HU209799B - Process for producing neomycin-sulfate - Google Patents

Abstract

The present invention relates to a process for the preparation of neomycin sulfate from fermentation broths by filtration of acidified fermentation broth, purification of acidic cation exchange resin or acidic cation exchange resin and anion exchange resin, washing of the ion exchange resin with water and aqueous ammonium hydroxide solution and elution with aqueous ammonium hydroxide solution and elution with an aqueous ammonium hydroxide solution. by processing an ion exchange resin solution from the ion exchange resin during the ion exchange of the neonatine-containing filtrate, and maintaining the process for producing neomycin sulfate in a range of 0.22-0.50 mol / l, which is controlled by A. adjust the pH of the fermentation broth or filtrate to 1.0-1.9 with sulfuric acid in the presence of oxalic acid, neutralize with ammonia, filter, or acidify the broth with sulfuric acid to pH 3.5-5.5; s rik, the ion exchange resin, eluting the bound active compound in a known manner, purified and neomycin sulfate in the eluate was recovered. EN 209 799 A Scope of the description: 8 pages

Description

The present invention relates to a process for the preparation of neomycin sulfate from fermentation broths by filtration of the acidified fermentation broth, purification of the acidic cation exchange resin, (and) anion exchange resin, washing of the ion exchange resin with water and aqueous ammonium hydroxide solution followed by aqueous ammonium hydroxide processing.

Neomycin, a member of the aminoglycoside group of antibiotics, is known to be produced by fermentation using various strains of Streptomyces and Micromonospora. Microorganisms biosynthesize several compounds during their life and produce a variety of by-products, decomposition products and dyes. These include paromamine, mono-N-acetylneomycin B, mono-N-acetylneomycin B, mono-N-acetylneomycin C, paramomycin I, paramomycin II, neamin, neomycin B and neomycin C. Among these compounds, neomycin B and C have a significant biological activity, but the effect of component B is significantly superior to that of neomycin C. Considering the significant use of this drug in both human and veterinary therapies, several solutions have been developed for isolation and purification from fermentation broth. These processes are basically divided into 3 main groups, namely, those using precipitation, extraction and ion exchange.

The description of the precipitation technology is given, inter alia, in U.S. Patent No. 785,382. United Kingdom, and U.S. Patent No. 2,686,749. U.S. Pat.

Of the extraction methods, reference is made to U.S. Pat. Czechoslovakia and Decree 160 456; Hungarian patent specification.

In practice, the most widely used solutions using ion exchangers process filtered fermentation broth. Among the various types of ion exchangers, the use of a weakly acidic cation exchanger of the carboxyl type is described in U.S. Patent No. 2,667,441. U.S. Patent No. 162,939; and No. 169,452. while the use of a highly acidic cation exchange resin of the sulfonic acid type is disclosed in U.S. Patent No. 3,005,815. U.S. Pat.

As mentioned above, fermentation produces a complex group of compounds, some of which have similar chemical properties to neomycin B. Paper and thin layer chromatography (and more recently, high pressure liquid chromatography) analyzes indicated that some of the accompanying materials were eluted from the ion exchange resins with neomycin B and contaminated the resulting crude product. Because of the additive effect of the development of fermentation technologies and strains, an increase in the amount of undesirable components has been observed, for these reasons, many attempts have been made to remove impurities, to obtain the active compound with the highest purity and with minimal loss.

For the purification of neomycin, 2,667,441,

U.S. Patent Nos. 944,939, 3,005,815 disclose that the highly acidic cation exchange resin repeats the ion exchange or uses a variety of additives to selectively bind and elute the active ingredient. No. 1,360,928. U.S. Patent No. 3,313,693; Ammonium molybdate, a

No. 313,694 U.S. Pat. a non-ionic detergent according to Czechoslovakia. No. 169,452. According to the Hungarian patent, it is expedient to wash impurities from the bound neomycin with acetic acid at a concentration of 1-3%. Another known possibility for a high pore cation exchange8 and sulfonic acid resin is the repeated ion exchange as described in U.S. Patent No. 2,827,417. The use of a condensation-type resin having a condensation type having virtually no ion-exchange capacity is described in U.S. Patent No. 162,943. according to the Hungarian patent specification.

