CN110746473A - Purification process for reducing content of lincomycin B component - Google Patents

Abstract

The invention discloses a purification process for reducing the content of a lincomycin B component. The process uses a back extraction solution of a solvent extraction solution or an aqueous solution of a lincomycin crude product as a purification object, adjusts the pH of the solution by NaOH, then adsorbs the solution on a chromatographic column filled with silica gel matrix, washes the solution by pure water, then resolves the solution by butanol or acetone, and then adjusts the acid by hydrochloric acid and crystallizes the solution. The purification process uses a high performance preparative liquid chromatography technology, and has the advantages of high separation yield, short separation period, good process stability and high automation degree. Compared with other purification processes, the process equipment occupies less land, has little environmental pollution and strong production controllability, and is beneficial to industrial scale application.

Description

Purification process for reducing content of lincomycin B component

Technical Field

The invention belongs to the technical field of pharmaceutical chemicals, and particularly relates to a purification process for reducing the content of a lincomycin B component.

Background

Lincomycin produces a certain amount of lincomycin B in the fermentation process. Because the component B has lower antibacterial activity and higher toxicity, the requirements on the component B are increasingly strict due to lincomycin and derivative products of the lincomycin, clindamycin phosphate and the like. The current means for reducing the content of the component B mainly comprise: 1. after the higher alcohol extraction, the component B is washed with alkaline water. The method has poor controllability, large yield loss and large amount of waste water. 2. And separating the hydrochloric acid back extraction solution or the crude product water solution by resin to remove the component B. The method has the advantages of long period, low efficiency and non-centralized analysis unit, and a large amount of wastewater can be generated after the resin is regenerated by acid and alkali. Therefore, a more efficient purification process for reducing the content of the lincomycin B component is needed.

Disclosure of Invention

In order to overcome some defects of the traditional purification process, the invention develops a high-efficiency purification process for reducing the content of the lincomycin B component.

The technical scheme of the invention is as follows:

a purification process for reducing the content of a lincomycin B component is characterized by comprising the following steps: using the back extraction solution of the solvent extract or the aqueous solution of the lincomycin crude product as a purification object, adjusting the pH of the solution by NaOH, adsorbing the solution on a chromatographic column filled with silica gel matrix, washing the solution by pure water, resolving the solution by butanol or acetone, adjusting the acid by hydrochloric acid, and crystallizing the solution.

The separation column of the invention can be filled into a high-pressure chromatographic column or a medium-low pressure column according to the particle size of the filler. Meanwhile, the fermentation filtrate or the water solution for recovering the crude product can be used for replacing the back extraction solution or the water solution for the crude product for purification and separation.

The purification process for reducing the content of the lincomycin B component is characterized by comprising the following steps of: adjusting pH of the solution to 7.5-8.5 with NaOH, and adsorbing on a chromatographic column filled with silica gel matrix; the sample loading mass of the lincomycin is controlled to be 7-13% of the mass of the filler.

The purification process for reducing the content of the lincomycin B component comprises the following steps and operations:

① adjusting pH of the back extract of the solvent extract or the aqueous solution of the lincomycin crude product to 7.5-8.5 with NaOH to obtain alkaline solution;

②, pumping the alkalines into a chromatographic column filled with silica gel matrix filler, and controlling the sample loading mass of lincomycin to be 7-13% of the filler mass;

③ washing the column with 0.5-2BV pure water, collecting the fraction by stages, and considering whether the fraction is used indiscriminately according to the content of component B after HPLC detection;

④ is resolved with 1.5-6BV acetone containing 0-10% water or butanol containing 0-20% water;

⑤ adding hydrochloric acid to adjust pH to below 2, directly crystallizing acetone solution, concentrating butanol solution, crystallizing or back extracting with acid water, and crystallizing with acetone;

⑥ the column was equilibrated with 2-5BV of pure water and the process could be repeated ① to ⑤.

