KR101844833B1 - Purification method for teicoplanin - Google Patents

Abstract

Problem: Large amount of solvent is required for recovery and purification of teicoplanin, large-scale facilities are required, and it takes time.
SOLUTION: The present invention makes it possible to precipitate teicoplanin by making the fermentation culture of teicoplanin in an acidic condition of pH 1.5 to 3.0, and recovering it in a high yield by a simple process using a water-miscible organic solvent. In addition, the present invention can purify teicoplanin with high purity by removing impurities including Teico 2 by washing the Teicoplanin of the modifier sample in an acidic solvent and eluting it.

Description

Purification method for teicoplanin < RTI ID = 0.0 >

The present invention relates to a method for efficiently purifying teicoplanin A2, which is a glycopeptide-based antibiotic.

Glycopeptide antibiotics are antibiotics that inhibit bacterial cell wall synthesis, and are mainly used for the treatment of Gram-positive bacterial infections. They are used as a remedy for methicillin-resistant Staphylococcus aureus (MRSA) infections. In particular, teicoplanin among glycopeptide antibiotics is considered as a therapeutic agent for serious drug-resistant infectious diseases such as vancomycin resistant enterococci (VRE) as well as methicillin-resistant Staphylococcus aureus (MRSA) Oral administration is also possible antibiotics. In particular, teicoplanin is an excellent antibiotic with the advantage of being able to use half-life and intramuscular injection higher than vancomycin.

Teicoplanin was first named Teichomycin A2, and Actoplanes teichomyceticus nov., A member of actinomycetes . sp. It was reported to be produced by ATCC 31121 [J. Antibiotics 31 (1978), p. 170-177, J. Antibiotics 31 (1978), p. 276-283. Later, teacomycin A2 was named teicoplanin and was found to be a mixture of several related compounds [J. Antibiotics 37 (1984), p. 615-620, J. Antibiotics 42 (1989), p. 361-366].

Teicoplanin, according to the Korean Pharmacopoeia, requires more than 80% of teicoplanin A2 complex and less than 15% of teicoplanin A3 group. Methods for obtaining teicoplanin that meet these quality criteria have been presented in various publications. In these methods, a method of obtaining teocopranin from a fermentation broth of Actinofrenes tecomycetixus is disclosed in J. of Antibiotics , 31 (3 (Korean Patent No. 10-0184644), and a method in which a culture solution is adjusted to an alkali by a method of filtration to collect the cells (Korean Patent No. 10-0184644) And a method of recovering and acquiring a resin adsorbed on teicoplanin (Korean Patent No. 10-0321304). In the case of organic solvent extracts obtained from the primary culture, the solvent is removed by concentration under reduced pressure or by adding water, or the content of the solvent is lowered to bind to a synthetic adsorbent resin, an ion exchange resin or an affinity resin, And then eluting it with a solvent.

Conventional teicoplanin refining technology requires the use of an excessive amount of organic solvent, and the adsorption power of the adsorbent resin is low to 1 to 30 g / L level, thereby increasing the amount of adsorbent resin required and increasing the volume of the eluting solvent inevitably, The burden follows.

In addition, according to the internationally accepted standard of medicine, 10% or less of teicoplanin A3 group and 5% or less of other groups should be satisfied as impurities. Studies have shown that teicoplanin transamination reaction produces Teico 2 during long-term culture or purification ( J. Antibiotics 49 (1995), pp 644-650). Teico 2 belongs to the guitar family as an impurity, and the removal of Teico 2 in the industrial production of Teicoplanin is very important in the process of improving the quality of Teicoplanin. However, Teico 2 is structurally similar to Teicoplanin, and its physical properties are similar, making it difficult to remove in the usual purification process.

Teicoplanin possesses acyl groups and is more hydrophobic than other glycopeptide antibiotics. Therefore, teicoplanin is usually contained in solids such as cells as well as in the culture supernatant at sub-neutral pH in the recovery after the end of fermentation. For appropriate recovery, the pH is adjusted to alkaline (pH 11.0) and filtrated or centrifuged to recover the culture supernatant, or the culture solution is recovered by adding an organic solvent.

This process often leads to the use of large quantities of solvents and the generation of large volumes of solutions and consequently an increase in the scale of industrial production facilities. Also, the process time required to adsorb a large volume of the solution is consumed.

Accordingly, it is an object of the present invention to provide a simple method of recovering and purifying teicoplanin which can minimize the volume of the process and effectively remove impurities. The present invention also provides a method for separating and purifying teicoplanin highly effective for the removal of Teicoplanin A3 group, which is an impurity, and Teico 2, which is an epimerized teicoplanin impurity belonging to other groups, in particular.