In order to make good use of the capacity of the cation exchange resin, alkaline earth metals are eliminated from the ion exchange solution. No. 150,474. According to the Hungarian patent, alkaline earth metals are removed by acidification of the fermentation broth with oxalic or phosphoric acid followed by filtration. No. 181,748. According to the Hungarian patent, fermentation broth is acidified with sulfuric acid in the presence of oxalic acid before filtration. No. 199,563. According to the Hungarian patent, fermentation broth is also acidified with sulfuric acid in the presence of oxalic acid. The removal of alkaline earth metals is accomplished by the addition of trisodium phosphate to the filtrate of the fermentation broth, and the filtrate is made alkaline and the precipitate is filtered off.

Given that the above methods do not, by themselves, provide a pharmaceutically acceptable product, activated carbon is also suggested for the clarification of aqueous neomycin solutions. On the other hand, there is a significant loss of active ingredient in the purifications carried out in this way, so it is advisable to limit the use of carbon to a minimum. A process for combining ion exchange with activated carbon clarification is described in U.S. Patent No. 199,563. The pH of the aqueous solution prior to application to the resin is improved by adjusting the pH of the antibiotic-containing aqueous solution to 9.5-12.

As previously mentioned, the filtrate of the fermentation juice is preceded by the addition of trisodium phosphate, which causes the alkaline earth metals to precipitate. Despite the removal of alkaline earth metals, the optimum load on a Lewatit CNP-80 weak acid cation exchange resin on a 1000 cm3 ammonium cycle is 37 grams, due to the reduced pH of the resin at alkaline pH.

In some cases, clarification is supplemented with chemical treatment that destroys the dyes to reduce the amount of clarifying carbon. Brominated water or Rongalite can be used for this purpose in accordance with U.S. Patent No. 56,365. or a hydrogen peroxide according to U.S. Patent No. 181,748. according to the Hungarian patent specification.

HU 209 799 A

In this latter process, up to 85% yield and an average product quality of 680 IU / mg can be achieved using anion exchange purification prior to treatment with hydrogen peroxide.

The quality requirements for neomycin (or neomycin sulfate), which can be used as the active ingredient of the drug, are updated from time to time in pharmacopoeias. In the past, apart from physical properties, only a few chemical (or biological) requirements (such as solution pH, specific rotation, dry matter bioavailability, neamin content) had to be met, whereas today there are specific limits for neomycin B and C components. rates.

Some buyers take advantage of market competition to determine specific requirements. Thus, it became necessary to produce a product with an active ingredient content of 710 IU / mg and less than 6.0% by weight of neomycin C.

After reviewing the purification procedures described above, reproduction experiments were performed in accordance with U.S. Patent No. 181,748. and U.S. Patent No. 199,563. according to Hungarian patents. Our experiments are illustrated in Fig. 1 / b. 1 / c., 2 / b., 4 / b. through examples. No. 181,748. hereinafter referred to as "anion exchange purification", U.S. Patent No. 199,563. hereinafter referred to as "alkaline ion exchange".

1 / b, 1 / c, 4 / b showing anion exchange purification. Examples 1 to 4 show that 85% yield and up to 680-690 IU / mg of product quality can be achieved by this procedure. The ratio of neomycin C to neomycin B cannot be reduced below 6% by weight. 1 / b. 1 / c. By way of example, it can be seen that more than 100 g of active ingredient can be applied to 1000 cm ^{3 of} resin in this process, although this results in a loss of quality and yield. 1 / b. and 4 / b. Comparison of Examples 1 to 4 points to the fact that otherwise reproducible methods do not produce the expected results for some substantially different fermentation broths, i.e. it is advisable to compare different purification methods from the same fermentation broths.

The alkaline ion exchange is illustrated in Fig. 2 / b. In the same manner as described in Example 1 to 2, it can be stated that this process achieves an active ingredient content of more than 700 IU / mg and no more than 80 g of active ingredient can be applied to 1000 cm ^{3 of} resin. This process is highly dependent on the amount of active ingredient applied to the column. The ratio of neomycin C to neomycin B in the product obtained cannot be reduced below 6%. The yield of the process is below 85%.

An overview of the purification procedures described above shows that neomycin C having an active ingredient content above 710 IU / mg and less than 6 wt% can be obtained only by repeated ion exchange and reduction of the drug loading of the ion exchange column, and in less than 85% yield.

A further problem with some of the above processes is that they do not reliably ensure consistent quality in the processing of different quality (active ingredient) fermentation broths, so in practice they are quite difficult to reproduce with the same result.

In fact, ion exchange purification is significantly disturbed if the active ingredient content of the fermentation broth, as well as the ratio of the active ingredient to the impurities and the metal content present, fluctuate over a relatively wide range. As a result of these changes, the individual solutions that we wish to carry out ion-exchange purification behave differently during the setting and purification elution, and sometimes the purification elution causes great losses of material. This loss increases with increasing column load.

