CN112409426B

CN112409426B - Preparation method of sisomicin sulfate

Info

Publication number: CN112409426B
Application number: CN202011342422.9A
Authority: CN
Inventors: 张德重; 朱丹; 严宝冬; 孙明
Original assignee: Heilongjiang Green Health Biotechnology Co ltd
Current assignee: Heilongjiang Green Health Biotechnology Co ltd
Priority date: 2020-11-25
Filing date: 2020-11-25
Publication date: 2023-10-13
Anticipated expiration: 2040-11-25
Also published as: CN112409426A

Abstract

The invention provides a preparation method of sisomicin sulfate, which comprises the steps of carrying out primary concentration on gentamicin production byproducts containing sisomicin, regulating pH by sulfuric acid, carrying out fixed bed dynamic adsorption on weak acid ion resin, washing by dilute ammonia water, resolving by concentrated ammonia, carrying out secondary concentration, regulating pH by sulfuric acid again, carrying out fixed bed dynamic adsorption on reversed phase resin, carrying out 10-30% alcohol chromatography, concentrating chromatographic liquid, adding 25% of analytically pure sulfuric acid to regulate pH to 4.0-5.0, carrying out salt conversion, adding 3-10% (W/V) of medicinal active carbon for decolorization, and carrying out spray drying.

Description

Preparation method of sisomicin sulfate

Technical Field

The invention belongs to the field of separation and purification of antibiotics, and particularly relates to a preparation method of sisomicin sulfate.

Background

Sisomicin (SISO) is an aminoglycoside antibiotic synthesized by Micromonospora, firstly, the American pioneer reports about research on sisomicin sulfate in 1970, the antibacterial spectrum is similar to gentamicin, has strong antibacterial effect on most gram positive bacteria and negative bacteria, has the characteristic of antibiotic post-effect on a plurality of pathogenic bacteria, and is widely applied to clinically treating septicemia, bacterial endocarditis, serious respiratory tract infection, kidney and urinary tract infection, skin tissue and soft tissue infection, burns, surgical peripheral bacterial infection and the like. The sisomicin bulk drug produced in China occupies a considerable share in the market, and accounts for about 80% of the world yield. The sisomicin can be directly used for producing sisomicin sulfate injection, and is also a synthetic parent of semisynthetic antibiotic Netilmicin (Netilmicin), and both sisomicin and Netilmicin are listed in the basic drug directory of China. The sisomicin sulfate in China is prepared by adopting the traditional fermentation, extraction and purification process, the obtaining way is single, the process is complex, the consumption of raw materials and power energy sources is large, the time consumption is long, and the production cost is high. The traditional production process of sisomicin sulfate is that actinomycin is used for fermenting raw materials such as starch, soybean cake powder and the like for 100-120 hours to obtain fermentation liquor containing sisomicin about 1000U/mL, and the production process comprises the following production processes of fermentation liquor acidification treatment, resin adsorption, ion exchange separation and purification, concentration, salt conversion decoloration, spray drying and the like.

The gentamicin is a group of multicomponent aminoglycoside antibiotics with similar structures, which are produced by actinomycin of micromonospora criminalis and micromonospora echinosporium, mainly comprises C-group complex (C1, C2, C1a and C2 a), and also comprises various small components with similar structures, such as sisomicin (SISO), minomycin (C2 b), gentamicin A, A1, A3, B, B1, X2 and the like, wherein the sisomicin can generally reach 100-200U/mL when the gentamicin is produced by domestic fermentation at present, and accounts for 5% -10% of the total amount of antibiotics in fermentation liquor. The Chinese pharmacopoeia of 2020 edition prescribes that the sisomicin sulfate is not more than 2.0%, the minocycline is not more than 3.0%, the single impurity is not more than 2.0%, the total impurity is not more than 4.5%, and the like, related substances such as sisomicin, gentamicin A, gentamicin B and the like are required to be removed in the production of the gentamicin sulfate, a large amount of byproducts are generated and are directly abandoned as impurities, so that the residual quantity of antibiotics such as sisomicin in wastewater is high, the environmental protection treatment difficulty is high, and a large amount of drug-resistant bacteria are generated after the antibiotics enter natural water, the ecological environment is destroyed, and the human health is endangered. The method for extracting antibiotics such as sisomicin from gentamicin sulfate production byproducts not only changes waste into valuable and improves enterprise benefits, but also reduces wastewater treatment difficulty, actively bears social responsibility for protecting environment, and has great popularization value.

