KR101682389B1 - Method for manufacturing highly pure crystallized Vacomycin and Solution for highly pure crystallized Vacomycin - Google Patents

Abstract

The present invention relates to a method for producing vancomycin crystallized at high purity and a solution for high purity vancomycin crystals. More particularly, the present invention relates to a method for producing vancomycin having high crystal quality and uniform crystallinity and improved purity of vancomycin And to a solution for high purity vancomycin crystallization.

Description

TECHNICAL FIELD [0001] The present invention relates to a method for producing vancomycin crystallized at a high purity and a method for producing a highly pure crystallized vancomycin,

The present invention relates to a method for producing vancomycin crystallized at high purity and a solution for high purity vancomycin crystals. More particularly, the present invention relates to a method for producing vancomycin having high crystal quality and uniform crystallinity and improved purity of vancomycin And to a solution for high purity vancomycin crystallization.

Glycopeptids Antibiotics are antibiotics in the form of saccharides attached to the backbone of nonribosomal peptides and include vancomycin, teicoplanin, telavancin, bleomycin, such as bleomycin, ramoplanin, and decaplanin.

Among them, vancomycin is the first antibiotic of the glycopeptide system and is a microorganism isolated from soil in the Borneo region by Eli Lilly in 1956, Amycolatopsis orientalis {synonym Streptomyces orientalis ( Streptomyces orientalis) orientalis , and the synonymous Nocardia orientalis .

Vancomycin is an antibiotic with a molecular weight of about 1449, which is composed of a heptapeptide in which seven amino acid residues are linked by a peptide bond and has an -o-o-vancosamine-glucosyl group attached thereto. Vancomycin inhibits bacterial growth by inhibiting the action of Pbp2, a major enzyme in bacterial peptidoglycan synthesis, by inhibiting cell wall synthesis, and is an antibiotic effective against Gram-positive bacteria, especially those with a thick peptidoglycan layer. That is, vancomycin binds to the acyl-D-alanyl-D-alanine of the peptidoglycan, a substrate of Pbp2, to interfere with the elongation of the novel peptidoglycan, As the cell wall, which is the last defense net of bacteria, is not completely synthesized, the bacteria can not withstand the osmotic pressure and eventually die.

Vancomycin is mainly used for the treatment of infectious diseases such as Staphylococcus, Streptococcus, Clostridium difficile, which are Gram positive bacteria. It is especially important for the treatment of methicillin-resistant Staphylococcus aureus (MRSA), which is fatal to surgery patients, elderly patients and weakly immunized persons. It is commonly used as an injectable or oral agent in the form of vancomycin hydrochloride. Vancomycin is also widely used for the treatment of endocarditis, which is allergic to penicillin or cephalosporin. It has been used for the treatment of cardiac surgery, orthopedic surgery, It is also used for preventive treatment in brain surgery such as ventriculoperitoneal shunt.

As described above, vancomycin, which is effective as an antibiotic, generally produces vancomycin obtained from microbial fermentation through various stages of separation and purification processes. In the case of vancomycin currently listed in US and European Pharmacopoeia, the content of vancomycin, the content of total impurities, The maximum content of flameproof is strictly regulated. In the United States, vancomycin content should be above 88% by USP (United States Pharmacopeia) HPLC method, and no substances other than vancomycin should be present in excess of 4%. In Europe, the European Pharmacopoeia (EP) has a vancomycin content of over 93% and, like the USP, strictly regulates substances with a content of vancomycin exceeding 4%. In order to satisfy these regulations, several stages of separation and purification process are required. In general, a crystallization process is introduced as a final purification stage in the production process of pharmaceuticals with high purity such as antibiotics.

Crystallization technology is a method of producing and dissolving solids from a liquid or gas mixture, which is a core technology in the field of controlling the physical properties and shape of crystalline materials as well as the separation and purification of specific materials from the mixture. This crystallization technology not only enhances the quality of the final product, but also produces high value-added products with low energy consumption and environmental friendliness. In addition, the fixed investment cost is low and the process is simple, so the application field is wide.

Such a crystallization technique includes all the techniques for growing crystals by generating crystal nuclei in a solution. The term "crystal nucleus" refers to the smallest crystal grains that are initially formed from a solution. Such nucleation refers to homogeneous nucleation, in which nuclei are formed by supersaturation in a liquid phase, and to homogeneous nucleation of the outer surface (external impurity particles, And heterogenous nucleation in which nuclei are formed with the help of the nucleus. Uniform nucleation can induce nucleation spontaneously due to high supersaturation, so nucleation rate is very fast. On the other hand, in the case of heterogeneous nucleation, the rate of nucleation is very slow due to the relatively low supersaturation. In general, a solute having a relatively high solubility such as a bioproduct is limited in heightening the degree of supersaturation. Therefore, in many cases, formation of crystal grains is caused by nonuniform nucleation. In the case of vancomycin, too, the nucleation time is very long and the rate of nucleation is shortened due to the increase of the surface area of the reactor.

On the other hand, ionic liquids have characteristics such as low vapor pressure, high thermal stability, and high ion conductivity, which is a conventional alternative to volatile organic solvents. It is an environmentally friendly material. Due to the unique properties of these ionic liquids, it has recently been applied to the crystallization of proteins and organic compounds. In particular, ionic liquids in protein crystallization processes have been reported to have a great influence on crystal morphology (nucleation, crystal growth, crystal morphology / stability, stability).