Thus, in the course of developing our invention, we aim to develop a purification process that can be used for highly reproducible, volatile fermentation broths, which provides a product with improved purity requirements at good yield and production capacity, or a lower quality product with simple technology. makes it possible. A further aim was to reduce the amount of mycelium produced during the fermentation of neomycin in large quantities in order to minimize environmental contamination.

The present invention is based on the discovery that: during ion exchange of neomycin, the selectivity of the ion exchange resin can be substantially influenced by keeping the ammonia concentration of the solution exiting the ion exchange resin above 0.22 mol / l, while maintaining the ion exchange solution at a pH close to neutral. The ammonia concentration in the resin effluent solution provides a broad range of 0.22-0.50 mol / l concentrations of nearly the same selectivity, regardless of whether the desired ammonia effluent concentration from the ion exchange resin is introduced by the introduction of ammonium ions before ion exchange or by alkaline earth metal bonded to the ion exchange resin. In the latter case, the surprising fact was also found that the required alkaline earth metal concentration can be assured by filtration of the fermentation broth at pH 3.5-5.0 and that the removal of the alkaline earth metal by step elution is not required, whereas the ammonia prior to ion exchange by over-acidifying the fermentation broth or the filtrate after filtration of the fermentation broth, followed by ammonia neutralization.

By controlling the ammonia concentration in the effluent solution of the ion exchange resin according to the present invention, it is achieved that impurities with a structure similar to neomycin B do not bind to the ion exchange resin and indeed the binding of neomycin C is only partial, thus purifying the active ingredient efficiency and better quality.

According to the present invention, during the ion exchange of the neomycin-containing juices, the ammonia concentration of the solution flowing from the ion exchange resin is kept in the range of 0.22-0.50 mol / l, which is ensured by prior isolation experiment with sulfuric acid in the presence of oxalic acid. to a certain extent further sa3

The solution is acidified and then neutralized with ammonia, filtered and the filtrate is subjected to ion exchange, or the fermentation broth is acidified with sulfuric acid and then filtered over a pH range of 3.5 to 5.5 to avoid the separation of the alkaline earth metals and ion exchange with the resulting filtrate. followed by elution of the active ingredient from the ion exchange resin in both cases after the ion exchange operation and purification of the eluate in a known manner.

The process of the present invention, termed ammonia-controlled ion exchange, was also compared to anion exchange purification and alkaline ion exchange. Comparisons should be made for ion exchanges starting from the same fermentation due to the non-measurable different impurity content of the fermentation broths. Thus, FIGS. and 1 / d. examples of ammonia-driven ion exchange are illustrated in FIG. and 1 / c. with examples of anion exchange purification, 3 / a. an example of ammonia-driven ion exchange is illustrated in FIG. 3 / b. with an example of purification without ammonia control, in FIG. an example of ammonia-controlled ion exchange is shown in FIG. 4 / b. with an anion-exchange purification example, in Fig. 2 / a. and 2 / c. examples of ammonia-driven ion exchange are illustrated in FIG. 2 / b. alkaline ion exchange example. It can be stated that the ammonium-controlled ion exchange can produce a higher yield, higher drug content and lower neomycin C content than the comparative examples.

With ammonia-controlled ion exchange, a product containing 720 IU / mg of neomycin C, reduced to 6 wt%, and reduced to 4 wt%, can be produced at 90% yield without the need for repeated ion exchange.

Advantages of the process according to the invention are as follows:

- The screened mycelium is suitable for secondary use as the content of the antibiotic is low and does not contain impurities such as oxalate.

- Neomycin sulphate can be obtained in good yield and reliably reproducible from fermentation broths with a relatively wide range of active ingredients, high in color and impurities.

- With lower quality requirements, our process is further simplified.

The invention is illustrated by the following non-limiting examples.

Example 1

1 / a. Example 1 (Ammonia-controlled ion exchange at a flow rate of 0.22 mol / L at an application rate of 108.8 g active ingredient / 1000 cm ³ resin.)

Streptomyces fradiae strain NR-8/51 (NCAIM B (P) 000 313) (deposited 04.12.1984) fermented with 75.9 dm3 of 778.5 g of neomycin B and C (of which 693.6 g / i. The pH of the fermentation broth, 1% w / w neomycin B and 84, 9 g (i.e. 10.9% w / w) neomycin C) was adjusted to 4.5 with 4.65 kg of 25% w / w sulfuric acid. After the addition of Perlite, the mycelium was filtered through a prefiltered vacuum drum filter while the layer was washed with water. The calcium content of the filtrate thus obtained was precipitated with 0.85 kg of oxalic acid in 10% w / w solution, then the pH was adjusted again from 1.3 to 4.5 with ammonia solution, and the oxalate precipitate was filtered off.

