The method of extraction of biologically active substances

 

(57) Abstract:

Usage: biotechnology, separation of biologically active substances. The essence of the invention: method of extraction of biologically active substances provides for filtering the culture fluid, passing the filtrate through a polysulfone or polysulfone or polyacrylonitrile and the subsequent allocation of the target product. 1 C.p. f-crystals, 4 PL.

The invention relates to biotechnology and relates to a method of extraction of biologically active substances (BAS).

An important step in the production of biologically active substances is picking them out at the end of the biosynthesis of the native solutions filtered culture fluid. Native solutions contain a large amount of ballast impurities that are dissolved in the form of colloids and suspensions. These impurities significantly degrade the performance of the allocation process and chemical cleaning and reduce the output of BAS. In this regard, almost all production of biologically active substances include the stage of pre-processing culture fluid or native solution.

The most common methods of protein purification from impurities are heat and acid coagulation [1] Known methods for cleaning afektivnim method of coagulation of impurities is the method of education of the filler directly into the liquid, when adding reagents, forming therein an insoluble precipitate [4] the Cleaning carried out by precipitation of proteins soluble salts of alkaline earth metals [5] Quaternary ammonium salts, detergents [6] the denaturation of proteins by acidification and heating [7]

The disadvantage of this method is the low output of BAS, a multi-stage, energy consumption and consumption of various chemicals in the processing phase, the culture fluid or a native solution. Loss of the target product may vary from 4 to 30%

The use of thermal coagulation of the culture fluid or a native solution leads to inactivation (destruction) of BAS.

A few more gentle way of handling the culture fluid or a native solution compared to the above is the processing method using chemical coagulants special type of high molecular weight polyelectrolytes, flocculants [8-11]

Closest to the claimed method according to technical essence and the achieved effect is the method of extraction of dentists, wherein before carrying out the extraction in the native solution as a demulsifier added 0.2% contact Petrova and acidified with it before adding mutilation kind of impurity, resulting in reduced yield and quality of the target product. This disadvantage is completely eliminated in the present method of allocation of BAS.

The aim of the present invention is to increase the output and quality of the target product.

This objective is achieved in that the culture fluid without heat and chemical treatments filtered and derived native solution (or hydrolyzed) is subjected to ultrafiltration by passing through a straight - chain or polymer based material polysulfonamide or polyacrylonitrile speeds ranging from 10 to 50 l/m2h depending on the type of BAS (see tab. 1).

The output of BAS at the stage of obtaining the intermediate product (concentrate the eluate, extract) the proposed method is from 53 to 97% for different substance that significantly exceed this figure by a known method prototype (see tab. 2). The reduction in the number of organic impurities for aminoglycoside antibiotics, including gentamicin is approximately 20% increase in the output of BAS in the present method in comparison with the known method the prototype for different BAS changed from 7.0 to 34.9% due to the exclusion of the preliminary processing stage of a cultural Jew

Table 4 presents the efficiency of the ultrafiltration purification compared to treatment with flocculants according to the method of the prototype.

New in the proposed method is the use of filtration of biologically active substances through hetero - or chain material based on polysulfone, polysulfonamide or polyacrylonitrile speeds ranging from 10 to 50 l/m2including passing the solution BAS through hetero - or chain material with a speed of less than 10 l/m2h is impractical, as it increases the filtering process, which leads to partial inactivation of the target product and the yield of biologically active substances. Increase speed transmission solution BAS more than 50 l/m2PM is associated with increased pressure, which is a solution for straight - chain or material. This in turn causes compression of the pores of the polymeric material, clogging their orgelmesse and reduce ultimately speed transmission solution.

The examples below illustrate the invention.

Example 1.

3 l of culture liquid of gibberellins (5,85 g by weight) was filtered under vacuum and get 6,0 l native solution from the leaching waters (5,62 g of gibberellins by weight). The output h is rlozano polymeric material based on polyacrylonitrile (PAN-20) at a rate of 50 l/m2including Get 7,25 l of filtrate (5.6 g of gibberellins by weight). The output stage receiving the filtrate 99.5% To the filtrate add 76 g of activated charcoal and spend the adsorption of gibberellins on coal under stirring for 4 hours then the coal is separated, washed with water and desorbed with gibberellin coal with aqueous acetone solution with mass fraction of 90% After distillation of the acetone from aqueous acetone, the eluate of gibberellins get 106 ml water concentrate (3.13 gibberellins by weight). The output of the active substance in the stages of sorption desorption on coal is 55.9 percent of the Output of the gibberellin content in the culture fluid 53.5%

The prototype under all other equal conditions, the yield of gibberellins on water concentrate content in the culture fluid is 40.5% Increase in the yield of substance of the proposed method in comparison with the prototype is 32,1% (Rel.).

