RU2334511C1 - Method of coproporphyrin iii extraction and purification - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: method is performed by the following way: unfiltered culture liquid is acidated to pH 2-2.5, sorb on cationite KU-23/100 in pulsing fluidisated mode with subsequent desorption using elution systems with pH 6.0±0.1, containing sodium citrate 1.5% and carbamide 0.5%. Coproporphyrin III is precipitaled on produced eluate, reducing pH to 3.5±0.1. Produced precipitate is extracted with hydrochloric acid solution, thereafter chromatographic cleaning of extract on molecular sorbent follows. End product is precipitaled on eluate. Then extraction and chromatographic cleaning are repeated, whereupon end product is precipitaled on eluate and transformed into potassium salt.

EFFECT: technology allows for production of water-soluble preparation based unfiltered culture liquid.

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Description

Usage: biotechnology, medicine. The inventive proposed a new method for producing coproporphyrin III. To isolate the target product in a water-soluble form, sorption of coproporphyrin III from the culture fluid is carried out on a KU-23/100 sorbent, followed by desorption using an eluent system containing sodium citrate and urea. From the obtained eluate, coproporphyrin III is isolated by precipitation when the pH is adjusted to 3.5 ± 0.1. Coproporphyrin III is isolated from the precipitate obtained by extraction with a 5-7% hydrochloric acid solution. The extract obtained is subjected to chromatographic purification on silica gel KSK. From the resulting eluate, coproporphyrin III precipitates by adjusting the pH to 3.5 ± 0.1. The resulting precipitate is again subjected to extraction with a 5-7% hydrochloric acid solution and chromatographic purification of coproporphyrin III is repeated. From the obtained eluate, the target product precipitates and is converted to a water-soluble form upon alkalization. This method with a selected set of technological methods allows to obtain a highly purified preparation of a water-soluble salt of coproporphyrin III with a purity of 90-95%.

The invention relates to the medical and microbiological industry, in particular to a technology for producing coproporphyrin III.

The technical solution was carried out by obtaining coproporphyrin III in a water-soluble form, which can be used in medicine for the diagnosis and treatment of cancer more effectively and with fewer side effects than the known water-insoluble forms.

An analysis of the patent and scientific and technical literature indicates that traditional methods of purification of coproporphyrin III include the stage of esterification of porphyrins and separation of their ether derivatives on alumina. This is due to the generally accepted opinion that various forms of porphyrins can be effectively separated as ether derivatives. Thus, highly purified water-insoluble forms of coproporphyrin III are obtained (US Pat. No. 4,436,663, ed. St. USSR No. 1482946). The methods for producing water-soluble forms according to the sources found are associated with the preparation of water-insoluble esters of porphyrins, their separation and their further conversion to water-soluble forms. There is also a way to obtain water-soluble coproporphyrin III by directly isolating it and transferring it to a water-soluble state by alkalization. So, the closest analogue (prototype) to obtain a water-soluble form of coproporphyrin III is RF patent No. 2078138, according to which the purification of the target product is carried out using sequential reprecipitation of porphyrins from the native solution with hydrochloric acid solutions, followed by chromatography on macroporous anion exchange resin with desorption with ammonia solution, precipitation of coproporphyrin III from the eluate when acidified and converted to a water-soluble form when alkalized. The described method is time consuming, because the stage of obtaining the native solution from the culture fluid by centrifugation is used. In addition, for chromatographic purification, it is proposed to use AN-80 anion exchange resin, according to which there is no evidence that this sorbent is approved for use in the medical industry, or aluminum oxide, which is a fine powder with high hydrodynamic resistance and therefore not suitable for industrial use scale.

The proposed method for the isolation and purification of coproporphyrin III is based on a combination of reprecipitation techniques by adjusting the pH to 3.5 ± 0.1 with chromatographic purification on two different sorbents. This technology allows to obtain a highly purified water-soluble form of coproporphyrin III directly from the culture fluid, bypassing the stage of separation of the native solution from biomass.