Thus a volume of 123 dm ³ containing 739.5 g of active ingredient. A filtrate solution of 0.12 mol / L ammonia was passed through 4 Lewatit CNP-80 cation exchange resins in a 1700 cm ³ ammonium cycle at 2500 cm ³ / hour. The concentration of ammonia leaving the ion exchange was 0.22 mol / l. After filling, the resin beds were washed with deionized water, followed by washing with 0.2 M ammonia solution. The passage of this ammonia wash was led to a channel up to the extinction peak, followed by continuous dilution to a cation exchange column adjusted to a concentration of 0.07-0.09 M ammonia. The active ingredient was eluted with 2% ammonia solution. A total of 14.16 dm ³ , 628.6 g of neomycin containing eluate was collected from the 4 columns.

This was passed through a column containing 600 cm ^{3 of} hydroxide in the form of a Varion ADM anion exchange resin at 600 cm 3 / h and then rinsed with 1800 cm 3 of deionized water at 600 cm 3 / h. The decolourised eluates and the deionized aqueous washer were combined and concentrated at 45 ° C under 0.9 bar vacuum to remove 40% w / w drug content. The residue contained 603.5 g of neomycin. The resulting evaporation residue (1510 cm ³ ) was adjusted to pH 6.2 with 428 cm ^{3 of} 5 M sulfuric acid, followed by the addition of 60 cm ^{3 of a} 3% solution of hydrogen peroxide and clarified with carbon.

After filtration of the activated carbon, the neomycin sulfate solution was diluted with deionized water to a concentration of 20% by weight of the active ingredient and then spray dried. Spray drying was carried out with air at 205 degrees Celsius at a solution delivery rate of 2 dm ³ / h. The temperature of the air leaving the dryer was 105 degrees Celsius.

The product thus obtained was 597.2 g of which 560.0 g of neomycin B sulfate and 32.8 g of neomycin C sulfate.

Relative neomycin C sulfate content: 5.8%

Active ingredient content: 700 IU / mg [g] d = + 56 degrees (C = 0.5, 0.1 N sulfuric acid)

Purity test: thin layer chromatography neamin content: 1.5% w / w.

Purification of the ammonia wash to another cation exchange column gave 20.8 g of product (15.0 g of neomycin B sulfate and 1.9 g of neomycin C sulfate) as described above.

The active substance content of the product is 660 IU / mg.

1 / b. Example 1 (Anion exchange purification at 54.1 g drug / 1000 cm ³ resin drug application)

1 / a. Parallel to Example 4, the same fer4

The fermentation broth from the menthol was also recovered in a known manner by the following procedure:

The pH of the fermentation broth with a volume of 75.9 dm ³ containing 778.5 g of neomycin B and C (of which 693.6 g of neomycin B and 84.9 g of neomycin C) was added with 0.85 kg of oxalic acid in 10% solution It was adjusted to 2.0 with 25% sulfuric acid. 3.4 kg of sulfuric acid solution was used for pH adjustment. After adding Perlite, the mycelium and the precipitated salts were filtered off with a pre-layer vacuum drum filter while the layer was washed with water.

The resulting filtrate was adjusted to pH 8.5 with concentrated ammonium hydroxide solution. The precipitate was removed from the solution by filtration using 0.15 kg perlite. A filtrate broth with a volume of 128 dm ³ , containing 736 g of the active ingredient, and 0.092 mol / l ammonia was passed through 8 Lewatit CNP-80 cation exchange resins in a 1700 cm ³ ammonia cycle. The ion exchange effluent concentration of the ion exchange is 0.20 mol / l. Hereinafter, FIG. method. Elution with the aqueous ammonia 2 wt% total of 14.9 dm ³ 691.0 g of neomycin-containing eluate collected. The 1600 cm3 evaporation residue after anion exchange contained 651 g of neomycin. 1 / a. The procedure was as follows, except that 421 cm ^{3 of a} 30% solution of hydrogen peroxide were used for decolorization to give 624 g of product, of which 556.0 g of neomycin B sulfate, 51.4 g of neomycin C sulfate.

Relative neomycin C sulfate content: 9.2%

Active ingredient content: 683.0 IU / mg [a] ® = + 55 degrees (C = 0.5, 0.1 N sulfuric acid)

Purity test: thin layer chromatography neamin content: 2.5% w / w.