Therefore, the inventors of the present invention invented a method for recovering teocopyranin in high yield by precipitating the filtrate of fermentation culture of teicoplanin at an acidic pH, more preferably at a pH of 1.5 to 4.0 with a strong acidity, Preferably a water-miscible organic solvent. The inventors of the present invention also invented a method of purifying teicoplanin with high purity by removing impurities including Teico 2 by washing the teicoplanin of the crude purification sample in an acidic solvent condition and eluting it under an acidic solvent condition .

In the present invention, the teicoplanin fermentation culture filtrate refers to a culture solution obtained by removing microbial cells and solids from a fermentation broth of teicoplanin-producing microorganism, preferably Actinofrenes tecomycetics. Removal of bacteria and solids can be performed by conventional biotechnologists such as centrifugation, drum filtration, filter press filtration, and nanofilter filtration. The fermentation culture filtrate of teicoplanin has a pH ranging from 6.0 to 11.5 according to a conventional method. In the present invention, the fermentation culture filtrate is treated with an acidic regulator, preferably an inorganic acid such as hydrochloric acid, sulfuric acid solution or the like, or an organic acid solution such as acetic acid or the like, and is acidic, preferably pH 1.5 to pH 4.0, more preferably pH 1.5 to 2.5 To precipitate tecopyran. ≪ / RTI > The precipitate can be obtained by a known method, for example, by using centrifugal force, using a filter, or by sedimentation. When the precipitation step is carried out at pH conditions deviating from the method of the present invention, the stability of teicoplanin can not be assured, and the purity is lowered and the yield is decreased because the separation degree of teico-2 is low.

In the present invention, the precipitate obtained by the above method can be recovered by mixing with a solvent capable of dissolving teocopyran. The solvent capable of dissolving teicoplanin may be selected from the group consisting of water and a C3-C4-alcohol, a C3-C4-ketone and a C3-C4-nitrile, and a mixed solvent thereof may be used. Water and water-miscible solvents such as acetone, isopropanol, methanol, ethanol, and a mixed solvent of at least one of them may be used. More preferably, 20 to 70% (v / v) water mixed solvent of isopropanol and 20-70% (v / v) water solvent mixed with acetone are used.

Using the method of the present invention, the purity of teicoplanin is 5-10% (w / v) in the culture filtrate, while the purity of the recovered teicoplanin solution is 20-40% (w / v) And the volume of the recovered teicoplanin solution is reduced to 1/5 to 1/20 (v / v) of the culture filtrate. At this time, the recovery rate of teicoplanin can be 60 to 80% (w / v).

In addition, the present invention provides a method for purifying teicoplanin to a level that satisfies pharmaceutical quality. The Teocoprene solution recovered by the method provided in the present invention can use an adsorption resin, an affinity resin or an ion exchange resin for purification to a higher level, and preferably an aromatic type, Methacrylic type, and polyamide type group, and more preferably aromatic type styrene / divinylbenzene polymer (meth) acrylate type, Resin can be selected and used.

The adsorption of teicoplanin is carried out in an organic solvent of 0 to 20% (v / v) and the pH is carried out in a neutral to alkaline range, preferably at a pH of 6.0 to 9.0, preferably at a pH of 7.0 to 8.5. After the adsorption, the washing is washed with acidic (pH 2.0-5.0) demineralized water and further washed with a mixed solvent of an acidic water-miscible organic solvent and water, wherein the water-miscible solvent is preferably a C3-C4-alcohol, C3 -C4-ketone, and C3-C4-nitrile. For further washing, a water-miscible solvent is mixed with water at 5 to 30% (v / v), more preferably a mixed solvent of 10 to 20% (v / v) is used. The washing solvent is carried out in an acidic range, preferably at a pH of from 1.5 to 5.0, more preferably at a pH of from 1.5 to 3.0. Washing is preferably carried out with a solvent three to 20 times the volume of the resin, more preferably 8 to 12 times.

The water-miscible solvent is preferably a C3-C4-alcohol, a C3-C4-alcohols, a water-miscible solvent, a water-miscible solvent of 20 to 70% (v / v) Ketone, and C3-C4-nitrile, more preferably 30 to 50% (v / v) acetone or a mixed solvent of isopropyl alcohol and water is selected. The elution solvent is carried out in the acidic range, preferably in the pH range of 1.5 to 5.0, more preferably in the pH range of 1.5 to 3.0. The elution is preferably carried out by adding an elution solvent 3 to 20 times the resin volume, more preferably 3 to 10 times. In the present invention, the flow rate during loading, washing and elution can be in the range of 0.5 BV / hr to 4 BV / hr, and the flow rate does not affect the constitution of the present invention.