In order to obtain vancomycin of high purity (> 97%) and high yield (> 95%) in the conventional vancomycin crystallization process, optimization of a number of conditions such as solvent selection, crystallization temperature, time and pH has been required. However, until now, it takes about 24 hours to produce crystals of vancomycin, which makes it difficult to mass-produce vancomycin by crystallization. In addition, the conventional crystallization method of vancomycin has a problem that the crystal size of vancomycin is uneven, the quality of crystal such as the presence of crushed crystals is lowered, and crystallization with high purity is difficult.

Accordingly, it is urgently required to study a method for producing vancomycin which is excellent in crystal quality, uniform, and crystallized in a short time.

SUMMARY OF THE INVENTION The present invention has been made in order to solve the above-mentioned problems, and it is an object of the present invention to provide a vancomycin which has a crystal quality uniform and a crystal quality within a short time, A method for producing vancomycin crystallized at a high purity and a solution for high purity vancomycin crystallization which can be obtained.

In order to solve the above-mentioned problems, the present invention provides a process for producing an imidazolium halide, which comprises (1) a step of adding 12 to 27% by volume of an imidazolium halide containing an alkyl group having 1 to 6 carbon atoms for shortening the crystallization time and improving crystal quality 1 < / RTI > solution of a vancomycin precursor; (2) preparing a second solution by mixing the first solution and the hydrophilic organic solvent in a volume ratio of 1: 3.3 to 3.8; And (3) crystallizing vancomycin by treating the second solution at 8 to 12 占 폚.

According to a preferred embodiment of the present invention, the imidazolium halide containing an alkyl group having 1 to 6 carbon atoms is 1-butyl-3-methyl-imidazolium tetrafluoroborate. imidazolium tetrafluoroborate, 1-butyl-3-methyl-imidazolium hexafluorophosphate, 1-butyl-3-methyl-imidazolium hexafluoroantimonate 1-ethyl-3-methyl-imidazolium hexafluoroantimonate, 1-ethyl-3-methyl-imidazolium tetrafluoroborate, (1-ethyl-3-methyl-imidazolium hexafluoroantimonate) and 1-ethyl-3-methyl-imidazolium hexafluoroantimonate And may include any one or more of them.

According to another preferred embodiment of the present invention, the imidazolium halide containing an alkyl group having 1 to 6 carbon atoms is 1-butyl-3-methyl-imidazolium tetrafluoroborate and 1-butyl- -Imidazolium < / RTI > hexafluorophosphate.

According to another preferred embodiment of the present invention, the solvent of the first solution is distilled water, and the imidazolium halide containing an alkyl group having 1 to 6 carbon atoms may be contained in the first solution in an amount of 18 to 22% by volume .

According to another preferred embodiment of the present invention, the pH of the first solution may be 4.4 to 4.7.

According to another preferred embodiment of the present invention, the electrical conductivity of the first solution may be 8.5 to 10.5 ms / cm.

According to another preferred embodiment of the present invention, the crystallization minimum time in the step (3) may be 11 to 16 hours.

According to another preferred embodiment of the present invention, the steps (1) to (3) may satisfy all of the following conditions (a) to (c).

(a) (1) comprises 1-butyl-3-methyl-imidazolium tetrafluoroborate in an amount of 18 to 22% by volume relative to the first solution, (b) the pH of the first solution is 4.4 to 4.7 And the electric conductivity is 9.5 to 10.5 ms / cm. (C) The minimum crystallization time in step (3) is 11 to 13 hours.

Further, in order to solve the above-mentioned problems, And an imidazolium halide including an alkyl group having 1 to 6 carbon atoms for improving crystallization time and crystal quality, wherein the imidazolium halide having an alkyl group having 1 to 6 carbon atoms and a solvent 12: 88 ~ 27: 73 by volume. The solution for crystallization of high purity vancomycin is provided.

According to a preferred embodiment of the present invention, the solution for crystallizing vancomycin may satisfy the following conditions (a) to (c).

(a) an imidazolium halide containing an alkyl group having 1 to 6 carbon atoms, such as 1-butyl-3-methyl-imidazolium-tetrafluoroborate and 1-butyl-3-methyl-imidazolium-hexafluoro (B) the pH of the solution for crystallizing vancomycin is 4.4 to 4.7; and (c) the conductivity of the solution for vancomycin crystallization is 8.5 to 10.5 ms / cm.

According to another preferred embodiment of the present invention, the solution for vancomycin crystals may contain an imidazolium halide containing an alkyl group having 1 to 6 carbon atoms and a solvent in a volume ratio of 18: 82 to 22: 78.

Hereinafter, terms used in the present invention will be described.

The term "alkyl group" used in the present invention includes both straight chain or branched chain, and the meaning of including an alkyl group is used to mean that the alkyl group is included as a substituent.

The term " vancomycin precursor " used in the present invention may be a fermentation broth obtained from a strain of nocardia orientalis, or a hydrochloric acid form of vancomycin in which cells are removed from the fermentation broth and impurities are removed by ion exchange resin.

The method of the present invention for producing vancomycin enables to obtain vancomycin having a crystal size uniformly, a crystal quality remarkably improved and a crystal purity remarkably improved even though the crystallization time is shortened as compared with the conventional method. In addition, the solution for high purity vancomycin crystallization of the present invention shortens vancomycin crystal formation time, produces crystals of uniform size, and enables high-purity vancomycin to be obtained with high crystal quality.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a photomicrograph of a crystal according to crystallization time for each concentration of an imidazolium halide containing an alkyl group having 1 to 6 carbon atoms. FIG.
2 is a video microscope photograph of vancomycin crystals obtained according to the pH of a first solution according to a preferred embodiment of the present invention.
3 is a video image of vancomycin crystals formed according to the electrical conductivity of the first solution according to a preferred embodiment of the present invention.
4 is an XRD pattern of vancomycin crystals formed according to the electrical conductivity of a first solution according to a preferred embodiment of the present invention.
5 is a video microscope photograph of vancomycin crystals according to the amount of hydrophilic organic solvent added.
6 is a video microscope picture of the vancomycin crystal obtained according to the crystallization temperature.