The invention takes the high performance preparative liquid chromatography as a purification means, and has the advantages of high separation yield, short separation period, good process stability and high automation degree. Compared with other purification processes, the process equipment occupies less land, has little environmental pollution and strong production controllability, and is beneficial to industrial scale application.

Drawings

FIG. 1 example 1 UV monitoring spectrum (210 nm);

FIG. 2 example 2 UV monitoring spectrum (210 nm);

FIG. 3 example 3 UV monitoring spectrum (210 nm);

FIG. 4 chromatographic assay of the feed solution before purification in example 3;

FIG. 5 chromatographic detection of the product obtained in example 3;

FIG. 6 example 4 UV monitoring spectrum (210 nm);

FIG. 7 example 5 UV monitoring spectrum (210 nm);

FIG. 8 example 6 UV monitoring spectrum (210 nm);

FIG. 9 example 7 UV monitoring spectrum (210 nm);

FIG. 10 UV monitoring spectrum (210nm) of example 8.

Detailed Description

The following embodiment is used to specifically describe a high-efficiency lincomycin purification process of the present invention.

Example 1

Taking hydrochloric acid back-extraction liquid of a solvent extraction solution (the pH is 2.9, the content of the component B in a normal crystal product is 3.0 percent), adding 20 percent NaOH to adjust the pH to be 8.2, filtering the solution through a 0.45 mu m filter membrane, and measuring the titer to 25 ten thousand (the external standard quantity is 275 mg/mL). 330g of 30-micron filler LK-1 is filled into a dynamic axial compression column (50-DAC, inner diameter 50mm, filling height-250 mm). The sample is loaded to 120mL (the mass of the sample accounts for 10.0 percent of the mass of the filler), the sample is washed by water to 300mL (about 0.9BV), and acetone is analyzed to 1560mL (about 4.7 BV). The loading and elution procedures were as follows:

TABLE 1

The phase A is taken as a sample loading liquid and then is taken as pure water; phase B is acetone

Collecting acetone analysis fraction for 16-38min, wherein lincomycin A yield is 100%, adding HCl for acidification, crystallizing to obtain 30.0g (crystallization yield is 90.9%), and HPLC detecting content of component B is 1.2%.

Example 2

Taking hydrochloric acid back-extraction liquid of a solvent extraction solution (the pH is 2.9, the content of the component B in a normal crystal product is 3.0 percent), adding 20 percent NaOH to adjust the pH to be 8.2, filtering the solution through a 0.45 mu m filter membrane, and measuring the titer to 25 ten thousand (the external standard quantity is 275 mg/mL). 330g of 30-micron filler LK-1 is filled into a dynamic axial compression column (50-DAC, inner diameter 50mm, filling height-250 mm). And (3) loading 120mL (the mass of the sample accounts for 10.0% of the mass of the filler), washing 450mL (1.4 BV) after loading, and resolving 1400mL (4.2 BV) by acetone. The loading and elution procedures were as follows:

TABLE 2

The phase A is taken as a sample loading liquid and then is taken as pure water; phase B is acetone

Collecting acetone analysis fraction for 19-38min, wherein lincomycin A yield is 95.07%, adding HCl for acidification, crystallizing to obtain 28.1g (crystallization yield is 89.8%), and HPLC detecting that B component content is 0.565%.

Example 3

The hydrochloric acid back extraction solution (pH is 2.9, the content of the B component in the normal crystal product is 3.0%) of the solvent extract is taken, 20% NaOH is added to adjust the pH to 8.1, and the mixture is filtered through a 0.45 mu m filter membrane, so that the titer is 20 ten thousand (external standard quantity is 214 mg/mL). 330g of 30-micron filler LK-1 is filled into a dynamic axial compression column (50-DAC, inner diameter 50mm, filling height-250 mm). 160mL of sample (sample mass accounts for 10.4% of the filler mass), 300mL of sample is washed with water (0.9 BV), and 1420mL of sample is analyzed by acetone (4.3 BV). The loading and elution procedures were as follows:

TABLE 3

The phase A is taken as a sample loading liquid and then is taken as pure water; phase B is acetone

Collecting acetone analysis fraction for 17-36.5min, wherein lincomycin A yield is 94.24%, adding HCl for acidification, crystallizing to obtain 27.5g (crystallization yield is 88.5%), and HPLC detecting content of component B is 0.361%.

