antibiotics is mainly carried out by fermentation. Their extraction from fermentation broth can be accomplished e.g. extraction (Czechoslovakia 84,708 and Hungarian Patent Specification 160,456), formation of a water-insoluble complex salt (U.S. Patent No. 2,944,939) or binding on an ion exchange resin. Among the ion-exchange resins, the weakly acidic carbolxyl-type ion-exchange resins are the most widely used in practice (U.S. Patent Nos. 2,667,441, 3,691,279, 3,962,827, 4,023,404 and 169,952).

The major problem in recovering any aminoglycoside antibiotic in high purity and its most labor-, material- and energy-consuming operation is the removal of related compounds, inactive substances and colorants.

Namely, strains producing the aminoglycoside-type antibiotics (Streptomyces fradiae, Streptomyces tenebrarius, Streptomyces kanamyceticus) are known to produce dyes with very similar properties to the active ingredients and to produce inactive UV-absorbing inactive components. they are difficult and difficult to separate. Contaminants degrade the chances of drug recovery, the efficiency of ion exchange, the selective separation of individual anibiotic components, and the efficiency of crystallization. Their presence not only reduces the cost-effectiveness of production, but also poses a problem for the mourning of human or animal 2 of sufficient purity! and compliance with the quality requirements for pharmaceutical products, in particular for the manufacture of pharmaceutical specialties.

Several approaches have been attempted to reduce the amount of contaminants that accompany various aminoglycoside antibiotics.

During the processing of the Nebramycin Complex fermentation broths, repeated ion exchange or purification on a sephadex column is used for separation and purification of the active compounds, which is combined with the purification of the eluates with a large amount of activated carbon (U.S. Pat. No. 3,691,279; U.S. Pat. No. 174,315; Japanese Patent Specification). However, even when large amounts of activated carbon are added, only a portion of the dyes and inactive components can be removed with considerable loss of material. However, contaminants remaining after clarification inhibit the efficient crystallization of the tombramycin and kanamycin bases, which results in a further decrease in yield.

There are even more problems with the processing of apramycin because the removal of the colorant that accompanies apramycin is even more difficult and requires a greater use of activated carbon.

For the purification of neomycin, the ion exchange on a highly acidic cation exchange resin is repeated according to U.S. Patent Nos. 2,667,441, 2,944,939, and 3,005,815, or various additives such as sodium fluorite are used in the French nonionic deuterium 1,360,928. 116,551 Czechoslovakians, EDTA US 3,313,694 and Wofatit EZ decolorizing resin according to Hungarian patent application 162,943, and treatment with bromine water, washing with 56,365 Polish, 2% acetic acid and hydrogen peroxide digestion according to Hungarian Patent Application No. 181,748.

It is known that the attempt to increase valuable components in the fermentation broth, changing the fermentation conditions, is accompanied by a surge of inactive components and dyes. Therefore, it is necessary to combine the above methods with increasing amounts of activated carbon treatment to assure an adequate quality of the finished product (e.g. 0.4-0.6 kg / kg for tobramycin, 2.0 kg / kg for apramycin, 0.3- 0.5 kg of activated carbon is used).

SUMMARY OF THE INVENTION The object of the present invention is to provide a process which makes the production of fermentable aminoglycoside antibiotics (tobramycin, kanamycin, apramycin, neomycin) simpler and faster than hitherto known complex and time-consuming processes, and provides a substantially higher yield of highly pure product.

SUMMARY OF THE INVENTION It is an object of the present invention that the binding of weakly basic aminoglycoside antibiotics to a carboxylic cation exchange resin at an alkaline pH range can be achieved at or above the pK value of the most basic group of each antibiotic.

Known applications in the ion exchange processing of aminoglycoside antibiotics known in the art-21

EN 199563 Maximum pH for nebramycin-type antibiotics is pH 9.0 (Hungarian Patent No. 174,815) and for neomycin is pH 8.5 (Hungarian Patent No. 181,748), not for the recovery of active ingredients and mainly for purification. is the difference with the generally lower pH of 6-8. It is consistent with the common practice for weakly acid cation exchange resins to operate in the pH range of 6 to 10 and to use a pH of one or two units lower than its pK for the most effective binding, considering the basicity of the substance to be bound. The degree of purification of the active compounds at the above-mentioned pH 9.0 and pH 8.5 ion exchange rates is insignificant, with other purification operations following the ion exchange (proper carbon clarification, use of a strongly basic anion exchange resin, hydrogen peroxide destruction). implemented.

It is also known that in recent years, in parallel with efforts to increase the active ingredient content of fermentation broth, the pH used in the production of nebramycinic neomycin-type antibiotics is shifted to the acidic pH range (pH 4.5).

The fact that the ion exchange can be carried out in the alkaline pH range of 9.5 to 12.0 is surprising, since the bonding of weak bases to the resin takes place in protonated form and the degree of protonation gradually decreases with increasing pH. In the pH range, the active ingredients would not be able to bind to the resin.

By using an alkaline pH range for ion exchange according to the invention, it is possible to create, in contrast to the practice so far, conditions under which only the active ingredient is bound to the resin by its very basic activity and its impurities and minor constituents. worthless components are squeezed out of it. Further processing of the substantially pure eluate containing the active ingredient, selectively separating the individual antibiotic components, or clarifying the solution to be crystallized or precipitated with significantly less activated carbon, can be accomplished by other treatment methods used to remove dyes (e.g. bromination, hydrogen peroxide treatment). ) become redundant and the product can be crystallized with much better efficiency.

Within the alkaline pH range, a range of 9.5-12.0, preferably 10.2-11.0, has been found to be optimal in terms of purification efficiency.