Hereinafter, the present invention will be described in more detail.

As described above, vancomycin is required to have high purity in various countries in order to be used in medicine. However, conventional crystallization of vancomycin requires about 24 hours for crystal formation, which makes it difficult to mass-produce vancomycin by crystallization, There is a problem that it is difficult to obtain vancomycin which crystallizes at a high purity, is excellent in crystal quality, and has a uniform crystal size.

In the present invention, (1) a vancomycin precursor is added to a first solution containing 12 to 27% by volume of an imidazolium halide containing an alkyl group having 1 to 6 carbon atoms for shortening the crystallization time and improving the crystal quality ; (2) preparing a second solution by mixing the first solution and the hydrophilic organic solvent in a volume ratio of 1: 3.3 to 3.8; And (3) crystallizing vancomycin by treating the second solution at 8 to 12 캜. The present invention has been made to solve the above-mentioned problems by providing a method for producing vancomycin crystallized at high purity.

As a result, vancomycin having a uniform crystal size, excellent crystal quality and significantly improved crystal purity can be obtained even though the crystallization time is drastically shortened as compared with the conventional method.

First, in step (1), a vancomycin precursor is added to a first solution containing 12 to 27% by volume of an imidazolium halide containing an alkyl group having 1 to 6 carbon atoms, which shortens the crystallization time and improves the crystal quality .

The first solution will be described in detail.

The first solution includes an imidazolium halide containing an alkyl group having 1 to 6 carbon atoms, and may include a solvent.

In the present invention, an imidazolium halide containing an alkyl group having 1 to 6 carbon atoms used for shortening the crystallization time and improving the crystal quality will be described first.

Conventionally, the solvent used in the crystallization process of vancomycin is usually distilled water, and it is common to dissolve the vancomycin precursor in distilled water and induce supersaturation of the solution to crystallize vancomycin. However, in this case, the time required for crystallization is prolonged so that the production of vancomycin is a limit to mass production, and the obtained crystals are broken or uneven.

Accordingly, the inventors of the present invention have studied the solution of the above problems. When the imidazolium halide containing an alkyl group having 1 to 6 carbon atoms is included in the solvent, the time required for the crystallization of vancomycin can be shortened, And the size of the crystal can be increased at the same time, leading to the present invention.

The imidazolium halide containing an alkyl group having 1 to 6 carbon atoms for use in the present invention may be a powder in the form of particles or a liquid, and may be a substance commonly referred to as an ionic liquid. An ionic liquid is a liquid ionic compound composed of a cation and an anion at normal temperature. Typically, the salt is dissolved in a solvent to have conductivity, but the ionic liquid has conductivity in that state. It is known that it is difficult to volatilize due to low steam pressure and has excellent thermal stability. Such an ionic liquid shortens the time required for crystallization of vancomycin, and the material diffusion rate is very slow in vancomycin crystal growth, so that a crystal of a certain shape grows cleanly, thereby improving crystal quality. The crystal size can be relatively increased while the number of crystals produced is small.

According to a preferred embodiment of the present invention, the alkyl group having 1 to 6 carbon atoms may be linear or branched, preferably two alkyl groups may be included in the imidazolium as a substituent, more preferably the number of carbon atoms May be 1 to 4. Accordingly, the imidazolium halide containing an alkyl group having 1 to 6 carbon atoms is preferably 1-butyl-3-methyl-imidazolium tetrafluoroborate, 1-butyl-3 1-butyl-3-methyl-imidazolium hexafluorophosphate, 1-butyl-3-methyl-imidazolium hexafluoroantimonate Ethyl-3-methyl-imidazolium tetrafluoroborate, 1-ethyl-3-methyl-imidazolium hexafluorophosphate (1-ethyl- 3-methyl-imidazolium hexafluorophosphate) and 1-ethyl-3-methyl-imidazolium hexafluoroantimonate (1-ethyl-3-methyl-imidazolium hexafluoroantimonate). Among the above listed materials, in view of improving the crystallinity of vancomycin and shortening the crystallization time, it is more preferable to use 1-butyl-3-methyl-imidazolium tetrafluoroborate and 1-butyl- Phosphate, and more preferably 1-butyl-3-methyl-imidazolium tetrafluoroborate for further improved crystallization time.

The imidazolium halide containing an alkyl group having 1 to 6 carbon atoms as described above is contained in an amount of 12 to 27% by volume based on the total volume of the first solution, and more preferably, in order to obtain a more uniform and uniform crystal, 18 to 22% by volume of an imidazolium halide containing an alkyl group having 1 to 6 carbon atoms relative to the total volume can be contained. If it is contained in an amount less than 12 vol%, the time required for vancomycin crystallization may be prolonged and the object of the invention may not be achieved or crystals may not be formed. If it is contained in an amount exceeding 27 vol%, the effect of shortening the time required for crystallization of vancomycin may be insignificant, and the quality of the crystal may be rather lowered and the crystallization efficiency may be lowered.