Disclosure of Invention

In view of the above, the invention aims to provide a preparation method of sisomicin sulfate, which is a full-line process method for preparing sisomicin sulfate by recycling gentamicin sulfate byproducts and extracting and preparing sisomicin sulfate.

Sisomicin is a water-soluble, multi-element basic aminoglycoside antibiotic containing double bonds, the molecular weight of the sisomicin is 447.26, the molecular weight of the sisomicin contains 4 amino groups and 1 methyl group, and 5-level dissociation equilibrium exists in an aqueous solution.

The ionic liquid can exist in different electrochemical states in aqueous solutions with different pH values, ions with different valence states are in a multistage dissociation equilibrium state, and gradual dissociation occurs along with the pH value of the solution from high to low. When ph=9.7, more than 99% of sisomicin is present in the 0-valent form; at ph=8.0, the valence 0 drops to 61.3%, while the valence +1 rises to 33.2%, and 5% of sisomicin starts to be at valence +2; at a pH of 7.2 to 7.4, about 75% of sisomicin is present in the form of +1 and +2 valences; when the pH value is 6.6-6.8, the +2 and +3 valence forms account for more than 75 percent, and the +4 valence is 5-10 percent; at ph=4.6, 96.2% of sisomicin is present in the +4 valence form, the remainder being +3 valence. The alkaline nature of sisomicin determines that the sisomicin is preferably extracted by an ion exchange method, and the sisomicin is subjected to exchange adsorption on resin under the condition of low pH of a solution; eluting the resin under the condition of alkaline pH.

In order to achieve the above purpose, the technical scheme of the invention is realized as follows:

the preparation method of sisomicin sulfate is characterized by comprising the following steps of:

a. carrying out nanofiltration concentration or film concentration on gentamicin production byproducts containing sisomicin to obtain primary concentrated solution;

b. adding 25% of analytically pure sulfuric acid into the primary concentrated solution under stirring to obtain primary concentrated solution with pH value of 8.0-9.5;

c. pumping the primary concentrate with the pH value of 8.0-9.5 obtained in the step b into a fixed bed filled with macroporous weak acid ion resin or SP series macroporous network resin for dynamic adsorption, and flushing and balancing the resin column by using purified water after the adsorption is finished to obtain a flushed resin column;

d. c, performing dilute ammonia elution on the washed resin column obtained in the step c until the content of the washed sisomicin is 15-25%, so as to obtain an eluted resin column;

e. c, resolving the eluted resin column obtained in the step d until the tail sample unit is smaller than 500U/ml to obtain resolving liquid with sisomicin content of 60-80%;

f. concentrating the analysis liquid obtained in the step e by nanofiltration or concentrating by a film to obtain secondary concentrated liquid;

g. adding 25% of analytically pure sulfuric acid into the secondary concentrated solution obtained in the step f under stirring to obtain secondary concentrated solution with the pH value of 7.0-9.0;

h. pumping the secondary concentrate with the pH value of 7.0-9.0 obtained in the step g into a fixed bed filled with reverse resin for dynamic adsorption, and flushing and balancing the resin column by using purified water after the adsorption is finished to obtain a flushed resin column;