From the passage of the ammonia wash, 1 / a. Example 26 gave 26 g of product. The product contained 19.5 g of neomycin R sulfate and 2.8 g of neomycin C sulfate.

The active ingredient content of the product is 648 IU / mg.

I / c. Example 1 (Anion exchange purification at 108.2 g drug / 1000 cm ³ resin drug application)

1 / b. Example 1 was followed by passing the 131 dm ³ filtrate broth containing 731 g of the active ingredient at 0.090 mol / dm3 ammonia concentration through 4 cation exchange resins in 1700 cm ^{3 of} ammonium cycles.

The product was obtained in an amount of 596 g of which 515 g of neomycin B sulfate, 50.9 g of neomycin C sulfate.

Relative neomycin C sulfate content: 9.9%

Active ingredient content: 679.0 IU / mg [α] β = + 54.2 degrees (C = 0.5, 0.1 N sulfuric acid)

Purity test: thin layer chromatography neamin content: 2.5% w / w.

From the passage of the ammonia wash, 1 / a. Example 28 gave 28 g of product having an active ingredient content of 642 IU / mg.

1 / d. Example I / A (Ammonia-controlled ion exchange at a flow rate of 0.23 mol / l of ammonia at 54.4 g active ingredient / 1000 cm ³ resin). The procedure described in Example 1 was followed with the exception that the 120 dm ³ filter cake containing 751 g of active ingredient, 0.13 M ammonia was passed through 8 cation exchange resins in 1700 cm ^{3 of} ammonium cycles. The concentration of ammonia leaving the ion exchange was 0.234 mol / l.

The product thus obtained was 642 g of which 597 g of neomycin B sulfate and 33.2 g of neomycin C sulfate.

Relative neomycin C sulfate content: 5.6%

Active ingredient content: 704.0 IU / mg [a] _b = +56.2 degrees (C = 0.5, 0.1 N sulfuric acid)

Purity test: thin layer chromatography neamin content: 1.5% w / w.

From the passage of the ammonia wash, 1 / a. Example 18 gave 18 g of product. The product contained 14 g of neomycin B sulfate and 1.5 g of neomycin C sulfate.

The active substance content of the product is 664 IU / mg.

Example 2

2 / a. Example 1 (Ammonium-controlled ion exchange at 0.46 mol / l effluent concentration of 105.9 g active ingredient)

Fermented with Streptomyces fradiae strain NR-8/51 (NCAIM B (P) 000 313) with a volume of 108 dm ³ containing 1515 g of neomycin B and C (of which 1288 g / i. E. 85% / neomycin B and 227 g / i. / neomycin C) was diluted with water to a concentration of 8000 μg / cm 3 and adjusted to pH 1.8 with 9.5 kg of 25% sulfuric acid. After the addition of Perlite, the mycelium was filtered through a pre-layered vacuum drum filter. The filtrate was washed with water during filtration.

The calcium content of the filtrate thus obtained was precipitated with 1.2 kg of oxalic acid in a 10% solution by maintaining the pH of the filtrate above 1.8 by adding concentrated ammonia solution, and finally adjusting the pH to 4.5 with the oxalate precipitate. filtered, the pH of the filtrate was adjusted to 6.5 with concentrated ammonia solution.

The resulting filtrate broth, 210 dm ³ , containing 1440 g of active ingredient and containing 0.33 M ammonia, was passed through 8 Lewatit CNP-80 cation exchange resins in a 1700 cm ³ ammonia cycle at a rate of 2500 cm ³ / h. The ion exchange effluent concentration of the ion exchange is 0.46 mol / l.

After filling, the resin beds were washed with deionized water, followed by washing with 0.2 M ammonia solution. The passage of this ammonia wash was led to a cation exchange column with a continuously rising ammonia content in the 0.07-0.20 mol / L concentration range to the channel until the extinction peak, followed by continuous pH adjustment to 4.5. The active ingredient was eluted with 2% ammonia solution. A total of 18 dm3 of eluate containing 1288 g neomycin was collected from the 8 columns. The eluates were concentrated at 50 degrees Celsius in a vacuum of 0.85 bar to reach 40% drug content and

HU 209 799 The demoniac has been decontaminated. The resulting evaporation residue (2853 cm ³ solution containing 1153 g of neomycin) was adjusted to pH 6.2 with 845 cm ^{3 of} 5 M sulfuric acid, followed by the addition of 58.0 cm ^{3 of a} 3% solution of hydrogen peroxide and charcoal. We clarified. After filtration of the activated carbon, the neomycin sulfate solution was poured into five volumes of anhydrous methanol.