Comparing ion exchange in the alkaline pH range with conventional ion exchange at a neutral pH of about 7.5, the difference in purity is illustrated by the difference in the extinction values of the eluates obtained after gradient elution chromatography:

E ^4% 1 cm (295 nm) pH: 7.5 1.4-1.6 pH: 10.2-10.8 0.4-0.4

The process of the invention can be carried out with any acidic cation exchange resin e.g. Lewatit CNP-80,

Amberlite IRC-50, Varion KCO, Wofatit CP-300, etc. and many adsorption resins can be used in the alkaline pH range, e.g. Diaion HP-20, Diaion HP-21, Diaion SP-207, XAD-1184 and so on.

The ion exchange or adsorption process in the alkaline pH range can be employed in several variants in combination with each known technological step in processing the filtered fermentation broth. In the case of production of tobramycin or kanamycin, in a preferred embodiment of the present invention, the ion exchange in the alkaline pH range is incorporated into the fermentation broth processing step so that the filtered, neutral (pH 7.5) fermentation broth is weakly acidic cation exchange resin. ion exchange followed by dissolution of the active compound with ammonium hydroxide, the solution containing carbamoyl tobramycin and carbamoylcannamycin is hydrolyzed in a strong alkaline medium with sodium hydroxide and then adjusted to a pH of 10.2-10.8 with sulfuric acid. and applied to the weakly acidic cation exchange resin. After elution of the active compounds with an aqueous solution of ammonium hydroxide, the resulting solution was concentrated in vacuo to ca. Concentrate to 30% solids, adjust the pH to 7.5 with sulfuric acid, and separate the tobramycin and kanamycin by stepwise elution chromatography. Solutions containing pure tobraimycin and pure kanamycin base are concentrated in vacuo, clarified with a small amount of activated carbon, the tobramycin base is crystallized and the kanamycin sulfate is precipitated from methanol.

In the case of ion exchange in the alkaline pH range as described above, not only the antibiotic bases but also the sodium ions in solution are bound to the ion exchange resin. Thus, during this operation, a desalting process (sodium sulphate removal) is also carried out. The practical importance of this is because large amounts of sodium sulphate must be removed prior to the step gradient elution chromatography, which results from the neutralization of the strongly alkaline hydrolyzate with sulfuric acid. Until now, the sodium sulphate deprotection has been accomplished by dripping the sodium sulphate-containing hydrolyzate into six volumes of methanol per volume of solution and filtering out the sodium sulphate. The aqueous methanolic solution of the active ingredient was de-solvented in vacuo. In addition to the labor, time, equipment and energy requirements of these operations, other environmental and safety concerns are raised, such as preventing the introduction of methyl alcohol into the atmosphere, destroying sodium sulfate contaminated with methyl alcohol, and regeneration of methyl alcohol.

The ion exchange purification in the alkaline pH range may also be used if an aqueous concentrate of the aminoglycoside antibiotic (e.g., aqueous solution of tobramycin, kanamycin, apramycin, neomycin, etc.) is obtained by ion exchange or chromatography other than the above procedure.

The present invention is also applicable if the antibiotic is recovered from the cementitious juice by other means than by binding to an ion exchange resin, e.g. by extraction or by complex salt formation. In this case, the processing shall:

GB 199563 An aqueous solution of the active ingredient is prepared in some way (eg, by evaporation of the solvent used for the extraction, or by dissolution of the complex salt in the active ingredient), the contents of which are read as described below.

The ion exchange in the alkaline pH range can also be used to recover and purify the active ingredient directly from the filtered fermentation broth. In this case, the pH of the filtered fermentation broth is adjusted to 10.2-10.8, and the active compound is bound at this pH. Most of the impurity components, dyes, are already removed from the weakly acidic cation exchange resin by the effluent solution and the washing water. The resin-bound active ingredient 15 can be readily resolved and further processed using known technology steps with good efficiency.

The efficiency of this variant of the process is further enhanced by the warming of the active compound in the filtered fermentation broth on an ion exchange resin and dissolution in an aqueous solution of ammonium hydroxide, preferably at 60 ° C.

Especially in the case of an eluate containing apramycin which is particularly rich in coloring agents, the process may be advantageously carried out by diluting the solution to an adsorption resin at a pH of 9.5 to 12.0, preferably 10.2 to 11.0. , Diaion-20, Diaion-21, XAD-1184, etc.), and the apramycin-containing solution and wash water flowing from the column are processed according to the already known technological steps.

The invention is further illustrated by the following examples, without limiting the scope thereof.

Example 1

Streptomyces tenebrarius K-26, strain NCAIMB (P%) 00169, is incorporated herein by reference. According to the Hungarian patent application No. 39,339, fermented 39 m ³ nebramycin containing phenol broth (total activity 40 by bioautographic measurement 1968 U / ml) is acidified to pH 2.1 with oxalic acid and filtered through a pre-drum filter while the filter layer is washed with water. The filtered juice was adjusted to pH 7.5 with concentrated ammonium hydroxide and the precipitate re-filtered through 45 pre-drum filters.

m ^{3 of} neutral filtered juice is applied to a Lewatit CNP-80 weakly acid cation exchange resin in a 3500 liter ammonium cycle at a rate of 2500 l / h. After curing the drug, the resin 50 bed is washed in a 2500 rpm bed with 12 m ³ deionized water.