Example 4

Taking hydrochloric acid back-extraction solution of the solvent extract (pH is 2.9, and the content of the component B in a normal crystal product is 2.8%), adding 20% NaOH to adjust the pH to 8.0, filtering the solution through a 0.45 mu m filter membrane, and measuring the titer to 20 ten thousand (external standard quantity is 214 mg/mL). 320g of 30 mu m filler LK-1 is filled into a dynamic axial compression column (50-DAC, inner diameter 50mm, filling height 250 mm). 170mL (sample mass accounts for 11.4% of filler mass), 450mL (1.4 BV) of water washing after sample loading, 1460mL (4.5 BV) of acetone resolution. The loading and elution procedures were as follows:

TABLE 4

The phase A is taken as a sample loading liquid and then is taken as pure water; phase B is acetone

Collecting acetone analysis fraction for 19.9-39min, wherein lincomycin A yield is 76.94%, adding HCl for acidification, crystallizing to obtain 23.2g (crystal yield is 82.7%), and HPLC detecting content of component B is 0.329%.

Example 5

Taking hydrochloric acid back-extraction solution of the solvent extract (pH is 2.9, and the content of the component B in a normal crystal product is 2.8%), adding 20% NaOH to adjust the pH to 8.0, filtering the solution through a 0.45 mu m filter membrane, and measuring the titer to 20 ten thousand (external standard quantity is 214 mg/mL). 320g of 30 mu m filler LK-1 is filled into a dynamic axial compression column (50-DAC, inner diameter 50mm, filling height 250 mm). 145mL of sample (sample mass accounts for 9.7% of filler mass), 450mL of sample after sample loading (1.4 BV) by water washing, and 1485mL (4.6 BV) by acetone resolution. The loading and elution procedures were as follows:

TABLE 5

The phase A is taken as a sample loading liquid and then is taken as pure water; phase B is acetone

Collecting acetone analysis fraction for 19.4-39min, wherein lincomycin A yield is 80.43%, adding HCl for acidification, crystallizing to obtain 21.3g (crystallization yield is 85.3%), and HPLC detecting to obtain B component content of 0.333%.

Example 6

Adding water into lincomycin crude product (with the content of the component B being 3.2%) to prepare a solution with the concentration of 230mg/mL, adding 20% NaOH to adjust the pH value to 8.1, and filtering the solution through a 0.45 mu m filter membrane. 600g of 60-micron filler LK-1 is filled into a dynamic axial compression column (50-DAC, the inner diameter is 50mm, and the filling height is 500 mm). The sample is 270mL (sample mass accounts for 10.3% of filler mass), 570mL (-1 BV) is washed by water after sample loading, and 3186mL (-5.3 BV) is resolved by acetone/water (93/7). The loading and elution procedures were as follows:

TABLE 6

The phase A is taken as a sample loading liquid and then is taken as pure water; phase B is acetone/water (93/7)

Collecting acetone analysis fraction for 28-70min, wherein lincomycin A yield is 95.24%, adding HCl for acidification, crystallizing to obtain 49.8g (crystallization yield is 87.2%), and HPLC detecting that B component content is 0.461%.

Example 7

Taking hydrochloric acid back-extraction liquid of a solvent extraction solution (the pH is 2.9, the content of the component B in a normal crystal product is 3.0 percent), adding 20 percent NaOH to adjust the pH to 8.10, filtering the solution through a 0.45 mu m filter membrane, and measuring the titer to 25 ten thousand (the external standard quantity is 275 mg/mL). 330g of 30-micron filler LK-1 is filled into a dynamic axial compression column (50-DAC, inner diameter 50mm, filling height-250 mm). The sample is loaded to 120mL (the mass of the sample accounts for 10.0 percent of the mass of the filler), and is washed by water to 450mL (about 1.4BV) after the sample loading, and butanol/water (8/2) is used for analyzing to 905mL (about 2.7 BV). The loading and elution procedures were as follows:

TABLE 7

The phase A is changed into pure water after starting to be a sample loading liquid; phase B is butanol/water (8/2)

Collecting butanol fraction for 24.2-44.5min, wherein lincomycin A yield is 81.4%, adding HCl for acidification, concentrating, crystallizing to obtain product 24.2g (crystallization yield is 90.1%), and HPLC detecting component B content is 0.661%.