The precipitated neomycin sulfate was filtered, slurried with 2x900 cm ^{3 of} anhydrous methanol and dried in vacuo to give 1210 g of product, 1080 g of neomycin B sulfate and 61 g of neomycin C sulfate.

Relative neomycin C sulfate content: 5.6%

Active ingredient content: 720; 0 IU / mg [<x] c? = +57.8 degrees C (0.5, 0.1 N sulfuric acid)

Purity test: thin layer chromatography neamin content: 1.5 m%:

Purification of the ammonia wash to another cation exchange column afforded 29.5 g of product, containing 23.8 g of neomycin B sulfate and 2.8 g of neomycin C sulfate as described above. Active substance content 672 IU / mg.

2 / b. Example 4 (Alkaline ion exchange at 111.4 g active ingredient / 1000 cm ³ resin and 37.1 g active ingredient / 1000 cm ³ resin application)

2a. In parallel with Example 1b, the active ingredient was obtained in a known manner from a fermentation broth from the same fermentation by the following procedure:

A fermentation broth of 108 dm ³ containing 1515 g of neomycin B and C (of which 1288 g of neomycin B and 227 g of neomycin C) was diluted with water to a concentration of 8000 pg / cm ³ and the pH of the fermentation broth was 1.2 kg of oxalic acid in 10% solution. 4.1 kg with 25% sulfuric acid was adjusted to 2.1. The acidified fermentation broth was filtered through a pre-vacuum suction filter and the layer was washed with water during filtration.

The pH of the thus obtained syrup was 202 dm ³ and 1400 g of active substance was adjusted to 8.3 with 1200 cm ^{3 of} concentrated ammonia solution. The metal ion content of the filtrate was 0.16 g / dm3, expressed as Ca ²⁺ . 2000 g of trisodium phosphate were added to the solution so that the pH of the solution was raised to 9.0. The pH of the neomycin-containing syrup was raised to 10.35 with 1800 cm <^{3> of a} 10 mol / l sodium hydroxide solution. Precipitates from the solution were filtered off. 205 dm ³ of filtrate juice containing 1370 g of active ingredient having a pH of 10.43 and a concentration of 6682 pg / cm ^{3 were obtained} . 30 dm ^{3 of} the alkaline filtrate was passed through a Lewatit CNP 80 resin on a 1800 cm 3 ammonium cycle at 2000 cm 3 / h. The concentration of ammonia leaving the ion exchange is 0.18 mol / l. After application of 12 dm ³ filter juice, 620 g / cm ³ neomycin concentration was measured as the ion exchange proceeded. The combined ion exchange effluent concentration of neomycin was 1610 pg / cm ³ . Seeing weak binding of neomycin, 10 dm ^{3 of} alkaline filtration juice in standing bed

At a rate of 2000 cm ³ / hour, Lewatit CNP-80 resin on a 1800 cm ³ ammonium cycle was passed. The resin bed was washed with a total of 6 dm ³ deionized water in a standing bed at 2000 cm3 / hour. The resin-bound drug was dissolved in a 2% (w / w) ammonia solution in a standing bed at a rate of 200 cm ³ / hour (a total of 2500 cm ³ required).

The pH of the eluate was adjusted to 9.20 5 mol / dm ³ sulfuric acid concentration. The solution was passed through a Lewatit CNP-80 resin in a 1000 cm ³ ammonium cycle at a rate of 300 cm ³ / hr. The resin bed was washed with 2 dm ³ deionized water and the active ingredient was eluted with 2% ammonia.

The neomycin-containing eluate was evaporated at 40 ° C to 200 cm 3. The pH was adjusted to 5 6.6 mol / dm ³ sulfuric acid was then decolorized with charcoal. The carbon bed was washed with 20 cm ^{3 of} deionized water. 236 cm ³ volume of the clarified solution is slowly added into five volumes of anhydrous methanol. The precipitate was filtered, slurried with 2 x 100 cm ^{3 of} anhydrous methanol and dried in vacuo at 50 ° C.

The product was obtained: 58.0 g of neomycin sulfate.

Relative neomycin C sulfate content: 6.2%.

Active ingredient content: 705.0 IU / mg [a] j = +58 degrees (C = 0.5, 0.1 N sulfuric acid)

Purity test: thin layer chromatography: neamin content: 1.5 m%.