The active compound was dissolved and separated first with 0.1 M ammonium hydroxide solution (37.5 m ³ ) and then with 0.3 M ammonium hydroxide solution (21 m ³ ). Elution was carried out in a 300 l / hr bed. During dissolution, the eluate is collected in 200 L units and analyzed by thin layer chromatography. The worthless, non-active color fractions are drained. 60

The apramycin-containing fractions (fraction A, volume 14,500 liters) were collected and concentrated under vacuum at 40 ° C to 30% solids. The concentrate was processed as in Example 5. 65

The carbamoyl-kanamycin and carbamoyl tobramycin containing fractions (the so-called fraction "B" with a volume of 19,500 liters) were concentrated in vacuo at 40 ° C.

The resulting concentrate "B" contains 227 liters of 38.5% solids, ie 87.40 kg of carbamoylcannamycin, carbamoyl tobramycin, and many minor components and colorants.

The 227 liter concentrate was hydrolyzed with 60 kg of sodium hydroxide for 10 hours at 100 ° C. The hydrolyzate was cooled to 25 ° C and diluted to 600 liters with deionized water. To the solution of pH 13.88 was added 180 liters of 5 M sulfuric acid and the pH was adjusted to 10.61. The solution was diluted to a volume of 3 m ³ (6.8% solids content) and passed through a Lewatit CNP-80 resin on a 3400 liter ammonium cycle at 300 L / hr. The resin bed is washed with 10 m ³ deionized water at 300 l / h. The washing water removes most of the dyes and minor components. The resin-bound drug is dissolved in a bed of 200 l / h with 2% ammonium hydroxide solution (8 m ^{3 in} total). The eluate is collected in m ³ aliquots. The drug-containing fractions were pooled by thin layer chromatography (total volume 4 m ³ ) and concentrated in vacuo at 40 ° C and de-ammonia.

The resulting concentrate contains 300 liters of 21.3% solids, i.e. a total of 63.90 kg of kanamycin, tobramycin, and a small amount of minor components and colorants.

The solution containing 300 L of kanamycin and tobramycin was diluted to 800 L and the pH was adjusted to 7.45 with 5 M sulfuric acid (60 L).

The solution is passed to a Varion KCO weak acid carboxyl type resin in a 1500 liter ammonium cycle at a rate of 25 L / h. The resin bed is first washed with 200 L of deionized water at 25 L / hr and then with 2 m ^{3 of} water at 100 L / hr. The active ingredient was separated in a standing bed of 9 m ^{3 of} 0.05 M, then 9 m ^{3 of} 0.10 M, and finally 39 m ^{3 of} 0.15 M ammonium hydroxide at 150 L / h. The eluate was collected in 200 L portions and examined by thin layer chromatography.

The worthless, non-active fractions are channeled.

The kanamycin-containing fractions were combined (7400 L volume), concentrated in vacuo at 40 ° C, and de-ammoniated.

The resulting concentrate contains 100 liters of 15% solids, i.e. a total of 15.00 kg of kanamycin base.

The typical parameter of the solution is:

E ^4% 1 cm (295 nm) - 0.430

The solution was adjusted to pH 5.7 with 20 L of 5 M sulfuric acid and clarified with 1.0 kg of activated carbon. The carbon bed is washed with 10 liters of deionized water.

The resulting solution contained 130 liters of 16.5% solids, i.e. a total of 21.45 kg of kanamycin sulfate.

The 130 liters of solution were added into six volumes of methanol (780 liters). The precipitated kanamycin sulfate crystals were filtered off and the precipitate was suspended in methanol (2 x 50 L) and dried at 50 ° C under vacuum.

HU 199563 A

Finished product: 21.30 kg kanamycin sulfate.

Yield: 546.15 g / m ²

The main quality characteristics of the recovered product are:

E ^4% i _C m (295 nm) - 0.145

Purity test: TLC (total size and intensity of foreign spots): 4.5%, Rf (24 'C): 0.15.

Biological action on dry matter: 625 pm / mg.

The tobramycin fractions obtained by chromatography (20.8 m ³ ) were evaporated in vacuo at 40 ° C and de-ammoniated.

The resulting concentrate contained 163 liters of 15.0% solids, i.e. a total of 24.45 kg of tombramycin base.

The typical parameter of the solution is:

E ^4% 1 cm (295 nm) - 0.570

The solution was clarified with 2 kg of activated carbon, the carbon bed was washed with 50 liters of deionized water, and the filtrate and the washings were evaporated in vacuo at 35 ° C to 35.5% solids.

The resulting concentrate contains 64 liters of 35.5% solids, i.e. a total of 22.72 kg of tobramycin base.

From this solution, the solid tobramine base product is obtained by crystallization from ethyl alcohol in a known manner.

Finished product: 17.55 kg tobramycin base active ingredient.

Yield: 450 g / m ³

The main quality characteristics of the recovered product are:

E ^4% 1 cm (295 nm) - 0.124

Purity test: TLC (individual spot size and intensity): 0.5%

Purity test: TLC (total size and intensity of foreign spots): 15% [a] ®D + 149 '(c - 1 water)

Example 2

The fermented broth of Example 39, 39 m ³ (total activity 1872 U / ml by bioautographic measurements), was obtained by filtration, ion exchange, elution and evaporation of the eluate containing 210 liters of 39% solids, ie a total of 81.90 kg of carbanoylcannamycin, carbamoyl tobramycin , as well as so-called "minor" components and colorants. Fraction B was hydrolyzed with 60 kg of sodium hydroxide for 10 hours at 100 ° C. After cooling to 25 ° C, the pH of the hirolysate was adjusted to 10.6 with 160 L of 5 M sulfuric acid and the solution was sparged with six volumes of 2,820 L of methanol. The precipitated sodium sulfate precipitate was separated by centrifugation from the aqueous methanol supernatant containing the active ingredient. The precipitate was washed with 1000 L of methanol. All methanolic solutions were removed in vacuo at 40 ° C and evaporated.