The proposed method for producing a water-soluble form of coproporphyrin III is as follows:

The pH of the culture fluid containing the target product is adjusted to 2-2.5 by adding 35% hydrochloric acid to it at the rate of 20 ml per 1 liter of culture fluid. Then sorption of coproporphyrin III from the culture fluid onto the macroporous cation exchanger KU - 23/100 is carried out in a pulsating fluidized mode. After washing the sorbent with water, desorption of coproporphyrin III is carried out with an eluent system containing 1.5% sodium citrate, 0.5% urea and a pH of 6.0 ± 0.1. The pH of the resulting eluate containing coproporphyrin III is adjusted to 3.5 ± 0.1 to precipitate the desired product. The precipitate is separated by centrifugation, after which coproporphyrin III is extracted from the precipitate with a 5-7% hydrochloric acid solution. The use of hydrochloric acid with a concentration of more than 7% leads to emulsification of the suspension (due to protein impurities), poor separation, and contamination of the extract. When the acid concentration is below 5%, the acid consumption increases and the duration of its effect on the porphyrin-containing intermediate product. The extract obtained is applied to a KSK silica gel column (you can also use silochrome s-80 or silochrome CX - 2.5) to separate ballast substances. Then the column is washed with a 5% hydrochloric acid solution. Coproporphyrin III is precipitated from the obtained eluate by adjusting the pH to 3.5 ± 0.1, and the precipitate is separated by centrifugation. Next, the porphyrin is extracted with a 5-7% hydrochloric acid solution and re-chromatographed on silica gel as described above. The pH of the resulting eluate is adjusted to 3.5 ± 0.1 to precipitate coproporphyrin III. The resulting precipitate is separated using a centrifuge and washed with distilled water with a pH of 3.5 ± 0.1 (pH adjusted with hydrochloric acid) to remove mineral salts. Then the washed precipitate of coproporphyrin III is converted into a water-soluble form by dissolving it in an aqueous solution of potassium hydroxide. The potassium hydroxide content in this solution should be equal to the equivalent amount of carboxyl groups of the resulting coproporphyrin III preparation. Then the solution is dried at a temperature of 80 ° C. The purity of the preparation thus obtained is 90-95%.

The feasibility of the proposed method is illustrated by the following examples.

Example 1. On the selection and purification of the target product received 5450 ml of culture fluid with a concentration of coproporphyrin III 209 mg / L. To this culture fluid was added a 35% solution of hydrochloric acid with a volume of 110 ml. The pH of the culture fluid is 2.3. Then this culture fluid was passed through a column of KU-23/100 cation exchanger in a pulsating, fluidized mode. Then this column was washed with 8 liter water. After this, the desorption process was carried out by the eluent system, which is an aqueous solution of sodium citrate - 1.5%, urea - 0.5% and a pH of 6.05. The volume of the eluate was 6820 ml. The content of coproporphyrin III in it was 134.1 mg / L. Then, the pH of the eluate was adjusted to 3.45, and a precipitate formed containing the desired product. The precipitate was isolated by centrifugation, the volume of the mother liquor was approximately 6820 ml, containing coproporphyrin III in the amount of 0.78 mg / l - this is the loss of the target product at this stage. Then, coproporphyrin III was extracted from the precipitate with a 5% hydrochloric acid solution. The volume of the extract was 6190 ml, the content of coproporphyrin III in which was 146.2 mg / L. The obtained extract was subjected to the first chromatographic purification on silica gel KSK. The volume of the eluate was 21,400 ml with a content of coproporphyrin III of 40.1 mg / L. Then the pH of the eluate was adjusted to 3.5 and the precipitate with coproporphyrin III was isolated by centrifugation. The volume of the mother liquor was 21,400 ml, in which the content of the target product was 0.41 mg / L. Coproporphyrin III contained in the mother liquor is loss at this stage. From the obtained precipitate containing coproporphyrin III, the target product was extracted with a 5% hydrochloric acid solution. The volume of the extract was 630 ml with the content of coproporphyrin III in the amount of 1335.8 mg / l. The resulting extract was subjected to second chromatographic purification on silica gel KSK. The volume of the eluate was equal to 3485 ml with a concentration of coproporphyrin III equal to 237.4 mg / L. The pH of the resulting eluate was adjusted to 3.55 with 25% ammonia water. Coproporphyrin III precipitated, which was isolated using a centrifuge. The volume of the mother liquor was 3485 ml. The content of the target product in the mother liquor was equal to 1,054 mg / l - this is the loss of coproporphyrin III at this stage. The resulting precipitate to remove mineral salts was washed with distilled water with a pH adjusted with hydrochloric acid to 3.5. The washed precipitate was converted to potassium salt by dissolving it in a potassium hydroxide solution and adjusting the pH of the solution to 7.8. The solution was dried at 80 ° C. The weight of the preparation of coproporphyrin III tetrakalium salt after drying was 1004.8 mg with a moisture content of 10.5% or without water: 899.3 mg with the content of the target product 902.0 μg / mg (90.2%).