i. eluting the washed resin column obtained in the step h by 10-30% of methanol, ethanol or isopropanol solution, and stopping collecting from eluting until sisomicin Mi Xingfeng appears to Jiang Wei% of sisomicin content, thus obtaining eluent with sisomicin content more than 97%;

j. concentrating the eluent obtained in the step i by nanofiltration or film concentration to obtain sisomicin concentrated solution;

k. adding 25% of analytically pure sulfuric acid into the sisomicin concentrated solution obtained in the step j under stirring, adjusting the pH value to be 4.0-5.0, carrying out salt conversion to obtain sisomicin sulfate solution, adding 3-10% (W/V) of medicinal active carbon into the sisomicin sulfate solution, indirectly heating by using steam to enable the temperature of the feed liquid to reach 65-75 ℃, and carrying out heat preservation for 45-60 minutes for decoloration to obtain salt conversion decolored liquid;

and I, filtering the salt-transferring decolorized solution obtained in the step k to obtain a carbon-removed solution, and spray-drying the carbon-removed solution to obtain sisomicin sulfate.

Preferably, the primary concentrate obtained in step a has a titer of 60000-100000U/ml.

Preferably, the flow rate of the primary concentrated solution in the step c is 0.5-1.0BV, the sample loading amount is 30-50% of the saturation degree of the resin, and the flow rate of the purified water is 1.0-2.0BV when the purified water is used for flushing and balancing the resin column, and the flushing and balancing amount is 2-3 times of the column volume of the purified water.

Preferably, the dilute ammonia concentration used in step d is from 0.05 to 0.10mol/ml and the flow is from 0.5 to 1.5BV.

Preferably, the concentration of ammonia used in step e is 3.0-3.5mol/ml and the flow rate is 0.5-1.0BV.

Preferably, the secondary concentrate obtained in step f has a titer of 60000-100000U/ml.

Preferably, the flow rate of the secondary concentrated solution in the step h is 0.5-1.0BV, the loading amount is 20-25% of the saturation degree of the resin, the flow rate of the purified water is 1.0-2.0BV, and the purified water is flushed and balanced by 1-2 column volumes.

Preferably, the flow rate of the methanol, ethanol or isopropanol solution in the step i is 0.2-0.5BV.

Preferably, the titer of the sisomicin concentrate in step J is 200000-300000U/ml.

Preferably, the carbon removal liquid obtained in the step l has a titer of 150000-250000U/ml, a color grade of less than or equal to 1 and a pH of 4.0-5.0.

Compared with the prior art, the preparation method of sisomicin sulfate has the following advantages:

the invention provides a brand new process method for preparing sisomicin sulfate from the gentamicin sulfate production byproducts, which has the advantages of simple process and convenient operation, can change waste into valuable, improve the enterprise benefit, reduce the wastewater treatment difficulty and have higher economic benefit and social value.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention. In the drawings:

FIG. 1 is a HPLC-ELSD spectrum of a gentamicin fermentation product described in example 1 of the present invention;

FIG. 2 is a schematic diagram of a HPLC-ELSD of a primary concentrate of gentamicin byproduct as described in example 1 of the present invention;

FIG. 3 is a HPLC-ELSD spectrum of a primary concentrate of gentamicin byproduct as described in example 2 of the present invention;

FIG. 4 is a HPLC-ELSD spectrum of sisomicin sulfate according to example 2 of the present invention.

Detailed Description

Unless defined otherwise, technical terms used in the following examples have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods are conventional methods unless otherwise specified.

The present invention will be described in detail with reference to the following examples and drawings.