The resulting aqueous hydrolyzate contains 190 liters of 43.5% solids, i.e. a total of 82.65 kg of kanamycin, tobramycin, many minor components and colorants, and a small amount of sodium sulfate.

The solution containing 190 liters of kanamycin and tobramycin was diluted to 800 liters and the pH was adjusted to 7.48 with 5M sulfuric acid.

The components were isolated as described in Example 1 on a Varion KCO resin in a 1500 liter ammonium cycle.

The tobramycin containing fractions (volume 21.2 m ³ ) were evaporated in vacuo and de-ammoniated.

The resulting concentrate contained 162 liters of 15.0% solids, i.e. a total of 24.30 kg of tobramycin base.

The typical parameter of the solution is:

E ^4% 1 cm (295 nm) - 1.540

A solution of 162 liters of 15.0% solids, i.e. a total of 24.30 kg of tobramycin base, having a pH of 10.74, is passed through a bed of Lewatit CNP-80 resin on a 1080 liter ammonium cycle at 10 1 / h speed. The resin bed is washed first with 10 L / hr (300 L total) and then with 50 L / L (1200 L total) in deionized water. Many colors and minor components are removed in the wash water. After washing with water, the resin-bound drug is dissolved in a 2% ammonium hydroxide solution in a standing bed at 100 L / h (a total of 4 m ³ solution is required). The resulting eluate containing 2000 liters of active ingredient was concentrated in vacuo at 40 ° C and deprotected.

The resulting concentrate contained 145 liters of 16.0% solids, i.e. a total of 23.20 kg of tobramycin base.

The typical parameter of the solution was E ^4% 1 cm (295 nm) - 0.548

The solution was clarified with 2 kg of activated carbon, the carbon bed was washed with 50 L of deionized water, and the filtrate and washings were evaporated to 35.5% solids in vacuo at 40 ° C.

The resulting concentrate contains 62 liters of 35.5% solids, i.e. a total of 22.01 kg of tobramycin base.

The solution gives a solid tobramycin base by known crystallization techniques.

Finished product: 16.80 kg of tobramycin base active ingredient.

Yield: 430.8 g / m ³

Important quality parameters of the recovered product:

E ^4% 1 cm (295 nm) - 0.132

Purity Test: TLC (individual size and intensity of foreign spots): 1%

Purity test: TLC (total size and intensity of foreign spots): 2%

Biological limit for solids: 990 pm / mg [a] ²⁰ D - + 147 '(c - 1, water)

Example 3

The filtration pH of 65 liters of neutral nebramycin obtained from 39 liters of fermented broth (total activity by 1840 U / ml by bioautographic measurement) was adjusted to 10.45 with 10M sodium hydroxide solution. The solution is passed through a standing bed at a flow rate of 2500 ml / h on a Lewatit CNP-80 resin in a 3500 ml ammonium cycle. After curing the drug, the resin bed is washed in a bed (2500 mL / hour) with deionized water (13 liters total). Many colors and inactive minor components are removed in the wash water.

The active ingredient is dissolved in 37 L of 0.1 M ammonium hydroxide solution and then 21 L of 0.3 M ammonium hydroxide at 300 mL / h. The so-called carbamoyl-kanamycin and carbamoyl-tobramycin-containing so-called. Fraction B was concentrated in vacuo at 40 ° C.

The resulting concentrate contained 180 ml of 36% solids, ie a total of 64.80 g of carbamoylcannamycin.

EN 199563 A and carbamoyl tobramycin, as well as low in color and inactive minor component.

Fraction B (180 ml) was hydrolyzed with sodium hydroxide (54 g) at 100 ° C for 10 hours and the solution diluted to 600 ml (Example 1) and adjusted to pH 10.58 with 5 M sulfuric acid (100 ml). . The solution was diluted to 3000 ml (pH 10.49) and passed through a bed of Example 1 at a flow rate of 300 ml / h on a Lewatit CNP-80 type resin in an ammonium cycle of 3400 ml. The resin bed is washed with 8 liters of deionized water in a standing bed at 300 ml / h.

The resin-bound drug was eluted in a bed of 200 ml / hr with 2% ammonium hydroxide solution (7 liters total required). The eluate containing the active ingredient (3500 ml) was concentrated in vacuo at 40 ° C and deprotected.

The resulting concentrate contained 200 ml of 29.2% solids, i.e. a total of 58.40 g of tobramycin and kanamycin.

The resulting concentrate (200 mL) was diluted to 500 mL, adjusted to pH 7.50 with 5 M sulfuric acid, and isolated as described in Example 1 on a Varion KCO resin on a 1500 mL ammonium cycle.

The tobramycin-containing fractions (15 L) were concentrated in vacuo.

The resulting concentrate contained 155 ml of 15.5% solids, i.e. a total of 24.03 g of tobramycin base.

The typical parameter of the solution is:

E ^4% 1 cm (295 nm) - 0.490

The solution was clarified with 2 g of activated carbon, the carbon bed was washed with 50 ml of deionized water, and the filtrate and washings were evaporated in vacuo at 35 ° C to 35.5% dry weight.

The resulting concentrate contained 62 ml of 35.5% solids, i.e. a total of 22.01 g of tobramycin base.

From the solution, a solid tobramycin base product is obtained by known crystallization techniques.

Finished product: 17.16 g of tobramycin base.