2 / c. Example 1 (Ammonia-controlled ion exchange at a flow rate of 0.47 mol / l of ammonia at 52.2 g active ingredient / 1000 cm ³ resin)

2a. repeated examples broth from the same fermentation with the exception that only half of the resulting 208 dm 1420 g of active ingredient, concentrations of filtered liquor 0.325 mol / 1 ammonia ³ volume that is 108 dm ³ -t (710 g) was passed through a 8 x 1700 cm Lewatit CNP-80 cation exchange resin in ³ ammonium cycles at 2500 cm ³ / h. The concentration of ammonia leaving the ion exchange was 0.47 mol / L. 2 / c. Example 2 gave 702 g of product having 633 g of neomycin B sulfate and 23 g of neomycin C sulfate.

Relative neomycin C sulfate content: 3.6%

Active ingredient content: 719 IU / mg [α] D = +58.2 degrees (C = 0.5, 0.1 N sulfuric acid)

Purity test: thin layer chromatography: neamin content: 1.5 m%.

15 g of product were obtained from the ammonia wash with an active ingredient content of 661 IU / mg.

Example 3

3 / a. Example 1 (Ammonia-controlled ion exchange with 0.24 mol / l effluent ammonia at an application rate of 7.7 g active ingredient / 1000 cm ³ resin, without washing with 0.2 mol / l ammonia solution)

HU 209 799 A

From a fermentation broth of 114 dm ³ volume containing 1852 g of neomycin B and C according to Fig. 1 / a. 184 dm ^{3 of} 1760 g of neomycin B and C (of which 1596 g / l, 90.7% w / w neomycin B and 164 g / l, 9.3% w / w neomycin C), 0.14 mol / l Concentrated filtrate at pH 4 was passed through 8 Lewatit CNP-80 cation exchange resins in 1700 cm ³ ammonium form. The concentration of ion-exchanging ammonia is 0.24 mol / l. After washing with water, the active ingredient was eluted with 2% ammonia solution. A total of 223 dm ^{3 of} 1670 g neomycin eluate was collected from the 8 columns. The eluate was concentrated in vacuo at 50 ° C and depleted of ammonia. The resulting 4570 cm ³ volume of the evaporation residue the pH was adjusted to 6.2 with 1360 cm ³ of 5 mol / dm ³ sulfuric acid concentration.

The neomycin sulfate solution was added to five volumes of anhydrous methanol. The precipitated neomycin sulfate was filtered, suspended in 2x900 cm ^{3 of} anhydrous methanol and dried in vacuo.

The product obtained weighs 1769 g, of which 1502 g neomycin B sulfate and 140 g neomycin C sulfate.

Relative neomycin C sulfate content: 9.3%

Active substance content: 628 IU / mg

3 / b. Example I (Ammonia-controlled ion exchange at 0.19 mol / l effluent ammonia concentration at 128.8 g drug / 1000 cm ³ resin drug application, 0.2 mol / l ammonia solution wash)

From a fermentation broth containing 113 dm 3 of 1834 g of neomycin B and C, see Figures 1 / b. The filtrate broth obtained in Example 1 (180 dm ³ ) containing 1752 g of neomycin B and C containing 0.08 mol / l ammonia, pH 4 was passed through 8 Lewisit CNP-80 cation exchange resins in the form of 1700 cm ^{3 of} ammonium. The concentration of ammonia leaving the ion exchange was 0.19 mol / l. 3 / a. Example 176 gave 1765 g of product, which contained 1588 g of neomycin B sulfate and 146 g of neomycin C sulfate.

Relative neomycin C sulfate content: 9.2%.

Active substance content: 577 IU / mg

Example 4

4 / a. (Ammonium-controlled ion exchange at 0.26 mol / l effluent ammonia concentration of 53.8 g drug / 1000 cm ³ resin drug)

Fermented with Streptomyces fradiae strain NR-8/51 (NCAIM B (P) 000313) with a volume of 76 dm ³ containing 1312 g of neomycin B and C (of which 1168 g / w. 89% / neomycin B and 144 g / w. 11%). neomycin C) was diluted with water to a titer of 11000 pg / cm ³ and then adjusted to pH 4.5 with 6.53 kg of 25% sulfuric acid.

The diluted fermentation broth is shown in Fig. 1 / d. Prepared according to Example 1, the ion exchange effluent ammonium concentration was 0.26 mol / l, yielding 1181 g of product containing 1078 g of neomycin B sulfate and 43.2 g of neomycin C sulfate.

