CN112645993A - Purification method of high-purity lincomycin hydrochloride - Google Patents
Purification method of high-purity lincomycin hydrochloride Download PDFInfo
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Abstract
The invention discloses a purification method of high-purity lincomycin hydrochloride, which comprises the steps of purifying a lincomycin crude product by adopting a reverse phase chromatography method, desalting, transferring salt, and drying to obtain the high-purity lincomycin hydrochloride. The method can quickly and effectively remove impurities, and meets the requirements of laws and regulations and markets on lincomycin hydrochloride raw material medicines. The method has the advantages of simple operation, low cost, high purity of the obtained product, high yield, complete impurity removal and suitability for industrial popularization and application.
Description
Technical Field
The invention belongs to the field of separation and purification of chemical drugs, and particularly relates to a purification method of high-purity lincomycin hydrochloride, which is used for specifically purifying a lincomycin crude product through reverse phase chromatography, desalting, transferring salt and drying to obtain high-purity lincomycin hydrochloride powder.
Background
Lincomycin Hydrochloride (Lincomycin Hydrochloride) is an Lincomycin amine antibiotic, has the chemical name of 6- (1-methyl-trans-4-propyl-L-2-pyrrolidine formamido) -1-thio-6, 8-dideoxy-D-erythro-alpha-D-galactopyranoside Hydrochloride monohydrate, and has the molecular formula of C18H34N2O6S·HCl·H2O, molecular weight 461.02, and the structural formula is shown as follows:
the lincomycin molecule is divided into A, B, C, D, K, S six components due to different substituent groups, and has expression of different degrees in the fermentation process, wherein the A component accounts for more than 90%; the component B accounts for 5-10%, the clinical curative effect of the component B is only 20% of that of the component A, and the toxicity of the component B is high; the total amount of the other 4 components is not more than 1%.
| Component name | Substituent group 1 | Substituent group 2 | Substituent group 3 |
| A | CH3 | C3H7 | CH3 |
| B | CH3 | C2H5 | CH3 |
| C | CH3 | C3H7 | C2H5 |
| D | H | C3H7 | CH3 |
| K | H | C3H7 | C2H5 |
| S | C2H5 | C3H7 | C2H5 |
TABLE 1 lincomycin Components substituents
At present, streptomyces is generally adopted to ferment under certain conditions, fermentation liquor is filtered after being acidified, filtrate is adjusted in pH by NaOH, butanol is used for extraction, hydrochloric acid is added for reaction after reduced pressure concentration to generate crude lincomycin hydrochloride crystals, and after the crude crystals are dissolved, activated carbon adsorption, secondary crystallization and drying are carried out to obtain the lincomycin hydrochloride crude product with the content of more than 90%.
The publicly reported method for purifying the lincomycin is mainly to obtain a lincomycin crude product by multiple extractions and macroporous resin column chromatography, and then to obtain the high-purity lincomycin hydrochloride by multiple crystallization. For example, Chinese patent CN103724380B discloses a lincomycin purification method, which adopts ceramic ultrafiltration membrane, extraction, back extraction and other processes to purify lincomycin, the process is complex, extraction liquid and back extraction liquid are easy to remain, the obtained lincomycin has low content and extremely many impurities, and the lincomycin cannot reach the standards of European and United states pharmacopoeia. For another example, CN104356179A discloses a lincomycin hydrochloride purification process, which comprises an adsorption step, a water washing step, an analysis step, a concentration step, a crystallization step, and drying to obtain a finished product. The process uses macroporous resin to purify lincomycin, removes lincomycin B component, has poor purification effect and high impurity content aiming at other impurities in the lincomycin crude product, and can obtain high-purity lincomycin hydrochloride only by using acetone for repeated recrystallization. And the purification period is long, the recovery rate is low, the loading capacity is low, the automation degree is low, and the stability between batches in the industrial process cannot meet the standard requirements of European and American pharmacopoeias. For this reason, it is necessary to develop an economical and efficient method for purifying lincomycin hydrochloride, which meets the requirements of european and us regulations.
Disclosure of Invention
The invention discloses a purification method of high-purity lincomycin hydrochloride, which comprises the steps of purifying a lincomycin crude product by adopting a reverse phase chromatography method, desalting, transferring salt, concentrating and drying to obtain the high-purity lincomycin hydrochloride. The process mainly utilizes the characteristic that lincomycin is in an ionic state under an acidic condition and in a molecular state under an alkaline condition, and utilizes the hydrophobic acting force between the lincomycin and the reversed-phase chromatographic packing under the molecular state to carry out separation. The method can effectively remove impurities and reach the standards required by European and American regulations.