Yield: 0.440 g / liter

The main quality characteristics of the recovered product are:

E ^4% 1 cm (295 nm) - 0.195

Purity Test: TLC (individual size and intensity of foreign spots): 1%

Purity test: TLC (individual spot size and intensity): 2.5%

Biological activity on dry matter: 985 pg / mg [a] ²⁰ o - +150 '(c - 1, water)

Example 4

67 liters of neutral nebramycin filtered juice obtained from 39 liters of fermentation (total activity by 2000 U / ml by bioautographic measurement) was passed through a heat exchanger and heated to 60 ° C with 3500 ml of ammonium-cycled Lewatit CNP- Type 80 resin is passed through a standing bed at 2500 ml / h. During ion exchange, the duplicated ion exchange column was maintained at 60 ° C, constant temperature. After curing, the resin bed was washed with 12 liters of 60 C deionized water in a bed at 2500 ml / h. Many colors and inactive minor components are removed in the wash water. The active ingredient was eluted with 37 liters of 0.10M ammonium hydroxide solution, followed by 19.5 liters of 0.3M ammonium hydroxide solution preheated to 60 ° C in a standing bed at 300 ml / h.

The so-called carbamoyl-kanamycin and carbamoyl-tobramycin-containing so-called. Fraction B was concentrated in vacuo at 40 ° C.

The resulting concentrate contains 154 ml of 36.2% solids, i.e. a total of 55.75 g of carbamoylcannamycin and carbamoyl tobramycin, as well as a small amount of coloring matter and minor components.

Fraction "B" (154 ml) was hydrolyzed with 45 g of sodium hydroxide for 10 hours at 100 ° C. After cooling, the solution was diluted to 600 mL and adjusted to pH 10.50 (100 mL) with 5 M sulfuric acid. The solution was diluted to 3000 mL (pH 10.45) and passed through Lewatit CNP-80 resin on a 3400 mL ammonium cycle as described in Example 1 at 300 mL / hr and washed on a standing bed at 300 mL / hr. and finally the active ingredient is dissolved in 2% ammonium hydroxide solution. The drug-containing fractions were concentrated in vacuo and demineralized.

The resulting aqueous hydrolyzate contains 190 ml of 25% solids, i.e. a total of 47.50 g of tobramycin, kanamycin, and very little coloring matter and minor component.

The hydrolyzate was chromatographed as in Example 1 on a Varion KCO resin in 1500 ml of ammonium cycle.

The tobromycin containing fractions were concentrated in vacuo at 40 ° C.

The resulting concentrate contained 191 ml of 13% solids, i.e. a total of 24.83 g of tobramycin base.

The typical parameter of the solution is:

E ^4% 1 cm (295 nm) - 0.430

The solution was clarified with 2 g of activated carbon, the carbon bed was washed with 50 ml of deionized water, and the filtrate and washings were evaporated in vacuo to 35.5% solids.

The resulting concentrate contained 66 ml of 35.5% solids, i.e. a total of 23.43 g of tobramycin base.

From this solution, the solid tobramycin base is obtained in a known manner by crystallization from a solvent.

Finished product: 18.33 g of tobramycin base.

Yield: 0.470 g / liter

The main quality characteristics of the recovered product are:

E ^4% 1 cm (295 nm) - 0.151

Purity Test: TLC (individual size and intensity of foreign spots): 1%

Purity test: TLC (total size and intensity of foreign spots): 2%

Biological action on dry matter: 996 gg / mg.

[α] ²⁰ D + 150 '(c-1, water)

Example 5

In Example 1, 14,500 liters of an eluate containing apramycin was obtained, the so-called. Fraction A was concentrated in vacuo at 40 ° C and de-ammoniated.

The resulting concentrate A contains 140 liters of 30% dry matter, or 42.00 kg apramycin base.

HU 199563 A Contains many coloring matter and minor components.

The 140 liter concentrate, pH 10.38, was diluted to 500 liters (pH 10.33) and passed through a bed of Lewatit CNP-80 resin on a 1800 liter ammonium cycle at 300 L / hr. The resin bed is washed in a bed of 300 l / h with 8 m ³ deionized water. Many colors and minor components are removed in the wash water.

The resin-bound drug is dissolved in a 2% ammonium hydroxide solution in a 200 l / h bed (total 5 m ³ solution required).

The apramycin-containing eluate (3 m ³ volume) was concentrated in vacuo at 40 ° C.

The resulting concentrate contained 200 liters of 16.8% solids, i.e. a total of 33.60 kg of apramycin base.

The 200 liter concentrate was adjusted to pH 5.74 with 22 liters of 5 M sulfuric acid and clarified with 5 kg of activated carbon. The carbon bed is washed with 50 L of deionized water and the filtrate and washings are concentrated in vacuo at 40 ° C.

The resulting concentrate contained 175 liters of 23% solids, i.e. a total of 40.25 kg of apramycin sulfate.

The typical parameter of the solution is:

E ^4% 1 _cm (295 nm) - 0.135

The 175 liters of solution were added to six volumes (1050 liters) of methanol. The precipitated apramycin sulfate crystals were filtered off, the precipitate was suspended in methanol (2 x 100 liters) and the product dried in vacuo at 50 ° C.

Finished product: 40.20 kg of apramycin sulfate.

Yield: 1030.8 g / m ³

The main quality characteristics of the recovered product are:

E ^4% 1 cm (295 nm) - 0.186

Purity test: TLC (total size and intensity of foreign spots): 5%

Biological Action on Solid: 572 pg / mg

Identification and purity test: The Rf of the TLC is the same as that of the reference (standard) substance. (Rf - 0.45 for 24 'C)

Example 6

12,400 liters and 8,400 liters of fermentation broth prepared according to Example 1 (2x39 m ³ fermentation broth (total activity by bioautographic measurement 1840 and 1780 U / ml)) were obtained. Fraction A was concentrated in vacuo at 40 ° C. The resulting concentrate contains 250 liters of 35.1% solids, i.e. a total of 87.75 kg apramycin base, as well as a large amount of coloring matter and minor components.