According to a preferred embodiment of the present invention, an imidazolium halide containing an alkyl group having 1 to 6 carbon atoms for obtaining a more pronounced and uniform vancomycin crystal may be contained in an amount of 18 to 22% by volume based on the total volume of the first solution have. More specifically, FIG. 1 is a video microscope photograph of the shape of crystals according to the crystallization time with respect to the concentration of imidazolium halide containing an alkyl group having 1 to 6 carbon atoms. In the case where the concentration is 10% by volume (A in FIG. 1) -Butyl-3-methyl-imidazolium tetrafluoroborate was used, crystals were obtained after 18 hours. When 1-butyl-3-methyl-imidazolium hexafluorophosphate was used, It can be confirmed that the crystal itself was not generated during the lapse of 18 hours. On the contrary, in the first solution, 15 vol%, 20 vol%, 1-butyl-3-methyl-imidazolium tetrafluoroborate or 1-butyl-3-methyl-imidazolium hexafluorophosphate as 25 vol% (B, C, D in FIG. 1), crystallization was obtained 12 hours before and 15 hours before crystallization, and the time required for crystallization was minimized. Particularly, 1-butyl-3-methyl-imidazolium tetrafluoro When borate was added in the above concentration, it was confirmed that crystals were already formed after 12 hours. It can be seen that the minimum time required for crystallization is very effective in shortening the crystallization time according to the time less than 12 hours.

Further, when the first solution contains 1-butyl-3-methyl-imidazolium tetrafluoroborate or 1-butyl-3-methyl-imidazolium hexafluorophosphate in an amount of 20 vol% C and D) crystals were very uniform and large in size, and thus vancomycin crystals were obtained which were remarkably superior to those in other concentrations.

Next, the solvent that can be contained in the first solution will be described.

The solvent is suitable for dissolving an imidazolium halide containing an alkyl group having 1 to 6 carbon atoms used in the present invention and can be used without limitation as long as it is a solvent which does not affect vancomycin precursor and vancomycin crystal formation, Distilled water can be used in terms of improving the quality of vancomycin crystals obtained.

The pH of the first solution containing an imidazolium halide containing an alkyl group having 1 to 6 carbon atoms as described above may preferably be 4.4 to 4.7. The pH of the first solution is one of the factors affecting the crystallization yield of vancomycin. In order to obtain the maximum crystallization yield of vancomycin, it is necessary to minimize the solubility of the vancomycin precursor in the first solution, Is more advantageous in terms of crystallization yield and crystallization quality. Preferably, the pH of the first solution may be 4.4 to 4.7, and if the pH is less than 4.4, vancomycin particles may not aggregate (gel) and vancomycin crystals may not be formed. If the pH is higher than 4.7 There may be a problem that crystals are not generated or crystals obtained may be crushed or the shape of the crystals may be irregular, and even if crystals are produced, uniform crystals can not be obtained. More specifically, FIG. 2 is a video microscope photograph of vancomycin crystals obtained according to the pH of the first solution according to a preferred embodiment of the present invention. The pH of the first solution is 4.0 (FIG. 2A), 5.5 2), it can be confirmed that no crystals are formed. When the pH is 5.0 (C in FIG. 2), a part of the crystals are produced but the crystals are not clear, the crystals are aggregated, . On the contrary, when the pH is 4.5, it can be confirmed that vancomycin crystals which are very distinct and uniform and have a remarkably high crystal quality are produced. The method of adjusting the pH of the first solution to the above-mentioned numerical value range can be performed by a method of adjusting the pH of a conventional solution, and as a non-limiting example, the pH of the first solution can be adjusted through hydrochloric acid or sodium hydroxide .

Further, the electrical conductivity of the first solution may be preferably 8.5 to 10.5 ms / cm, and more preferably 9.5 to 10.5 ms / cm. The electrical conductivity is a factor affecting the crystal yield and crystal quality of vancomycin, particularly contributing to the formation of distinct, uniform, and large crystals. The first solution can be determined for electrical conductivity through an imidazolium halide containing an alkyl group having 1 to 6 carbon atoms. If the electrical conductivity is such that the imidazolium halide containing an alkyl group having 1 to 6 carbon atoms If it is not feasible to do so, a further water-soluble salt may be further added to control the desired electrical conductivity value. Such a water-soluble salt may be any salt that does not affect the pH, As a non-limiting example, sodium chloride, magnesium chloride, potassium chloride, lithium chloride, and the like may be used alone or in combination of two or more. If the electric conductivity of the first solution is less than 8.5 ms / cm, unclear crystals are obtained and the crystallization efficiency is lowered. If the electric conductivity exceeds 10.5, clear crystals are produced, The larger the number of crystals, the larger the number of crystals than the crystal growth, and the size of crystals may not be suitable for producing large crystals. 3 is a video image of vancomycin crystals formed according to the electrical conductivity of the first solution according to a preferred embodiment of the present invention, wherein the electrical conductivities are 11 ms / cm (C in FIG. 3) and 12 ms / cm (D in FIG. 3), the crystal size is smaller and the crystal quality is lowered as compared with the case where the electrical conductivity is 9 ms / cm (A in FIG. 3) and 10 ms / , And the electric conductivity is 10 ms / cm (B in Fig. 3) is 9 ms / cm (A in Fig. 3), the crystal size is large and the most distinct and uniform crystal is obtained.

This result can be more clearly understood from the results of XRD analysis of the obtained vancomycin crystals. FIG. 4 is an XRD pattern of vancomycin crystals formed according to the electrical conductivity of the first solution according to a preferred embodiment of the present invention, (FIG. 4A), 10 ms / cm (FIG. 4B), 11 ms / cm (FIG. 4C) and 12 ms / In particular, when the electric conductivity is 9 ms / cm (A in FIG. 4) and 10 ms / cm (B in FIG. 4), the peak is clearly observed in the range of 5 ° to 10 °. Especially, Is 10 ms / cm (B in Fig. 4), the peak is narrow and thus the crystallinity is very excellent.