Example 2. On the selection and purification of the target product received 5930 ml of culture fluid with a concentration of coproporphyrin III 251 mg / L. A 35% hydrochloric acid solution of 120 ml was added to this culture fluid. The pH of the culture fluid became 2.1. Then this culture fluid was passed through a column of KU-23/100 cation exchanger in a pulsating, fluidized mode. After that, this column was washed with water of 9 liters. Then the desorption process was carried out by the eluent system, which is an aqueous solution of sodium citrate - 1.5%, urea - 0.5% and a pH of 6.1. The volume of the eluate was 7230 ml. The content of coproporphyrin III in it was 175.8 mg / L. Then, the pH of the eluate was adjusted to 3.5, and a precipitate containing the desired product precipitated. The precipitate was isolated by centrifugation, the volume of the mother liquor was approximately 7230 ml containing coproporphyrin III in an amount of 0.63 mg / l - this is the loss of the target product at this stage. Then, coproporphyrin III was extracted from the precipitate with a 5% hydrochloric acid solution. The volume of the extract was 6740 ml, the content of coproporphyrin III which is equal to 146.2 mg / L. The obtained extract was subjected to the first chromatographic purification on silica gel KSK. The volume of the eluate was 25100 ml with a content of coproporphyrin III - 48.3 mg / L. Then the pH of the eluate was adjusted to 3.5, and the precipitate with coproporphyrin III was isolated by centrifugation. The volume of the mother liquor was approximately 25100 ml, in which the content of coproporphyrin III is 0.65 mg / L. The target product contained in the mother liquor is loss at this stage. From the obtained precipitate containing coproporphyrin III, the target product was extracted with a 5% hydrochloric acid solution. The volume of the extract was 780 ml with the content of coproporphyrin III in the amount of 1524.1 mg / l. The resulting extract was subjected to second chromatographic purification on silica gel KSK. As a result, an eluate with a volume of 6790 ml and a concentration of coproporphyrin III of 171.4 mg / L was obtained. The pH of the resulting eluate was adjusted to 3.5 with 25% ammonia water. Coproporphyrin III precipitated, which was isolated using a centrifuge. The volume of the mother liquor was 6790 ml. The content of the target product in the mother liquor was equal to 1.45 mg / l - this is the loss of coproporphyrin III at this stage. The resulting precipitate to remove mineral salts was washed with distilled water with a pH adjusted with hydrochloric acid to 3.5. The washed precipitate of coproporphyrin III was converted to potassium salt by dissolving it in a solution of potassium hydroxide and adjusting the pH of the solution to 7.8. The solution was dried at 80 ° C. The weight of the dried preparation of coproporphyrin III tetrakalium salt was 1379.6 mg with a moisture content of 12.4% or without moisture: 1208.5 mg with a target product content of 943.6 μg / g (94.2%).