The concentrate (250 L) was adjusted to pH 5.6 with 50 L (5 M) sulfuric acid and the solution was sparged with six volumes (1800 L) of methanol. The precipitated apramycin sulfate crystals were filtered off, suspended in twice 200 liters of methanol and dried in vacuo at 50 ° C.

Crude product obtained: 105.3 kg apramycin sulfate.

The product is dark purple.

Dissolve 105.3 kg of crude apramycin sulfate in 1000 L of deionized water and adjust the pH of the solution to 9.52 with 20 kg of sodium hydroxide. The solution was diluted to a volume of 3 m ³ (pH 9.51) and passed through a bed of Lewatit CNP-80 resin on a 3400 liter ammonium cycle at 300 rpm. The resin bed is washed with 10 m ³ deionized water in a standing bed at 300 rpm. The washing water removes most of the dyes and minor components. The resin-bound drug is dissolved in a bed of 200 l / h with 2% ammonium hydroxide solution (7 m ^{3 in} total). The apramycin-containing eluate (3 m ³ volume) was concentrated in vacuo at 40 ° C and de-ammoniated.

The resulting concentrate contains 342 liters of 20% solids, i.e. a total of 68.40 kg of apramycin base, and a small amount of coloring matter and minor components.

The 342 liter concentrate was adjusted to pH 5.70 with 40 L of 5 M sulfuric acid and clarified with 10 kg of activated carbon. The carbon bed is washed with 50 L of deionized water, and the filtrate and washings are concentrated in vacuo at 40 ° C.

The resulting concentrate contained 390 liters of 22% solids, i.e. a total of 85.80 kg of apramycin sulfate.

The typical parameter of the solution is:

E ^4% i cm (295 nm) - 0.210

The 390 liter solution was sparged into six volumes of methanol (2340 liters). The precipitated crystals of apramycin sulfate were filtered off, the precipitate was suspended in methanol (2 x 200 L) and the product dried in vacuo at 50 ° C.

Finished product recovered: 85.60 kg apramycin sulfate.

Yield: 1.097.4 g / m ³

The main quality characteristics of the recovered product are:

E ^4% 1 cm (295 nm) - 0.296

Purity test: TLC (total size and intensity of foreign spots): 7.5%

Biological Action on Solid: 591 pg / mg.

Identification and purity test: The Rf of the TLC is the same as that of the reference (standard) substance. (Rf - 0.46 at 24 'C)

Example 7

68 liters of neutral nebramycin filtered juice obtained from 39 liters of fermentation broth (total activity by 1930 U / ml by bioautographic measurement) fermented according to Example 1 was passed through Example 351 on a Lewatit CNP-80 type resin in 3500 ml of an ammonium cycle. in bed at 2500 ml / hour. The resin is washed in a standing bed with deionized water, and the resin-bound active ingredient is eluted with 37 L of 0.1 M, followed by 18.5 L of 0.3 M ammonium hydroxide at 300 mL / hr.

Thin layer chromatography was used to separate the so-called apramycin containing. Fraction Jx with a volume of 9500 ml and a pH of 10.48.

The resulting eluate contained 9500 ml of 0.5% solids, i.e. a total of 47.50 g of apramycin base, as well as many coloring agents and minor components.

A purple solution of 9500 ml of apramycin base, pH 10.48, was passed through a standing bed at 500 ml of diaion SP-207 adsorption resin.

Apramycin-containing solution flowing through the resin is pale yellow in color.

The resin bed was washed with 250 mL deionized water.

The resin-clarified apramycin solution and wash water were evaporated in vacuo at 40 ° C.

HU 199563 A

The concentrated solution contains 310 ml of 15% solids, ie a total of 46.5 g of apramycin base, as well as a small amount of coloring matter and minor components.

The pH of the 310 ml solution was adjusted to 5.65 with 26 ml of 5 M sulfuric acid and clarified with 5 g of activated carbon. The carbon bed is washed with 50 ml deionized water. The filtrate and wash water were evaporated in vacuo at 40 ° C.

The resulting concentrate contained 230 ml of 23.5% solids, i.e. a total of 54.05 g of apramycin sulfate.

Solution characteristic:

E ^4% 1 cm (295 nm) - 0.380

230 ml of the solution was added into six volumes of methanol (1380 ml). The precipitated apramycin sulfate crystals were filtered off, the precipitate was washed with methanol (2 x 100 mL) and the product was dried under vacuum at 50 ° C.

Finished product: 53.5 g apramycin sulfate.

Yield: 1.372 g / liter

The main quality characteristics of the recovered product are:

E ^4% 1 cm (295 nm) - 0.490

Purity test: TLC (total size and intensity of foreign spots): 9.5%

Identification Test: The Rf of the TLC is the same as that of the reference (standard) substance (Rf - 0.46 at 24 'C).

Biological Action on Solid: 551 pg / mg.

Example 8

A 196.459. according to Hungarian Patent Application No. 4,195, fermented with 40 liters of neomycin (active ingredient content 10.200 pg / mg) with oxalic acid and sulfuric acid at pH 2.1, fermented with streptomyces fradiae strain NR-8/51 (accession no .: NCAIMB (P) 000313). filter through a pre-vacuum vacuum drum filter and wash with water during filtration. 72 liters of sour filtered juice (active ingredient content: 5,600 pg / ml) were adjusted to pH 4.3 with concentrated ammonium hydroxide and passed through a bed of Lewatit CNP-80 resin (4x1800 ml) on a standing bed of 2500-2500 ml / hour. The resin beds were washed with 5-5 liters of deionized water in a floating bed at 2500-2500 ml / h.