Next, the vancomycin precursor introduced into the first solution will be described.

The vancomycin precursor may be a fermentation liquid itself obtained from a strain of nocardia orientalis, or a vancomycin having a purity of less than 97% in hydrochloride form in which cells are removed from the fermentation broth and impurities are removed from the fermentation broth. The process of removing impurities from the fermentation broth may be a conventional vancomycin purification method. As a non-limiting example, the fermentation broth in which the cells are removed is continuously passed through a cation exchange resin, an anion exchange resin and a porous cation exchange resin, After that, alumina and weakly acidic cation exchange resin can be used to remove impurities such as pigment and protein.

The above-described vancomycin precursor may be included in an amount of 5 to 20 parts by weight based on 100 parts by weight of the first solution described in step (1) according to the present invention. If the vancomycin precursor is contained in an amount exceeding 20 parts by weight, the yield may be lowered, and even if the amount is less than 5 parts by weight, the yield may be lowered.

The step (1) according to the present invention may preferably be performed at room temperature, and if the temperature range is satisfied, the energy saving effect may be obtained.

Next, in step (2), the first solution and the hydrophilic organic solvent are mixed at a ratio of 1: 3.3 to 3.8 by volume to prepare a second solution.

In step (2), a hydrophilic organic solvent is added to lower the solubility of the solution mixed with the first solution and the vancomycin precursor through step (1), thereby causing the vancomycin dissolved in the mixed solution to be converted into a supersaturated state So that the crystal quality of vancomycin is excellent and the crystal size can be maximized. The hydrophilic organic solvent may reduce the solubility of the first solution and may be used without limitation in the case of an organic solvent which can be well mixed with the solvent of the first solution. If the solvent of the first solution is distilled water, For example, ethanol, methanol, acetone, etc. may be used in combination with one or more of them. In order to clarify the shape of the vancomycin crystal and to enlarge the particle size thereof It is preferable to use acetone.

The amount of the hydrophilic organic solvent to be added to the first solution in the step (2) according to the present invention is such that the hydrophilic organic solvent is mixed in the ratio of 1: 3.3 to 3.8 by volume relative to the first solution. If the amount of the hydrophilic organic solvent is less than 3.3 parts by volume In this case, vancomycin crystals exhibit an uneven needle shape, and a clear and uniform crystal can not be obtained, and when added in an amount exceeding 3.8 volume ratio, crystals may not be formed. Specifically, FIG. 5 is a video microscope photograph of vancomycin crystals according to the amount of hydrophilic organic solvent to be added, wherein acetone is added to the first solution at a volume ratio of 2.5 (B in FIG. 5) And it can be confirmed that the crystal quality of the crystal is not good, and when the acetone is added to the first solution at 4.0 volume ratio (E in FIG. 5), it can be confirmed that crystals of vancomycin are not produced, It is confirmed that the crystals of vancomycin are distinct and homogeneous when they are prepared by charging the first solution at a volume ratio of 3.5 volume (D in FIG. 5).

Next, in step (3), the second solution may be treated at 8 to 12 캜 to crystallize vancomycin.

Usually, the solubility of a solute in a solvent depends on the temperature, and the solubility of the solid generally decreases with decreasing temperature, so the temperature is one of the important factors influencing the crystallization yield of vancomycin. Thus, the quality of the vancomycin crystal obtained according to the temperature upon crystallization varies, and the second solution is treated at 8 to 12 ° C to obtain the most distinct and uniform crystal form. If the second solution is treated at a temperature lower than 8 占 폚, no vancomycin crystals are formed, aggregation phenomenon of the vancomycin particles sticking to each other (gelling phenomenon) may occur, and if the temperature is exceeded, There may be a problem that vancomycin crystals having poor quality, such as crystals of crushed or crushed into small needle-like crystals, coagulate with each other. Specifically, FIG. 6 is a video microscope photograph of vancomycin crystal obtained according to the crystallization temperature. It can be confirmed that no crystal was formed when the crystallization temperature was 5 ° C (FIG. 6A) 6 C), it can be confirmed that the shape of the crystal is broken into needles, whereas when the crystallization temperature is 10 ° C (FIG. 6B), it is confirmed that the crystal is clear, uniform, and the crystal quality is remarkably excellent .

The minimum crystallization time of vancomycin in the case of carrying out the steps (1) to (3) according to the present invention may be specifically varied depending on various factors derived from the explanation of steps (1) to (3) , Preferably 11 to 13 hours, which can drastically shorten the time required for crystallization of vancomycin (usually 24 hours) by the conventional method, thereby making the mass production of vancomycin more smooth .

The vancomycin crystals produced by the step (3) can be further washed with the hydrophilic organic solvent used in step (2) to remove impurities on the crystal surface, and the crystals can be filtered using a conventional crystal filtering method Can be separated.

Meanwhile, according to a preferred embodiment of the present invention, the method for producing vancomycin crystals of steps (1) to (3) according to the present invention can satisfy all the conditions (a) to (c).

First, as a condition (a), the first solution of step (1) may contain 18 to 22% by volume of 1-butyl-3-methyl-imidazolium tetrafluoroborate, It is more preferable to use 1-butyl-3-methyl-imidazolium tetrafluoroborate to improve the quality of crystals than to use crystals of vancomycin obtained only when 18 to 22% by volume is contained in the first solution Can be distinct and uniform and the crystal quality can be improved.

Next, as a condition (b), the pH of the first solution may be 4.4 to 4.7 and the electrical conductivity may be 9.5 to 10.5 ms / cm. When the pH range and electrical conductivity are satisfied, the shape of the obtained vancomycin crystal is clear Crystallization is not carried out toward increasing the number of crystal nuclei, but crystallization is carried out toward increasing the crystal size. Thus, vancomycin having improved crystal quality can be obtained.