Example 1

1) Taking gentamicin sulfate production byproducts containing sisomicin (as shown in figure 1, the content of sisomicin in a gentamicin fermentation product used in the embodiment is 5.12%, the gentamicin sulfate byproducts are calcium-magnesium plasma removed by separating dilute hydrochloric acid through macroporous cationic resin in the gentamicin production process, connecting 711 anion resin decoloration columns in series, flushing purified water until no chloride ions exist, collecting the obtained impurity parts washed by dilute ammonia, and obtaining primary concentrated solution with concentration unit of 18642U/ml through nanofiltration concentration, wherein the impurity parts comprise a large amount of gentamicin structural analogues such as gentamicin B and sisomicin, a small amount of ammonia solution of gentamicin main components C1a, C2 and the like, calcium-magnesium ions, most of pigments and the like are removed, the total titer of antibiotics in the byproducts is 600-1500U/ml, as shown in figure 2;

2) Slowly adding 25% of analytically pure sulfuric acid into the primary concentrated solution under stirring to adjust the pH to 8.5;

3) Pumping the primary concentrated solution with the pH value of 8.5 into a fixed bed filled with D11 resin for dynamic adsorption (column volume of 50L and height-diameter ratio of 20:1), wherein the flow is 0.5BV, the sample loading amount is 30% of the resin saturation, after the sample loading is finished, the resin column is washed and balanced by using purified water, the flow is 2BV, and the washing and balancing of 3 times of the column volume of the purified water can be stopped;

4) Eluting the resin column with 0.05mol/ml dilute ammonia at a flow rate of 0.5-1.5BV until the content of the washed sisomicin is 15-25%;

5) Resolving the resin column eluted by dilute ammonia, resolving the resolving solution by adopting 3.5mol/ml ammonia water with the flow of 0.5-1.0BV until the tail sample unit is less than 500U/ml, and obtaining resolving solution with the sisomicin content of 62.43%;

6) Concentrating the analysis solution by nanofiltration and concentrating by a film to obtain a secondary concentrated solution with a concentration unit of 89736U/ml;

7) Slowly adding 25% of analytically pure sulfuric acid into the secondary concentrated solution under stirring to adjust the pH to 8.26;

8) Pumping the secondary concentrate with the pH value of 8.26 into a fixed bed filled with reverse phase resin NM200 for dynamic adsorption (column volume of 50L, height-diameter ratio of 20:1), wherein the flow rate is 0.8BV, the sample loading amount is 20% of the resin saturation, after the sample loading is finished, the resin column is washed and balanced by using purified water, the flow rate is 1.5BV, and the washing and balancing of 2 times of the column volume of purified water can be stopped;

9) Eluting the resin column with 20% methanol solution at a flow rate of 0.3BV, starting to collect sisomicin from elution until sisomicin Mi Xingfeng appears, and stopping collecting until sisomicin content is reduced to 85%, thereby obtaining eluent with sisomicin content of more than 97.06%;

10 Concentrating the eluent by nanofiltration or film concentration to obtain a concentrated solution of sisomicin with a concentration unit of 224382U/ml;

11 Under stirring, slowly adding 25% of analytically pure sulfuric acid to adjust the pH to 4.7, and performing salt conversion to obtain sisomicin sulfate solution; adding 5% (W/V) medicinal active carbon into sisomicin sulfate solution, indirectly heating with steam to 65-75deg.C, and decolorizing under the condition of maintaining temperature for 45-60 min to obtain salt-transferring decolorized solution.

12 Filtering the salt-converted decolorized solution to obtain a carbon-removed solution, detecting the carbon-removed solution, wherein the final titer is 177874u/ml, the color grade is less than or equal to 1, the pH is 4.7, the bacterial endotoxin is qualified, and performing spray drying to obtain sisomicin sulfate.