Example 8

Taking hydrochloric acid back-extraction liquid of a solvent extraction solution (the pH is 2.9, the content of the component B in a normal crystal product is 3.0 percent), adding 20 percent NaOH to adjust the pH to 8.10, filtering the solution through a 0.45 mu m filter membrane, and measuring the titer to 25 ten thousand (the external standard quantity is 275 mg/mL). 330g of 30-micron filler LK-1 is filled into a dynamic axial compression column (50-DAC, inner diameter 50mm, filling height-250 mm). 120mL of sample (sample mass accounts for 10.0% of filler mass), 150mL of sample after sample loading (0.5 BV) is washed by water, and 980mL (3.0 BV) of butanol/water (8/2) is resolved. The loading and elution procedures were as follows:

TABLE 8

The phase A is changed into pure water after starting to be a sample loading liquid; phase B is butanol/water (8/2)

Collecting butanol fraction for 15.6-41min, wherein lincomycin A yield is 86.2%, acidifying with HCl, concentrating, crystallizing to obtain 25.4g (crystallization yield is 89.3%), and content of component B is 0.842% by HPLC.

The above embodiments are merely illustrative of the technical ideas and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.

Claims (6)

1. A purification process for reducing the content of a lincomycin B component is characterized by comprising the following steps: using the back extraction solution of the solvent extract or the aqueous solution of the lincomycin crude product as a purification object, adjusting the pH of the solution by NaOH, adsorbing the solution on a chromatographic column filled with silica gel matrix, washing the solution by pure water, resolving the solution by butanol or acetone, adjusting the acid by hydrochloric acid, and crystallizing the solution.

2. A purification process for reducing the content of lincomycin B component according to claim 1, wherein: the silica gel matrix filler is filled into a high-pressure chromatographic column or a medium-low pressure column according to the particle size.

3. A purification process for reducing the content of lincomycin B component according to claim 1, wherein: and (4) replacing the back extraction solution or the aqueous solution of the crude product with the fermentation filtrate or the aqueous solution of the recovered crude product for purification and separation.

4. A purification process for reducing the content of lincomycin B component according to claim 1, wherein: the solution was adjusted to a pH of between 7.5 and 8.5 with NaOH and then adsorbed on a silica gel-based packed chromatographic column.

5. A purification process for reducing the content of lincomycin B component according to claim 1, wherein: the sample loading mass of the lincomycin is controlled to be 7-13% of the mass of the filler.

6. The purification process for reducing the content of the lincomycin B component according to claim 1, which specifically comprises the following steps:

① adjusting pH of the back extract of the solvent extract or the aqueous solution of the lincomycin crude product to 7.5-8.5 with NaOH to obtain alkaline solution;

②, pumping the alkalines into a chromatographic column filled with silica gel matrix filler, and controlling the sample loading mass of lincomycin to be 7-13% of the filler mass;

③ washing the column with 0.5-2BV pure water, collecting the fraction by stages, and considering whether the fraction is used indiscriminately according to the content of component B after HPLC detection;

④ is resolved with 1.5-6BV acetone containing 0-10% water or butanol containing 0-20% water;

⑤ adding hydrochloric acid to adjust pH to below 2, directly crystallizing acetone solution, concentrating butanol solution, crystallizing or back extracting with acid water, and crystallizing with acetone;

⑥ the column was equilibrated with 2-5BV of pure water and the process could be repeated ① to ⑤.

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