Next, as the condition (c), when the conditions (a) and (b) are satisfied and the conditions of the steps (1) to (3) according to the present invention (amount of the hydrophilic organic solvent, crystallization temperature) are satisfied, crystallization of vancomycin Is 11 to 13 hours, the crystallization time can be remarkably shortened as compared with the conventional method, which enables the mass production of vancomycin.

On the other hand, And an imidazolium halide including an alkyl group having 1 to 6 carbon atoms for improving crystallization time and crystal quality, wherein the imidazolium halide having an alkyl group having 1 to 6 carbon atoms and a solvent 12: 88 ~ 27: 73 by volume.

The solution for crystallization according to the present invention is a solution capable of obtaining vancomycin crystals having a high crystallinity in a high purity within a short period of time and having an increased crystal size when a vancomycin precursor is added thereto and includes an alkyl group having 1 to 6 carbon atoms Containing imidazolium halide and solvent in a ratio of 12: 88 to 27: 73 by volume, more preferably an imidazolium halide containing an alkyl group having 1 to 6 carbon atoms in order to obtain more pronounced and uniform crystals, and The solvent may be included in a volume ratio of 18:82 to 22:78. If the imidazolium halide containing an alkyl group having 1 to 6 carbon atoms is contained in an amount of less than 12 parts by volume, the time required for vancomycin crystallization may be prolonged and the object of the invention may not be achieved or crystals may not be formed. In addition, if it exceeds 27 volume ratio, the effect of shortening the time required for crystallization of vancomycin may be insignificant, and the crystal quality may be rather lowered and the crystallization yield may be lowered.

Specific examples of the imidazolium halide containing the solvent and the alkyl group having 1 to 6 carbon atoms are as described above.

According to a preferred embodiment of the present invention, the solution for crystallizing vancomycin may satisfy the following conditions (a) to (c).

(a) an imidazolium halide containing an alkyl group having 1 to 6 carbon atoms, 1-butyl-3-methyl-imidazolium tetrafluoroborate and 1-butyl-3-methyl-imidazolium hexafluorophosphate (B) the pH of the solution for crystallizing vancomycin is 4.4 to 4.7; and (c) the conductivity of the solution for vancomycin crystallization is 8.5 to 10.5 ms / cm.

In the case of the solution for the crystallization of vancomycin satisfying all of the conditions (a) to (c), it is possible to obtain a vancomycin crystal having an excellent crystallinity and a large crystal size, uniformity and crystal shape, , When the imidazolium halide containing an alkyl group having 1 to 6 carbon atoms in the above condition (a) is 1-butyl-3-methyl-imidazolium tetrafluoroborate, a more excellent vancomycin crystal can be obtained in terms of crystallinity There is an advantage to have. The critical values for the pH value range of the condition (b) and the electrical conductivity value range of the condition (c) are the same as those described above, and the description thereof is omitted.

The present invention will now be described more specifically with reference to the following examples. However, the following examples should not be construed as limiting the scope of the present invention, and should be construed to facilitate understanding of the present invention.

≪ Example 1 >

First, 1-butyl-3-methyl-imidazolium tetrafluoroborate and distilled water were mixed at a volume ratio of 20: 80 to prepare a first solution. A 1N hydrochloric acid aqueous solution was added to adjust the pH of the mixed solution to 4.5, and sodium chloride was added to the mixed solution so that the electric conductivity of the mixed solution was 12 ms / cm to prepare a first solution. The microorganism isolated from the soil in the first solution is Norcardia orientalis (10) parts by weight based on 100 parts by weight of the first solution at room temperature (25 ° C) in a hydrochloric salt state of 88% purity in which impurities such as pigment and protein prepared by the conventional method originated from orientalis are removed, Respectively. Thereafter, the acetone was slowly dropped into the mixed solution with stirring dropwise, and the added acetone was mixed with the first solution at a ratio of 1: 3.5, thereby preparing a second solution. Then, the second solution was crystallized at 10 DEG C for 12 hours to prepare crystallized vancomycin.

≪ Examples 2 to 30 &

The procedure of Example 1 was repeated to prepare vancomycin which was crystallized by changing conditions such as kind and content of ionic liquid used as shown in Table 1 below.

≪ Comparative Examples 1 to 18 >

Crystallized vancomycin was prepared in the same manner as in Example 1 except that the conditions such as kind and content of ionic liquid used were changed as shown in Table 2 below.