Relative neomycin C sulfate content: 4.0%

Active ingredient content: 718 IU / mg [oc] = +57.8 degrees (C = 0.5, 0.1 N sulfuric acid)

Purity test: thin layer chromatography revealed that the neamin content was 1.5 m%.

Passing the ammonia wash over to another cation exchange column and working up the above gave 48 g of product containing 40.4 g of neomycin B sulfate and 4.3 g of neomycin C sulfate.

Active substance content: 665 IU / mg

4 / b. Example 1 (Anion exchange purification at an application rate of 53.8 g active ingredient / 1000 cm ³ resin at a flow rate of 0.21 mol / l)

4a. Example 1b. Prepared according to Example 1, the concentration of ion exchange effluent measured at 0.21 mol / dm ³ gave 1203 g of product containing 1019.6 g of neomycin B sulfate and 68.4 g of neomycin C sulfate.

Relative neomycin C sulfate content: 6.7%

Active ingredient content: 681 IU / mg [a] = +54.1 degrees (C = 0.5, 0.1 N sulfuric acid)

Purity test: thin layer chromatography revealed that the neamin content was 2.0 m%.

Depletion of the ammonium salt yielded 53 g of product with 38 g of neomycin B sulfate and 6.1 g of neomycin C sulfate. The active substance content of the product is 630 IU / mg.

Example 5 (Ammonia-controlled ion exchange at a flow rate of 0.30 mol / l of ammonia at 65.3 g active ingredient / 1000 cm ³ resin application)

The fermentation broth (75.9 dm ³ ) containing 1555 g of neomycin was diluted with 79.6 dm ³ water and adjusted to pH 4.5 with 5.6 g of 25% sulfuric acid. After the addition of Perlite, the mycelium was filtered through a pre-layered vacuum drum filter by heating to 45 degrees Celsius.

The resulting filtrate with a volume of 176.9 dm ³ , containing 1399.5 g of active ingredient, containing 3.8 g / l of metal ion (expressed as Ca ²⁺ ) and containing 0.03 mol / l ammonium ion in 14 Lewisite cycles of 1700 cm ³ We passed through a CNP80 type cation exchange resin. The ion exchange effluent concentration of the ion exchange is 0.30 mol / l. After filling, the resin beds were washed with deionized water, followed by washing with 6 dm ³ of 0.2 M ammonia solution per column. The passage of this ammonia wash to the extinction peak was channeled to a channel followed by continuous dilution to a cation exchange column adjusted to a concentration of 0.10-0.15 mol / dm ³ ammonia. The active ingredient was eluted with 1.2 M ammonia. The eluates are shown in Fig. 1 / a. was passed continuously through a column containing 600 cm ^{3 of} Varion ADM type anion exchange resin. The decolourised eluates were concentrated at 50 ° C in a 0.9 bar vacuum to 3600 cm 3 and de-ammoniated. The resulting evaporation residue was adjusted to pH 6.2 with 965 cm ^{3 of} 5 M sulfuric acid, and then 70 cm ^{3 of a} 3% w / w hydrogen peroxide solution were added and the solution clarified with carbon.

HU 209 799 A

After filtration of the activated carbon, the neonacin sulfate solution was diluted with deionized water to 20% active ingredient and then spray-dried. Spray drying was carried out with air at 205 degrees Celsius at a solution delivery rate of 2 dm ³ / h. The temperature of the air leaving the dryer was 105 degrees Celsius.

The amount of product thus obtained was 1368 g.

It contains 630 IU / mg

Leading the ammonia wash to another cation exchange column is shown in FIGS. The product was obtained in the same manner as in Example 1b, 38.8 g having an active ingredient content of 610 IU / mg.

Claims (1)

A process for the preparation of neomycin sulfate from fermentation broths by filtration of the acidified fermentation broth, purification of the acid cation exchange resin or acid cation exchange resin and anion exchange resin, washing the ion exchange resin with water and aqueous ammonium hydroxide solution, eluting with aqueous ammonia and characterized in that, during the ion exchange of the neomycin-containing filtrate, the concentration of ammonia in the solution flowing from the ion-exchange resin is kept in the range 0.22-0.50 mol / l, the supply of which is controlled by A.) adjust the pH of the fermentation broth or filtrate to 1.0-1.9 with sulfuric acid in the presence of oxalic acid, then neutralize to pH 4-7 with ammonia, filter, B.) or the fermentation broth is acidified to pH 3.5-5.5 with sulfuric acid solution, filtered, the active compound bound to the ion exchange resin is eluted in a known manner and the eluate is purified and the neomycin sulfate is recovered.