In order to achieve the purpose, the invention adopts the following scheme:
step one, purifying a lincomycin crude product by adopting a reverse phase chromatography method to obtain a lincomycin purified solution;
step two, adopting macroporous resin for desalting to obtain a lincomycin desalting solution;
and step three, obtaining lincomycin hydrochloride through salt conversion, concentration and drying.
More specifically, the reverse phase chromatography in step one is carried out as follows:
(1) column assembling: using polymer reverse phase filler as stationary phase of chromatographic column, adding 1-3CV 0-100% (V/V) content organic solvent-water solution, homogenizing, wet loading column with height not less than 25cm and loading pressure 0-10 MPa;
(2) column balancing: equilibrating the chromatographic column with 2-6CV buffered saline solution at a process line flow rate of no more than 10 cm/min;
(3) loading: dissolving the lincomycin crude product in a buffer saline solution to obtain a stock solution with the concentration of 200-300g/L, loading the stock solution to a chromatographic column with the loading capacity of 20-100g/L, wherein the process linear flow rate is not higher than 10 cm/min;
(4) impurity washing: washing impurity of the chromatographic column by using an organic solvent-buffer saline solution with 5-10CV content of 5-15% (V/V), wherein the linear flow velocity in the impurity washing process is not higher than 10 cm/min;
(5) and (3) elution: eluting lincomycin adsorbed on a chromatographic column by using an organic solvent-buffer saline solution with the content of 6-14CV (variable solvent/volume) of 15-40% (V/V), wherein the linear flow rate of an elution process is not higher than 10cm/min, and collecting an eluent component to be a lincomycin purified solution;
(6) regeneration: regenerating the chromatographic column by using an organic solvent-water solution with the content of 6-10CV (80-100 percent (V/V)), wherein the linear flow velocity in the regeneration process is not higher than 10 cm/min; and after the regeneration is finished, the balance step of the purification process in the next period is carried out.
The macroporous resin desalination in the second step is carried out according to the following steps:
(A) concentration: concentrating the lincomycin purified solution collected in the first reverse phase chromatography step, reducing the content of the organic solvent to 1-10% (V/V), and adding alkali to adjust the pH to 10-12;
(B) loading: loading the concentrated solution onto a macroporous resin column with a loading capacity of 20-150g/L and a linear flow velocity of not higher than 5 cm/min;
(C) washing: respectively washing the macroporous resin column with 2-6BV of aqueous alkali with pH value of 10-12 and pure water, wherein the linear flow rate is not higher than 5 cm/min;
(D) and (3) elution: eluting lincomycin with 2-6BV (V/V) organic solvent-water solution with the content of 50-100% (V/V), wherein the linear flow rate is not higher than 5cm/min, and collecting eluent, namely lincomycin desalting solution;
(E) column balancing: and (3) rebalancing the macroporous resin column by using 2-6BV of pure water, wherein the linear flow velocity is not higher than 5cm/min, and desalting in the next period.
The salt conversion drying in the third step is carried out according to the following steps
(a) Salt conversion: adding the lincomycin desalted solution in the second step into hydrochloric acid solution with the concentration of 0.1-4mol/L, adjusting the pH value to 3.5-4.5, wherein the reaction temperature is not more than 20 ℃;
(b) concentration: concentrating the collected liquid by 1-20 times by nanofiltration, distillation or reduced pressure distillation;
(c) and (3) drying: and carrying out vacuum drying to obtain powdery lincomycin hydrochloride.