The first solution The second solution crystallization
Temperature crystallization
time Imidazolium halide
(Kind / volume% ¹⁾ ) pH Electrical conductivity
(Ms / cm) Hydrophilic organic solvent
(Type / volume ratio ^{2 )} ) Example 1 X ^{3 )} / 20 4.5 12 Acetone / 3.5 10 12 Example 2 X / 20 4.5 12 Acetone / 3.5 10 15 Example 3 X / 20 4.5 12 Acetone / 3.5 10 18 Example 4 X / 15 4.5 12 Acetone / 3.5 10 12 Example 5 X / 15 4.5 12 Acetone / 3.5 10 15 Example 6 X / 15 4.5 12 Acetone / 3.5 10 18 Example 7 X / 25 4.5 12 Acetone / 3.5 10 12 Example 8 X / 25 4.5 12 Acetone / 3.5 10 15 Example 9 X / 25 4.5 12 Acetone / 3.5 10 18 Example 10 Y ^{4 )} / 20 4.5 12 Acetone / 3.5 10 12 Example 11 Y / 20 4.5 12 Acetone / 3.5 10 15 Example 12 Y / 20 4.5 12 Acetone / 3.5 10 18 Example 13 Y / 15 4.5 12 Acetone / 3.5 10 12 Example 14 Y / 15 4.5 12 Acetone / 3.5 10 15 Example 15 Y / 15 4.5 12 Acetone / 3.5 10 18 Example 16 Y / 25 4.5 12 Acetone / 3.5 10 12 Example 17 Y / 25 4.5 12 Acetone / 3.5 10 15 Example 18 Y / 25 4.5 12 Acetone / 3.5 10 18 Example 19 X / 20 4 12 Acetone / 3.5 10 18 Example 20 X / 20 5 12 Acetone / 3.5 10 18 Example 21 X / 20 5.5 12 Acetone / 3.5 10 18 Example 22 Y / 20 4 12 Acetone / 3.5 10 18 Example 23 Y / 20 5 12 Acetone / 3.5 10 18 Example 24 Y / 20 5.5 12 Acetone / 3.5 10 18 Example 25 X / 20 4.5 9 Acetone / 3.5 10 18 Example 26 X / 20 4.5 10 Acetone / 3.5 10 18 Example 27 X / 20 4.5 11 Acetone / 3.5 10 18 Example 28 Y / 20 4.5 9 Acetone / 3.5 10 18 Example 29 Y / 20 4.5 10 Acetone / 3.5 10 18 Example 30 Y / 20 4.5 11 Acetone / 3.5 10 18 * 1) is the ratio of the total volume of the first solution
* 2) is the volume ratio for the first solution
* 3) X is 1-butyl-3-methyl-imidazolium tetrafluoroborate
* 4) Y is 1-butyl-3-methyl-imidazolium hexafluorophosphate

The first solution The second solution crystallization
Temperature crystallization
time Imidazolium halide
(Kind / volume% ¹⁾ ) pH Electrical conductivity
(Ms / cm) Hydrophilic organic solvent
(Type / volume ratio ^{2 )} ) Comparative Example 1 X ^{3 )} / 10 4.5 12 Acetone / 3.5 10 12 Comparative Example 2 X / 10 4.5 12 Acetone / 3.5 10 15 Comparative Example 3 X / 10 4.5 12 Acetone / 3.5 10 18 Comparative Example 4 Y ^{4 )} / 10 4.5 12 Acetone / 3.5 10 12 Comparative Example 5 Y / 10 4.5 12 Acetone / 3.5 10 15 Comparative Example 6 Y / 10 4.5 12 Acetone / 3.5 10 18 Comparative Example 7 X / 20 4.5 12 Acetone / 2.0 10 18 Comparative Example 8 X / 20 4.5 12 Acetone / 2.5 10 18 Comparative Example 9 X / 20 4.5 12 Acetone / 3.0 10 18 Comparative Example 10 X / 20 4.5 12 Acetone / 4.0 10 18 Comparative Example 11 Y / 20 4.5 12 Acetone / 2.0 10 18 Comparative Example 12 Y / 20 4.5 12 Acetone / 2.5 10 18 Comparative Example 13 Y / 20 4.5 12 Acetone / 3.0 10 18 Comparative Example 14 Y / 20 4.5 12 Acetone / 4.0 10 18 Comparative Example 15 X / 20 4.5 12 Acetone / 3.5 5 18 Comparative Example 16 X / 20 4.5 12 Acetone / 3.5 15 18 Comparative Example 17 Y / 20 4.5 12 Acetone / 3.5 5 18 Comparative Example 18 Y / 20 4.5 12 Acetone / 3.5 15 18 * 1) is the ratio of the total volume of the first solution
* 2) is the volume ratio for the first solution
* 3) X is 1-butyl-3-methyl-imidazolium tetrafluoroborate
* 4) Y is 1-butyl-3-methyl-imidazolium hexafluorophosphate

<Experimental Example 1>

An electron microscope (SV-35 Video Microscope system, Some Tech., Korea) was used to measure the crystallization form and size of the vancomycin obtained in the above Examples and Comparative Examples. The vancomycin precipitates obtained from the crystallization process were observed at high magnification (× 100). Observed vancomycin precipitates were identified as moving images in the IT-Plus system (Some Tech., Korea), confirming the morphology and size of the vancomycin precipitates and are shown in FIGS. 1 to 3 and FIGS. 5 to 6.

More specifically, FIG. 1 is a video microscope photograph of the shape of crystals according to the crystallization time with respect to the concentration of imidazolium halide containing an alkyl group having 1 to 6 carbon atoms. In the case where the concentration is 10% by volume (A in FIG. 1) -Butyl-3-methyl-imidazolium tetrafluoroborate was used, crystals were obtained after 18 hours. When 1-butyl-3-methyl-imidazolium hexafluorophosphate was used, It can be confirmed that the crystal itself was not generated during the lapse of 18 hours. On the contrary, in the first solution, 15 vol%, 20 vol%, 1-butyl-3-methyl-imidazolium tetrafluoroborate or 1-butyl-3-methyl-imidazolium hexafluorophosphate as 25 vol% (B, C, D in FIG. 1), crystallization was obtained 12 hours before and 15 hours before crystallization, and the time required for crystallization was minimized. Particularly, 1-butyl-3-methyl-imidazolium tetrafluoro When borate was added in the above concentration, it was confirmed that crystals were already formed after 12 hours. It can be seen that the minimum time required for crystallization is very effective in shortening the crystallization time according to the time less than 12 hours. Further, when the first solution contains 1-butyl-3-methyl-imidazolium tetrafluoroborate or 1-butyl-3-methyl-imidazolium hexafluorophosphate in an amount of 20 vol% C and D) crystals were very uniform and large in size, and thus vancomycin crystals were obtained which were remarkably superior to those in other concentrations.