Each column was washed with 10-10 liters of 0.2 M ammonium hydroxide solution in a floating bed at 1000-1000 ml / h and then dissolved in a standing bed at 200-200 ml / h with 2% ammonium hydroxide solution (one column). elution requires 2500-2500 ml of solution).

Fractions of the eluate containing neomycin were pooled. A total of 6 liters of eluate is collected from the 4 columns.

The 6 L eluate was concentrated in vacuo at 40 ° C and de-ammoniated.

The resulting concentrate contains 830 ml of 27.8% solids, i.e. a total of 230.7 neomycin bases, as well as many colorants and minor components.

The typical parameter of the solution is:

E ^4% 1 cm (430 nm) - 6.4

The eluate containing 820 ml of neomycin base was diluted to 8000 ml with deionized water. The pH of the solution is 10.65.

The diluted eluate, pH 10.65, is passed through a Lewisit CNP-80 type resin in a 4800 ml ammonium cycle at 500 ml / h. The bed of the resin bed was washed with 4000 ml deionized water at 500 ml / hour. The resin solution and wash water remove a great deal of color and minor components. This is also indicated by the fact that the color of the solution is dark brown.

The column solution and wash water were evaporated in vacuo at 40 ° C.

The resulting concentrate contains 22 ml of 20% solids, i.e. a total of 4.4 g of coloring matter and minor component base.

The pH of the concentrate was adjusted to 6.5 with 5M sulfuric acid (the substances in the concentrate were converted to the sulfate salt and then sparged with 5 volumes of methanol (115 ml). The precipitate was filtered off, suspended in 2 x 10 ml of methanol and on.

6.14 g of coloring matter and minor component sulfate were obtained.

The main quality characteristics of the recovered product are:

Biological action on dry matter: 121 IU / mg.

Purity Test: Thin layer chromatography does not contain neomycin.

During loading and washing, most of the dyes and minor components from the neomycin were removed by the above purification step.

4800 ml of resin-bound neomycin was eluted in a standing bed at 300 ml / hour with 2% ammonium hydroxide solution (9 liters total required).

The neomycin-containing eluate (5 L) was concentrated in vacuo at 40 ° C.

The resulting concentrate contained 845 ml of 26.7% solids, i.e. a total of 225.6 g of neomycin base, and very little coloring matter and minor component.

The typical parameter of the solution is:

E ²⁶ · ⁷ * i cm (430 nm) - 1.63

The pH of the 845 ml solution was adjusted to 6.75 with 5 M sulfuric acid (150 ml) (formed with neomycin sulfate) and the solution clarified with 20 g of activated carbon. The carbon bed is washed with 100 ml deionized water. The filtrate and the washings were combined to give 1020 ml of a 34.2% solids solution containing 348.8 g of neomycin sulfate in total.

The typical parameter of the solution is:

E ³⁴ · ^3% 1 cm (430 nm) - 0.205

The solution containing 1020 ml of neomycin sulphate was added into 5 volumes of methanol (5,100 ml). The precipitate was filtered off, suspended in methanol (2 x 200 ml) and dried at 50 ° C in vacuo.

Yield: 345.0 g

The main quality characteristics of the recovered product are:

Biological action on dry matter: 718 IU / mg

Purity test: Nylon content less than 2% [α] ² % - +56 '(c = 0.5, 0, ln sulfuric acid) by thin layer chromatography

Example 9

The pH of 10 liters of acidic neomycin filtered juice (active ingredient content 6734 pg / ml) fermented and filtered according to Example 8 is adjusted to 8.3 with 50 ml of concentrated ammonium hydroxide.

HU 199563 A

The metal content of the filtered juice is 0.17 g / liter

To the solution is added 100 g of trisodium phosphate to raise the pH of the solution to 9.0.

10 ml of 10 M sodium hydroxide solution raise the pH of the filtered neomycin-containing juice to 10.35.

The solution precipitates well and is filtered off.

The resulting 10.13 liters of alkaline filtered juice contained 6600 gg / ml. The filtered juice at pH 10.88 in a standing bed is passed through a Lewatit CNP-80 resin in a 1800 ml ammonium cycle at 2000 ml / h. The resin bed is washed with a total of 6 liters of deionized water in a standing bed at 2000 ml / h. The filtered juice and washing water leaving the column contain very large amounts of coloring matter and minor components.

The resin-bound drug was dissolved in a 2% ammonium hydroxide solution in a standing bed at a rate of 200 ml / h (2500 ml total required).

The volume of the neomycin-containing ammonium eluate is 1350 ml and contains 38.52 g of neomycin base. The pH of the eluate was 11.40.

The eluate containing the ammonium hydroxide was adjusted to pH 9.18 with 50 mL of 5 M sulfuric acid.

The solution is passed through a standing bed at a flow rate of 300 ml / h through a Lewatit CNP-80 resin of 1000 ml ammonium cycle. The resin bed was washed with 2 L of deionized water at 300 mL / hr with 2% ammonium hydroxide solution (2 L total required).

The neomycin-containing eluate (volume 1200 ml) was concentrated in vacuo at 40 ° C and de-ammoniated.

The resulting concentrate contained 200 ml of 18.8% solids, i.e. a total of 37.60 g of neomycin base, and very little coloring matter and minor component.