Example 2.

The filtrate of gibberellins was prepared as in example 1 and then spend the sorption of gibberellins on nonionic macroporous sorbent Porous So Desorption of the active substance is carried out with an aqueous solution of acetone (with a mass fraction of 50% ). After distillation of the acetone from the eluate get 215 ml of water concentrate (5, and is 96.1 per cent (on prototype 40,5%).

Example 3.

1500 ml of hydrolysate vitamin B12(51.3 mg) is passed through heterochain polymer-based material is polysulfone (PS-50) with a speed of 25.5 l/m2am Getting 1875 ml of the filtrate (51,04 mg). The output of vitamin B12at the stage of receiving the filtrate 99.5% Obtained filtrate passes through the carboxylic cation exchange resin SG-1M, sorbed vitamin B12desorbed from the cation exchange resin with an aqueous solution of ammonia with a concentration of 0.6-0.7 mol/L. Get 275 ml of ammonia eluate of vitamin B12(49.5 mg). The output of BAS on stage sorption-desorption content in the hydrolysate is 97% Yield according to the prototype of the 90% Increase of vitamin B12the proposed method in comparison with known amounts of 7.8% (Rel.).

Example 4.

3 liters of the culture fluid of erythromycin (10,56 million units) filtered under vacuum and gets 6 l native leaching solution with water (there is a 10.03 million units ). The output of BAS on stage filtration of the culture fluid is 95% of the native solution erythromycin passed through heterochain polymer based material polysulfonamide (PSA) with the speed 21,4 l/m2'clock Gain of 7.5 L. of the filtrate (9,98 million units). The output of BAS at the stage of obtaining the filter is 96 ml butylacetate extract erythromycin (9,48 million units). The output of BAS in the extraction step is 95% Yield in butylacetate extract content from erythromycin in culture liquid is 89.8% of the Output of the prototype 67.9% of the Increase in the output of erythromycin on the proposed method in comparison with the known is 32.2% (Rel.)

Example 5.

6.0 liters native solution of oxytetracycline (50.6 million units) pass through a heterochain polymer based material polysulfonamide (PSA) with about speed 15.1 l/m2am Getting a 7.62 l of filtrate antibiotic (50.3 million units). The output of oxytetracycline on the stage of receiving the filtrate 99.5% of the obtained filtrate is precipitated catasrophy complex antibiotic at pH=8,5, then the complex 6% solution of oxalic acid at pH=1,0-1,2. Receive 1830 ml concentrate oxytetracycline (47,85 million units) Output antibiotic concentrate decomposed complex content oxytetracycline in native solution is a 94.6% (on prototype 84,6%). Increasing the yield of the antibiotic on the proposed method in comparison with known amounts of 11.8% (Rel.).

Example 6.

The filtrate oxytetracycline, obtained in example 5, is passed through nonionic macroporous sorbent Porous So Absorbed antibiotic desmin. units). The output of the antibiotic in the eluate from the content in the native solution is to 86.4% From the obtained eluate conduct the deposition of oxytetracycline base, bypassing the stage of deposition of the intermediate product Attleboro complex. The increase of antibiotic compared with known amounts to 15% (Rel.).

Example 7.

3 l of culture liquid kanamycin (8,67 million units) treated with oxalic acid, and then filtered under vacuum. Get a 5.5 litre native solution from the leaching waters (8,32 million units) Output stage filtration of the culture fluid is 96% of the native solution of kanamycin alkalinized and filtered. The output of the antibiotic at the stages of pre-treatment and double filtration is 95% native Filtered solution of kanamycin passed through chain polymer material based on polyacrylonitrile (PAN-20) at a rate of 27.2 l/m2hours Get to 6.75 l of filtrate (7,87 million units). The output stage receiving the filtrate kanamycin 99.5% of the obtained filtrate antibiotic adsorb on the cation exchanger KB-4P-2, then desorbed with kanamycin cation exchanger 2n. aqueous solution of ammonia. Get 152 ml. of ammonia eluate (7,52 million units). Output stages which is 86.7% of the prototype output kanamycin is 73.5 per cent increase in the output of kanamycin on the proposed method in comparison with known amounts of 18.0% (Rel.)