FIG. 2 is a video microscope photograph of vancomycin crystals obtained according to the pH of the first solution according to a preferred embodiment of the present invention, wherein the pH of the first solution is 4.0 (FIG. 2A), 5.5 D), it can be confirmed that no crystals were formed. When the pH was 5.0 (C in FIG. 2), a part of the crystals were produced but the crystals were not clear, the crystals were aggregated and the size of the crystals was not uniform can confirm. On the contrary, when the pH is 4.5, it can be confirmed that vancomycin crystals which are very distinct and uniform and have a remarkably high crystal quality are produced.

FIG. 3 is a video image of vancomycin crystals formed according to the electrical conductivity of the first solution according to a preferred embodiment of the present invention, wherein the electrical conductivities are 11 ms / cm (C in FIG. 3), 12 ms / 3), the crystal size is smaller and the crystal quality is lowered as compared with the case where the electrical conductivity is 9 ms / cm (A in Fig. 3) and 10 ms / cm It can be confirmed that the crystal size is larger and the most distinct and uniform crystal is obtained as compared with the case where the conductivity is 10 ms / cm (B in Fig. 3) is 9 ms / cm (A in Fig. 3).

FIG. 5 is a video microscope photograph of vancomycin crystals according to the amount of hydrophilic organic solvent to be added, wherein the crystals of vancomycin prepared by adding acetone to the first solution at a volume ratio of 2.5 (B in FIG. 5) had a needle shape It can be confirmed that the quality of the crystals is not good and it can be confirmed that crystals of vancomycin are not produced when acetone is produced by putting it in 4.0 volume ratio (E in FIG. 5) to the first solution, (D in FIG. 5), the vancomycin crystals are clearly distinct and homogeneous.

6 is a video microscope photograph of the vancomycin crystal obtained according to the crystallization temperature, it can be confirmed that no crystal was formed when the crystallization temperature was 5 ° C (FIG. 6A), and the crystallization temperature was 15 ° C C), it can be confirmed that the shape of the crystal is broken into needles, whereas when the crystallization temperature is 10 ° C (FIG. 6B), it is confirmed that the crystal is clear, uniform, and crystal quality is remarkably excellent.

<Experimental Example 2>

To determine the crystallinity of the crystallized vancomycin for Examples 3, 12, 25-30, an X-ray diffractometer (MiniFlex 600, Rigaku) was used. The WINHRD 3000 program was run to adjust the power to 40 kV and 15 mA, respectively, and the range of 2? Was 5 to 80. The results are shown in FIG.

4 is an XRD pattern of vancomycin crystals formed according to the electrical conductivity of a first solution according to a preferred embodiment of the present invention. The XRD pattern of the vancomycin crystal is shown in FIG. 4 as an XRD pattern having an electrical conductivity of 9 ms / 4), 11 ms / cm (C in FIG. 4) and 12 ms / cm (D in FIG. 4), the electric conductivity is 9 ms / ) And 10 ms / cm (B in FIG. 4), the peak was clearly observed at an azimuth angle of 5 ° to 10 °, and particularly when the electrical conductivity was 10 ms / cm (B in FIG. 4) It can be confirmed that the crystallinity is very excellent.

Claims (11)

(1) at least one of 1-butyl-3-methyl-imidazolium tetrafluoroborate and 1-butyl-3-methyl-imidazolium hexafluorophosphate which improve crystallization time shortening and crystal quality Introducing the vancomycin precursor into a first solution having a pH of 4.4 to 4.7 and an electrical conductivity of 8.5 to 10.5 ms / cm comprising 12 to 27% by volume of the compound;
(2) preparing a second solution by mixing the first solution and the hydrophilic organic solvent in a volume ratio of 1: 3.3 to 3.8; And
(3) treating the second solution at 8 to 12 占 폚 to crystallize vancomycin. delete delete The method according to claim 1,
Wherein the solvent of the first solution is distilled water and at least one compound selected from the group consisting of 1-butyl-3-methyl-imidazolium tetrafluoroborate and 1-butyl-3-methyl-imidazolium hexafluorophosphate is dissolved in the first solution To 18% by volume to 22% by volume of the total volume of the vancomycin. delete delete The method according to claim 1,
Wherein the minimum crystallization time in the step (3) is 11 to 16 hours. The method according to claim 1,
Wherein the steps (1) to (3) above satisfy both of the following conditions (a) to (c):
(a) In step (1), 1-butyl-3-methyl-imidazolium tetrafluoroborate is contained in an amount of 18 to 22% by volume based on the first solution
(b) the pH of the first solution is 4.4 to 4.7 and the electrical conductivity is 9.5 to 10.5 ms / cm
(c) The minimum crystallization time in step (3) is 11 to 13 hours Butyl-3-methyl-imidazolium tetrafluoroborate and 1-butyl-3-methyl-imidazolium hexafluorophosphate which improve crystallization time and crystal quality, and a solvent In a volume ratio of 12: 88 to 27: 73, the pH of the solution is 4.4 to 4.7, and the electrical conductivity of the solution is 8.5 to 10.5 ms / cm. delete 10. The method of claim 9,
The solution for crystallizing vancomycin is prepared by dissolving at least one compound selected from 1-butyl-3-methyl-imidazolium tetrafluoroborate and 1-butyl-3-methyl- imidazolium hexafluorophosphate with a solvent in a ratio of 18: : 78 volume ratio. The solution for crystallization of high purity vancomycin.

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