The typical parameter of the solution is:

E ¹⁸ · ⁸ * i cm (430 nm) - 0.980

The 200 ml concentrate was adjusted to pH 6.60 with 18 ml of 5 M sulfuric acid (formed with neomycin sulfate) and then clarified with 3 g of activated carbon. The carbon bed is washed with 20 ml deionized water.

The filtrate and washings were combined to give 235 ml of a 24.6% solids solution containing a total of 57.81 g of neomycin sulfate.

The typical parameter of the solution is:

E ²⁴ - ^6% t cm (430 nm) - 0.120

The solution containing 235 ml of neomycin sulfate was added to 5 volumes of methanol (1175 ml). The precipitate was filtered off, suspended in methanol (2 x 100 mL) and dried at 50 ° C under vacuum.

The product obtained was 57.75 g of neomycin sulfate.

Quality characteristics of the recovered product:

Biological action on dry matter: 710 µg / ml

Purity test: thin layer chromatography indicated that the neamin content is less than 2%.

(a) ²⁰ D - +58 '(c = 0.5, 0.1 n sulfuric acid)

Example 10

The fermentation broth containing 39 m ^{3 of} Nebramycin (Stevia activity by bioautographic measurement 1850 U / ml) fermented according to Example 1 was acidified to pH 2.0 with oxalic acid and filtered through a pre-drum filter while the filter layer was washed with water.

The pH of the filtered juice was adjusted to 4.5 with concentrated ammonium hydroxide solution (150 L) and the resulting 66 m ³ filtered juice was transferred without filtration to a bed of Lewatit CNP-80 resin in a 3500 liter ammonium cycle with 2500 1 / hours.

After the drug has been cured, fractions A and B are separated as in Example 1.

Fraction B containing carbamoyl kanamycin and carbamoyl tobramycin (22 m ³ ) was concentrated in vacuo. The concentrate (299 liters) contains 35.6% solids, totaling 106.44 kg of carbamoyl kanamycin, carbamoyl tobramycin, and many minor components and colorants.

The 299 liter concentrate was hydrolyzed with 75 kg of sodium hydroxide for 10 hours at 100 ° C. After hydrolysis, the hydrolyzate was cooled to 25 ° C. Dilute to 600 liters. To a solution of pH 13.52 is added 190 liters of 5 M sulfuric acid, the pH is adjusted to 10.65, and the solution is diluted to 4000 liters with deionized water.

The diluted solution, having a pH of 10.53 and a solids content of 6%, was passed through a Lewatit CNP-80 type resin in a 3400 liter ammonium cycle at 300 rpm as described in Example 1. After washing with deionized water, dissolve the resin-bound active compound in a total of 8 m ^{3 of} 2% ammonium hydroxide solution. The 5 m ³ eluate containing the valuable active ingredient is evaporated in vacuo and de-annealed.

The resulting concentrate contains 217 liters of 36.8% solids, i.e. a total of 79.86 kg of kanamycin, tobramycin, and a small amount of minor components and colorants.

The 217 liters of hydrolyzate is further processed as in Example 1 below.

The final product obtained is 23.00 kg of kanamycin sulfate

Yield: 590.00 g / m ³

Important quality characteristics of the recovered product:

E ^4% 1 cm (295 nm) - 0.143

Purity test: TLC (total size and intensity of foreign spots): 4%, Rf (24 'C): 0.15

Biological action on dry matter: 628 mg / mg.

Another finished product obtained was 19.89 kg of tobramycinbase active ingredient. Yield: 510.00 g / m ³

The main quality characteristics of the recovered product are:

E ^4% 1 cm (295 nm) - 0.125

Purity test: TLC (individual spot size and intensity): 0.5%

Purity test: TLC (total size and intensity of foreign spots): 1.5%

Biological action on dry matter: 985 gg / mg.

[α] ²⁰ ο - +150 '(c - 1, water)

By using the present invention, the manufacturing process of nebramycin and neomycin antibiotics is simplified and streamlined by the use of the same type of ion exchange operations. The technology line is richer, easier and less expensive to instrument.

High purification of active ingredients increases system capacity, significantly increases throughput, halves the specific use of activated carbon, and significantly reduces material and energy consumption.

This procedure can be used against quality standards for pharmaceutical technology (for9

EN 199563 For producing potent exportable products that meet stricter requirements.

Identification and purification by thin layer chromatography (TLC)

Layer: Kieselgel G Whatman K5 Plate Running Agent 2 (5 M sodium chloride in water containing 30% v / v ethyl alcohol)

(a) 0.5% solution of sodium hypochlorite

b) 96% ethyl alcohol

(c) Cadmium iodide starch reagent (1.1 g cadmium iodide and 1.5 g starch in 100 ml water).

Claims (4)

PREPARATION OF THE PREFERRED EMBODIMENTS 1. A method of producing nebramycin and neomycin antibiotics from a fermentation broth or by extracting and / or purifying an antibiotic containing aqueous solution of any other origin using a carboxyl-type cation exchange resin and / or an adhesive resin once or more, the cation exchange resin being ammonium. by eluting the hydroxide in an aqueous solution and treating the solution containing the antibiotic containing the eluate and the adsorption resin in a known manner, characterized in that the pH of the aqueous solution containing the antibiotic is at least one time before administration to the resin; preferably 10.2 to 11.0. 2. The method of claim 1, wherein the carboxyl-type ion exchange resin is a Lewatit CNP-80 resin. 3. The method of claim 1 wherein the adsorption resin is a Diaion SP-270 resin. Method according to claim 1 or 2, characterized in that the ion exchange and elution are carried out at 60 ° C when the antibiotic is recovered from the fermentation broth.