The stability of teicoplanin can not be assured when the process of washing, washing or elution is carried out under alkaline conditions out of the pH range suggested by the method of the present invention, Can not be lowered, and the separation degree of Teico 2 is low, thereby decreasing the purity and decreasing the yield. Therefore, in the present invention, washing with water, washing and elution using an acidic solvent provides an effect of removing chromaticity, securing stability of teicoplanin, and increasing the yield of the obtained product. With the above-described purification method of the present invention, teicoplanin having a purity of 85% (w / w) or more can be obtained.

Teckoprenine obtained by the method of the present invention may be partially removed by a method such as activated carbon treatment or crystallization, or may be obtained as a solid material, and may be obtained as a powder through freeze drying or vacuum drying . The method provided by the present invention can be provided at any stage of the teicoplanin A2 purification.

Takoeplanin having a high yield can be obtained by a simple process by using a method of removing impurities and recovering teocopyranin by precipitating teocopranin by adjusting the culture filtrate of the present invention to an acidic condition.

In addition, in the present invention, leaching and washing of teicoplanin using an adsorbent resin and an acidic solvent provide an effect of removing chromaticity, securing stability of teicoplanin, and increasing the yield of the obtained product. With the purification method of the present invention, teicoplanin having a purity of 85% or higher can be obtained.

1 is a structure of teicoplanin to be separated and purified through the present invention.
FIG. 2 shows a typical structure of Tequo 2, which is an impurity to be removed through the present invention.
3 is an HPLC chart of a sample of teicoplanin, A is an HPLC graph of a sample of the pre-column-adjusting agent, and B is an HPLC graph of a sample purified by the method of the present invention.

Hereinafter, the constitution of the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that the embodiments of the invention described below are intended to illustrate the present invention and that the scope of the present invention is not limited by these embodiments.

≪ Example 1 >

The content and purity of various intermediate solutions or end products obtained by carrying out the process of the present invention were determined by HPLC method. Mobile phase A was prepared by dissolving 7.80 g of sodium dihydrogenphosphate dihydrate in 1.650 L of distilled water, adding 300 mL of acetonitrile, adjusting the pH to 6.0 with sodium hydroxide solution, adding distilled water, adjusting to 2 L, and degassing. Mobile phase B was prepared by dissolving 7.80 g of dihydrogen phosphate dihydrate in 550 mL of distilled water, adding 1.4 L of acetonitrile, adjusting the pH to 6.0 with sodium hydroxide solution, adjusting to 2 L with distilled water and deaeration. Chromatographic peaks were detected at UV 254 nm and C18 column (THERMO Hypersil GOLD, 250 mm x 4.6 mm, 5 μm) was used. The flow rate was 1.8 ml / min and the gradient during chromatography was as shown in Table 1 below.

Mobile phase Time (minutes)  A (%)  B (%) 0 to 32 100 -> 70 0 -> 30 32 to 40 70 -> 50 40 -> 50 40 ~ 42  50 -> 100 50 -> 0

≪ Example 2 >

Fermented in a 5 L jar fermenter to obtain teicoplanin. L of fermentation, 5 g / L of peptone, 5 g / L of yeast extract, 5 g / L of calcium carbonate, 1.2 g / L of sodium chloride, 1 g / L of maltodextrin, 20 g / 0.5 g / L, and 0.1 g / L of calcium chloride, and cultured at a temperature of 33 DEG C at a stirring speed of 600 to 800 rpm and aeration amount of 0.5 to 1 < RTI ID = 0.0 > vvm for 6 days. The final pH of the fermentation culture was maintained at 7.8 or less. The concentration of teicoplanin incubated at this time was about 4.1 g / L.