Example 2

1) Taking the primary concentrated solution prepared in the example 1;

2) Slowly adding 25% of analytically pure sulfuric acid into the primary concentrated solution under stirring to adjust the pH to 8.8;

3) Pumping the primary concentrated solution with the pH value of 8.5 into a fixed bed filled with D11 resin for dynamic adsorption (column volume of 500L and height-diameter ratio of 25:1), wherein the flow is 0.5BV, the sample loading amount is 30% of the resin saturation, after the sample loading is finished, the resin column is washed and balanced by using purified water, the flow is 1.0-2.0BV, and the washing and balancing of 2-3 times of the column volume of purified water can be stopped;

5) Resolving the resin column eluted by dilute ammonia, resolving the resolving solution by adopting 3.5mol/ml ammonia water with the flow of 0.5-1.0BV until the tail sample unit is less than 500U/ml, and obtaining resolving solution with the sisomicin content of 72.19%;

6) Concentrating the above analysis solution by nanofiltration or film concentration to obtain a secondary concentrated solution with concentration unit of 60000-100000U/ml, as shown in figure 3, wherein the content of sisomicin in the secondary concentrated solution is 72.19%;

7) Slowly adding 25% of analytically pure sulfuric acid into the secondary concentrated solution under stirring to adjust pH to 7.0-9.0;

8) Pumping the secondary concentrate with pH of 7.0-9.0 into a fixed bed filled with reverse phase resin NM200 for dynamic adsorption (column volume of 500L, height-diameter ratio of 25:1), wherein the flow is 0.5-1.0BV, the sample loading amount is 20% -25% of the resin saturation, after the sample loading is finished, the resin column is washed and balanced by purified water, the flow is 1.0-2.0BV, and the washing and balancing of 1-2 column volumes of purified water can be stopped;

9) Eluting the resin column with 20% methanol solution at a flow rate of 0.3BV, and collecting from eluting until sisomicin Mi Xingfeng appears until sisomicin content is reduced to 85%, and stopping collecting to obtain eluent with sisomicin content of more than 97%;

10 Concentrating the eluent by nanofiltration or film concentration to obtain a concentrated solution of sisomicin with a concentration unit of 200000 ～ 300000U/ml;

11 Under stirring, slowly adding 25% of analytically pure sulfuric acid to adjust the pH to 4.0-5.0, and performing salt conversion to obtain sisomicin sulfate solution; adding 3-10% (W/V) medicinal active carbon into sisomicin sulfate solution, indirectly heating with steam to 65-75deg.C, and decolorizing under the condition of maintaining temperature for 45-60 min to obtain salt-transferring decolorized solution.

12 Filtering the salt-converted decolorized solution to obtain carbon-removed solution, detecting the carbon-removed solution, wherein the final titer is 150000 ～ 250000u/ml, the color grade is less than or equal to 1, the pH is 4.0-5.0, the bacterial endotoxin is qualified, and performing spray drying to obtain sisomicin sulfate, wherein the content of the sisomicin sulfate detected in the process shown in figure 4 is 97.42%.

Example 3

1) Taking the primary concentrated solution prepared in the example 1;

3) Pumping the primary concentrated solution with the pH value of 8.5 into a fixed bed filled with macroporous weak acid cationic resin HZ-0156 for dynamic adsorption (column volume of 500L, height-diameter ratio of 25:1), wherein the flow rate is 0.5BV, the sample loading amount is 30% of the resin saturation, after the sample loading is finished, the resin column is washed and balanced by using purified water, the flow rate is 1.0-2.0BV, and the washing and balancing of 2-3 times of the column volume of purified water can be stopped;

5) Resolving the resin column eluted by dilute ammonia, resolving the resolving solution by adopting 3.5mol/ml ammonia water with the flow of 0.5-1.0BV until the tail sample unit is less than 500U/ml, and obtaining resolving solution with the sisomicin content of 60% -80%;

6) Concentrating the analysis solution by nanofiltration or film concentration to obtain a secondary concentrated solution with a concentration unit of 60000-100000U/ml;

8) Pumping the secondary concentrate with pH of 7.0-9.0 into a fixed bed filled with reversed phase resin MOTO HZ20 for dynamic adsorption (column volume of 500L, height-to-diameter ratio of 25:1), wherein the flow is 0.5-1.0BV, the sample loading amount is 20% -25% of the resin saturation, after the sample loading is finished, the resin column is washed and balanced by purified water, the flow is 1.0-2.0BV, and the washing and balancing of 1-2 column volumes of purified water can be stopped;