Example 3. On the selection and purification of the target product received 5340 ml of culture fluid with a concentration of coproporphyrin III 236 mg / L. A 35% hydrochloric acid solution of 110 ml was added to this culture fluid. Its pH became equal to 2.0. Then this culture fluid was passed through a column of KU-23/100 cation exchanger in a pulsating, fluidized mode. Then this column was washed with 8.5 liters of water. After this, the desorption process was carried out by the eluent system, which is an aqueous solution of sodium citrate - 1.5%, urea - 0.5% and a pH of 6.1. The volume of the eluate was 7050 ml. The content of coproporphyrin III in it was 155.8 mg / L. Then, the pH of the eluate was adjusted to 3.5, and a precipitate containing the desired product precipitated. The precipitate was isolated by centrifugation, the volume of the mother liquor was approximately 7050 ml, containing coproporphyrin III in the amount of 1.05 mg / l - this is the loss of the target product at this stage. Then, coproporphyrin III was extracted from the precipitate with a 5% hydrochloric acid solution. The volume of the extract was 6830 ml, the content of coproporphyrin III in which was 158.9 mg / L. The obtained extract was subjected to the first chromatographic purification on silica gel KSK. The volume of the eluate was 22600 ml with a content of coproporphyrin III - 45.9 mg / L. Then the pH of the eluate was adjusted to 3.5, and the precipitate with coproporphyrin III was isolated by centrifugation. The volume of the mother liquor was 22600 ml, in which the content of the target product was equal to 0.83 mg / L. Coproporphyrin III contained in the mother liquor is loss at this stage. From the obtained precipitate containing coproporphyrin III, the target product was extracted with a 5% hydrochloric acid solution. The volume of the extract was 700 ml with a content of coproporphyrin III in an amount of 1445.3 mg / L. The resulting extract was subjected to second chromatographic purification on silica gel KSK. The volume of the eluate was 4740 ml with a concentration of coproporphyrin III equal to 210.3 mg / L. The pH of the resulting eluate was adjusted to 3.5 with 25% ammonia water. Coproporphyrin III precipitated, which was isolated using a centrifuge. The volume of the mother liquor was 4740 ml. The content of coproporphyrin III in the mother liquor was 0.97 mg / l - this is the loss of the target product at this stage. The resulting precipitate to remove mineral salts was washed with distilled water with a pH adjusted with hydrochloric acid to 3.5. The washed precipitate was converted to potassium salt by dissolving it in a potassium hydroxide solution and adjusting the pH of the solution to 7.6. The solution was dried at 80 ° C. The weight of the preparation of coproporphyrin III tetrakalium salt after drying was 1196.0 mg with a moisture content of 11.9% or without water: 1053.7 mg with the target product content of 911.1 μg / mg (91.1%).

Information sources

1. US patent 4436663, 1984.

2. Patent of the Russian Federation No. 2078138, 1993.

3. Copyright certificate No. 1482946, 1978.

Claims (1)

The method of isolation and purification of coproporphyrin III from the culture fluid, including sorption on a macroporous sorbent and extraction with a 5-7% hydrochloric acid solution, characterized in that the unfiltered culture fluid is acidified to pH 2-2.5, sorption on KU brand cation exchange resin is carried out -23/100 in a pulsating fluidized mode followed by desorption using an eluent system with a pH of 6.0 ± 0.1 containing 1.5% sodium citrate and 0.5% urea, coproporphyrin III is precipitated from the resulting eluate, bringing the pH to 3 5 ± 0.1, the resulting precipitate ext Agirov hydrochloric acid solution, the extract was purified by chromatography on a molecular sorbent deposition produce the desired product from the eluate, followed by repeated extraction and chromatographic purification, the desired product is precipitated from the eluate and converted into its potassium salt.