≪ Example 3 >

To the 2.8 L of teicoplanin-containing culture medium fermented in Example 2, water of about 0.5 times the volume of the culture medium was mixed and mixed with dilute sodium hydroxide solution (2N NaOH) to adjust the pH to 11.0, v) diatomaceous earth was added and filtered to remove the cells and solids, and 4 L of the culture filtrate containing teicoplanin was recovered. A dilute hydrochloric acid solution (5N HCl) was mixed with 100 ml of the culture filtrate to adjust the pH to 8.5. At this time, the concentration of teicoplanin in the culture filtrate was 2.62 g / L and the purity was about 12%. The culture filtrate was dispensed into each test tube in an amount of 10 ml each, and diluted hydrochloric acid solution (5N HCl) was mixed to adjust the pH to 2.0 to 7.0. After adjusting for 12 hours at refrigeration condition (6 ℃), it was visually confirmed that precipitation occurred. The precipitate was collected by centrifugation (3000 g, 10 minutes). The recovered precipitates showed a brownish pattern, and the amount of precipitate was higher as the pH became acidic. However, the amount of precipitate was about the same at pH 4.0 or less. The retention rate of teicoplanin present in the precipitate was evaluated by HPLC analysis of the teicoplanin remaining in the supernatant by centrifugation (Example 1) (Table 1). As a result, the residual teicoplanin remaining in the supernatant at pH 2.0 It was confirmed that coplanin was about 25% (about 75% in sediment). From the above results, it was confirmed that when the culture filtrate was adjusted to acidic pH using a pH adjusting agent, teicoplanin was easily obtained.

process
pH The concentration of teicoplanin in the supernatant (g / L) The amount of teicoplanin in the supernatant (%) The amount of Teicoplanin in the sediment (%) Absorbance of supernatant
Abs 450 Amount of sediment * pH 8.5
(Non-treatment) 2.62 100.0 0 3.670 - pH 2.0 0.65 24.9 75.1 0.920 +++++ pH 3.0 0.74 28.4 71.6 0.925 ++++ - pH 4.0 1.04 39.6 60.4 1.300 ++++ - pH 5.0 1.86 70.9 29.1 1.880 ++ - pH 6.0 2.38 91.0 9 2.420 + - pH 7.0 2.55 97.4 2.6 3.580 +

* Indicates the amount of relative sediment

<Example 4>

2 L of the culture filtrate containing teicoplanin recovered in Example 1 was added with a dilute hydrochloric acid solution (5 N HCl), adjusted to pH 2.0, allowed to stand at room temperature for 6 hours, Lt; / RTI &gt; As a result of observing during standing, a large amount of precipitate was generated after 6 hours at room temperature, and it was confirmed that a further more precipitate was formed when it was further left in the refrigerator. The precipitate was recovered by centrifugation (3000 g, 10 minutes), and the resulting precipitate was dissolved in acetone or a mixed solvent of isopropyl alcohol and water as shown in Table 3 below and analyzed by HPLC Relative recovery rate was measured. Each solvent was mixed with 0.1 times the volume of the culture filtrate, adjusted to pH 8.5, stirred for about 1 hour and centrifuged to recover the supernatant. The average of the test 3 was measured, and it was confirmed that the maximum standard deviation was less than ± 4 and was relatively uniformly dissolved. The highest recovery was obtained with 50% water mixed solvent in each solvent, and the best dissolution condition was acetone 50%.

Acetone
(v / v%) Relative Tacoplanin recovery (%) Isopropyl alcohol
(v / v%) Relative Tacoplanin recovery (%) 20 85.2 10 86.0 30 86.7 20 88.9 40 87.9 30 91.5 50 100.0 40 96.7 60 94.6 50 98.3 70 95.6 70 97.9

&Lt; Example 5 >

100 ml of the culture filtrate collected in Example 1 was mixed with dilute hydrochloric acid solution (5 N HCl) to adjust the pH to 2.0, and then allowed to stand at room temperature for 6 hours and then centrifuged to separate the precipitate and the precipitate supernatant. Water (5) (0.1 times), 7.5 (0.15 times) and 10 ml (0.2 times) was added to the recovered precipitate, and the mixture was adjusted to pH 8.5 with dilute sodium hydroxide (5N NaOH). The same volume of acetone as the water used here was added to the mixture to make a final 50% acetone (pH 8.5). After one hour of mixing, the precipitate was separated and removed by centrifugation, and the supernatant containing teicoplanin was recovered and analyzed by HPLC. The recovery of teicoplanin was measured by comparing with the culture filtrate (Table 4). The total recoveries were 60 ~ 69%, and recovery of at least about 80% and maximum of about 95% of teakoplanin was confirmed in the dissolution step (recovery rate from the precipitate) of 0.1 to 0.2 times.