Example 8.

6 l native solution dentists (46,03 million units) pass through a heterochain polymer based material polysulfonamide (PSA) with a speed of 10.3 l/m2am Getting 7.5 l of filtrate dentists (45.8 million units). The output of the antibiotic on the stage of receiving the filtrate 99.5% of the obtained filtrate antibiotic extracted with butyl acetate, and then spend reextraction and second butylacetate extraction. Get 625 ml butylacetate extract phenoxymethylpenicillin (43,6 million units). The output of the antibiotic in the extraction step is 95.2% of the content in the filtrate and 94.7% of content in native solution. The output of the antibiotic by known techniques in the extract from the native solution to thermal and acid treatments is 70.2 per cent increase in the output of the antibiotic on the proposed method in comparison with the known method is 34,9% (Rel.).

Example 9.

1 l native solution of gentamicin (0.54 g) content of organic impurities of 23.7 mg/ml and protein 67,5 mg/ml and passed through heterochain polymer based material polysulfonamide (PSA) at a rate of 21 l/m2am Getting 1.1 l of filtrate antibiotic (0,476 g) with codereal 97.2% of the content of the antibiotic molecules in the solution. The reduced content of organic impurities and protein in the filtrate gentamicin is 12% and 20%, respectively. The obtained filtrate antibiotic is passed through the cation exchanger, KB-2, gentamicin is desorbed from the cation exchanger 2 n aqueous solution of ammonia. Get 5.5 ml of the eluate antibiotic (0.45 g). The release of gentamicin in the eluate from the content in the native solution is 83,3% by a known method 58.2% Increase in the yield of the antibiotic to 43.1% (Rel.).

Example 10.

1 l native solution sizomitsin (0.34 g) is treated analogously to example 9. Get water-ammonia eluate of sizomitsin (0.32 g). The output of the antibiotic in the eluate from the content in the native solution is 94.0% prototype 87,6% Increase in the yield of the antibiotic by 7.3% (Rel.).

Example 11.

10 l native solution dentists (75.0 million units) pass through a heterochain polymer based material polysulfonamide (PSA) with the speed of 18.6 l/m2am Getting 10.5 l of filtrate dentists (74,625 million units ). The output of the antibiotic on the stage of obtaining ultrafiltrate 99.5% of the obtained filtrate antibiotic precipitated sulfuric acid solution with a mass fraction of 3% to 6% at a pH of from 2.0 to 2.3. Get 46,3, technical FM is lizovyvatj from water-acetone mixture. Get 30 g FMP acid activity 1680 μg/mg (50.4 million units), exit at the stage of recrystallization 75% Yield from native solution 67,2% Yield from the culture fluid 63,8% by known techniques 55,0% Increase in output compared with the known technology is 16% (Rel).

Sources of information

1. Zhukovskaya S. A. Medical industry of the USSR, 1966, N 3, p 18.

2. Kavati Soha, Akazawa Takaki, Japan patent N 10179 from 19.12.61. RICH. 1968, N 18P AIRPLANES.

3. Linde, H. W. Lohr GDR patent N 31481 from 13.10.61, RICH, 1965, N P.

4. Zhukovskaya S. A. Boiko, I. D. Medical industry of the USSR, 1964, No. 1, S. 38.

5. Stroud S. W. Ransley N. M.R. German patent N 941686 from 19.04.56, RICH, 1958, N 6, S. 389.

6. Musil Grech 1959, 92034, Oct 15, CA 1960, 54, N 8, 79886.

7. U.S. patent N 2509010, 1950, Manyf. Chemist, 1950, 21, N 11, 501.

8. Vernikov L. M. Zhukovskaya S. A. Antibiotics, 1968, No. 11, C. 982-991.

9. Vernikov L. M. Zhukovskaya S. A. Antibiotics, 1972, No. 4, S. 375-382.

10. Vernikov L. M. Zhukovskaya S. A. Antibiotics, 1974, No. 8, S. 697-710, S. 795-8-1.

11. Vernikov L. M. Zhukovskaya S. A. Antibiotics, 1978, No. 4, S. 298-304.

12. A. S. USSR N 158391, CL C 12 P 1/06, 1962 (prototype).

1. The method of extraction of biologically active substances, including filtering the culture liquid is tion is passed through straight - chain or polymeric material with a speed of 10 to 50 l/m2h

2. The method according to p. 1, characterized in that the polymer material used polysulfone, or polysulfonamide, or polyacrylonitrile.

 

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