9) Eluting the resin column with 20% ethanol solution at a flow rate of 0.3BV, and collecting from eluting until sisomicin Mi Xingfeng appears until sisomicin content is reduced to 85%, and stopping collecting to obtain eluent with sisomicin content of more than 97%;

12 Filtering the salt-transferring decolorized solution to obtain a carbon-removed solution, detecting the carbon-removed solution, wherein the final titer is 150000 ～ 250000u/ml, the color grade is less than or equal to 1, the pH is 4.0-5.0, the bacterial endotoxin is qualified, and performing spray drying to obtain sisomicin sulfate.

Example 4

1) Taking the primary concentrated solution prepared in the example 1;

2) Slowly adding 25% of analytically pure sulfuric acid into the primary concentrated solution under stirring to adjust the pH to 8.6;

3) Pumping the primary concentrated solution with the pH value of 8.5 into a fixed bed filled with resin LX55 for dynamic adsorption (column volume of 500L, height-diameter ratio of 25:1), wherein the flow rate is 0.5BV, the sample loading amount is 30% of the resin saturation, after the sample loading is finished, the resin column is washed and balanced by using purified water, the flow rate is 1.0-2.0BV, and the washing and balancing of 2-3 times of the column volume of purified water can be stopped;

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.

Claims

1. The preparation method of sisomicin sulfate is characterized by comprising the following steps of:

k. adding 25% of analytically pure sulfuric acid into the sisomicin concentrated solution obtained in the step j under stirring, adjusting the pH value to be 4.0-5.0, carrying out salt conversion to obtain sisomicin sulfate solution, adding 3-10% of W/V medicinal active carbon into the sisomicin sulfate solution, indirectly heating by using steam to enable the temperature of the feed liquid to reach 65-75 ℃, and carrying out heat preservation for 45-60 minutes for decoloration to obtain salt conversion decolored liquid;

2. The method for preparing sisomicin sulfate as claimed in claim 1, wherein: the primary concentrate obtained in step a has a titer of 60000-100000U/ml.

3. The method for preparing sisomicin sulfate as claimed in claim 1, wherein: and c, the flow rate of the primary concentrated solution is 0.5-1.0BV when the primary concentrated solution is adsorbed, the sample loading amount is 30-50% of the saturation degree of the resin, and when the purified water is used for flushing and balancing the resin column, the flow rate is 1.0-2.0BV, and the flushing and balancing volume of the purified water is 2-3 times of the column volume.

4. The method for preparing sisomicin sulfate as claimed in claim 1, wherein: the concentration of the dilute ammonia used in the step d is 0.05-0.10mol/ml, and the flow is 0.5-1.5BV.

5. The method for preparing sisomicin sulfate as claimed in claim 1, wherein: the titer of the secondary concentrate obtained in step f is 60000-100000U/ml.

6. The method for preparing sisomicin sulfate as claimed in claim 1, wherein: in the step h, the flow rate of the secondary concentrated solution is 0.5-1.0BV, the sample loading amount is 20-25% of the saturation degree of the resin, the flow rate of the purified water is 1.0-2.0BV, and the purified water with 1-2 column volumes is washed and balanced.

7. The method for preparing sisomicin sulfate as claimed in claim 1, wherein: the flow rate of the methanol, ethanol or isopropanol solution in the step i is 0.2-0.5BV.

8. The method for preparing sisomicin sulfate as claimed in claim 1, wherein: the titer of the sisomicin concentrated solution in the step J is 200000-300000U/ml.

9. The method for preparing sisomicin sulfate as claimed in claim 1, wherein: the carbon stripping liquid obtained in the step (l) has the titer of 150000-250000U/ml, the color grade of less than or equal to 1 and the pH value of 4.0-5.0.