Teicoplanin recovery (%) Culture filtrate 100.0 Precipitation supernatant 27.1 Sediment dissolved **
(50% acetone)
0.1 times 60.5 (82.9) * 0.15 times 60.6 (83.1) 0.2 times 69.2 (94.9)

* Numbers in parentheses indicate the recovery rate of teicoplanin from the sediment

** 50% acetone usage volume compared to culture filtrate volume

&Lt; Example 6 >

In the same manner as in Example 3, a teicoplanin dissolution supernatant was obtained. At this time, the solvent used for the dissolution was 50% acetone, and the volume was 0.1 times of the culture filtrate. The solvent was removed by concentrating the solution under reduced pressure to prepare a sample having a purity of about 30% and a concentration of about 80 g / L of Teicoplanin A2. The amount of teicoplanin absorbed after overloading the column packed with the resin shown in Table 5 below was calculated. As a result, the adsorption amount of ion - binding resin was very low, and HP 20, which is a porous adsorbent resin, was 30 g / L. In the case of a synthetic adsorbent having high binding ability, adsorption was possible up to 83 g / L. The use of high-bond performance resins can dramatically reduce the amount of solvent used in the column process, as the total resin usage is reduced.

Suzy Maximum adsorption amount of teicoplanin (g / L) SK1B 9 PA408 8 PK208 3 WK60L 3 CM650C 2 HP20 30 NM100 80 NM200 65 CG161M 83 CG161C 80

&Lt; Example 7 >

The mixture was mixed with about 0.5 times the volume of the culture medium to 2.8 L of the final Teckoplanin-containing culture medium, mixed with dilute sodium hydroxide solution (2N NaOH) to adjust the pH to 11.0 and mixed with 7% (w / v) Of diatomaceous earth was added and filtered to remove the cells and solids, and 4 L of the culture filtrate containing teicoplanin was recovered. At this time, the purity of Teicoplanin A2 was about 17% and the concentration was 1.4 g / L. A dilute hydrochloric acid solution (5 N HCl) was added to 1 L of a culture filtrate containing about 1400 mg of teicoplanin, and the mixture was adjusted to pH 2.0. The mixture was allowed to stand overnight in a refrigerator (6 ° C) . The precipitate was mixed with 50 ml of water, adjusted to pH 8.5 with dilute caustic soda (5N NaOH), and then about 50 ml of acetone was added thereto, followed by stirring in a 50% acetone solution (pH 8.5) for about 1 hour. After stirring, the undissolved solids were removed using a filter and the soluble supernatant containing teocopyran was recovered. Analysis of this supernatant by HPLC (A in FIG. 3) revealed that the purity of teicoplanin A2 was about 29% and the total amount was about 930 mg. In this case, the impurity was 63% in Teikopranin A3 group, 7%, which was concentrated under reduced pressure to prepare acetone at 15% or less. This was loaded onto a glass column (1.5 x 20 cm) filled with approximately 5 mL of CG161m (Amberchrom, Rome and Hass). The glass column was washed with 50 mL of deionized water (pH 4.5), and further washed with 50 mL of 15% isopropyl alcohol (pH 2.0). After washing, teicoplanin was eluted with 25 ml of 30% isopropyl alcohol (pH 2.0). The eluate was separated by 5 ml each, and the teicoplanin was recovered. The flow rate of the column work was between 0.5 and 4 BV / hr. The initial fractions containing teicoplanin were mixed and adjusted to pH 8.5. The HPLC analysis revealed that the purity of teicoplanin was 1.12% in the teicoplanin A3 group and that in the teicoplanin A2 group Purity was 95.39%, and teicoplanin purity of the other group was 3.49%, and teicoplanin of very good purity was eluted (Fig. 3B). The yield was 65.9%. This method of the present invention was particularly useful for the removal of other groups including Teico 2.

&Lt; Example 8 >

Teocopyanine was purified in the same manner as in Example 7. Except that the pH of 15% isopropyl alcohol and 30% isopropyl alcohol was adjusted to pH 1.5, pH 2.5, and pH 3.0, respectively. At this time, teicoplanin of 0.9 ~ 1.5%, 88 ~ 94% in the A2 group and 3.0 ~ 6.5% in the other group was recovered from the recovered Teicoplanin A3 group and teicoplanin of very good purity was eluted. The yield was 60-75%.

Claims (8)

a) adsorbing crude tetraplanin to at least one adsorption resin selected from the group consisting of an aromatic type adsorption resin, an aromatic chemically modified type adsorption resin and a methacrylic type adsorption resin;
b) washing the adsorbent resin with acidic water having a pH of 1.0 to 4.5;
c) 5 to 30% (v / v) water-washing the adsorbent resin by adjusting the miscible organic solvent to a pH of 1.5 to 3.0;
d) 20 to 70% (v / v) water-miscible (at least one) selected from the group consisting of C3-C4-alcohols, C3-C4-ketones and C3- Fractionating eluted teicoplanin with an organic solvent; And
e) selecting the Teocopranin A2 group in the eluted fractions.
The method according to claim 1,
wherein the adsorbent resin of step a) is a styrene / divinylbenzene polymer adsorbent resin. delete delete delete delete delete delete

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