Furthermore, the reverse phase filler in the first step is polymer microspheres made of polystyrene-divinylbenzene, and the crosslinking degree ranges from 80% to 99% (W/W). The polymer microsphere has a particle size of 30-150 μm and a pore size of
The specific surface area range is 800-1500m2(ii)/g, including but not limited to, LX-161 series reverse phase polymer chromatography packing manufactured by New science and technology materials, Inc. of Xian blue, to West (Xiao) science and technology (abbreviated as blue, to Xiao science and technology);
the organic solvent is a solvent which can be mutually dissolved with water in any proportion, and comprises but is not limited to one or a mixture of more of methanol, ethanol, acetonitrile, isopropanol and acetone;
the buffer salt solution: preparing a phosphate buffer solution with the concentration range of 0.5-5.0%, and adjusting the pH value to 5-9 by using ammonia water or sodium hydroxide;
the alkali refers to organic alkali with alkalinity, inorganic alkali and organic and inorganic salt which shows alkalinity in aqueous solution, and includes but is not limited to one or a mixture of more of methylamine, pyridine, ethylenediamine, triethylamine, tetramethylammonium, tetraethylammonium, sodium hydroxide, potassium hydroxide, sodium phosphate, ammonium phosphate, sodium acetate and ammonium acetate;
the macroporous resin refers to polymer beads formed by polyethylene benzene-divinylbenzene. The polymer pellet has a particle diameter of 0.1-1.5mm and a pore diameter of 0.1-1.5mm
Specific surface area of not less than 100m2(ii) a degree of crosslinking in the range of 15-80% (W/W), including but not limited to the model number LX-20 resin manufactured by seian blue dawn scientific and technical materials gmbh;
the concentration of the hydrochloric acid solution is 0.1-4 mol/L.
Compared with the prior art, the invention has the following advantages:
the method of the invention uses the reversed phase chromatography medium as the chromatographic column packing, and uses the proper elution condition to ensure that the purity of the lincomycin hydrochloride product reaches more than 99.6 percent, the yield reaches more than 88 percent, and the method has the advantages of short purification period, simple operation and low cost, and is suitable for industrialized popularization and application.
Drawings
FIG. 1 is a flow chart of a purification process of lincomycin hydrochloride.
Detailed Description
The present invention will be described in further detail with reference to examples of specific embodiments. However, the present invention is not limited to the following specific examples. In the following examples, a 10X 600mm stainless steel column (packed with blue-Dawn technical reverse phase chromatography packing LX-161 series, produced by Seisan blue-Dawn technical materials Co., Ltd., degree of crosslinking of 80 to 99% (W/W)) was used in the reverse phase chromatography step, and the polymer microspheres had an average particle diameter of 30 to 150 μm and a pore diameter of 30 to 150. mu.m
The specific surface area is 800-2Per g), the amount of the polymer reverse phase chromatography packing is60g, homogenizing by using 100-300mL of 40-70% ethanol aqueous solution (V/V), and carrying out wet column packing under the pressure of 5 Mbar; in the step of desalting with macroporous resin, 10 × 600mm glass column (filled with blue-Daizhi macroporous resin LX-20, produced by Xian blue-Daizhi New Material Co., Ltd., polymer pellet with particle diameter of 0.1-1.5mm and pore diameter of 0.1-1.5 mm) is used
Specific surface area of not less than 100m2(ii)/g, degree of crosslinking 30 to 70% (W/W)), 45mL of the macroporous resin, 100mL of pure water, mixing, and loading under normal pressure.
Example 1
The solution was prepared as follows:
mobile phase A: diluting 34g of phosphoric acid with 900ml of water, adjusting the pH value to 7 by using ammonia water, and then fixing the volume to 1L;
mobile phase B: adding 950mL of mobile phase A into 50mL of ethanol, uniformly mixing to prepare 5% (V/V) phosphate-ethanol solution, and fixing the volume to 1L;
mobile phase C: adding 250mL of ethanol into 750mL of mobile phase A, uniformly mixing to prepare a 25% (V/V) phosphate-ethanol solution, and fixing the volume to 1L;
dissolving 2.35g of lincomycin crude product into a crude product solution by using the mobile phase A, metering the volume to 10mL, and filtering by using a 0.45um filter membrane to obtain a filtered solution with the concentration of 235g/L and the volume of 10 mL.
LX-161S (the average particle size of polymer microspheres is 30-50um) is used as a polymer reversed-phase filler, the flow rate in the whole chromatographic purification process is 4.7ml/min, and a mobile phase A is used for balancing a chromatographic column for 40 min; pumping 10mL of filtered lincomycin crude product solution into the chromatographic column, wherein the loading capacity is 50g/L (the pure lincomycin mass/the volume of the polymer reverse phase filler column), eluting with a mobile phase B for 80min, eluting with a mobile phase C for 120min, and collecting 564mL of eluent; regenerating with 80% ethanol water solution for 60 min; concentrating the collected eluent to 20mL by rotary evaporation at 45 ℃ and 100 mbar;
the flow rate of the whole macroporous resin desalting process is 0.75ml/min, pure water is balanced for 120min, and the pH value of the concentrated solution obtained in the previous step is adjusted to 10.5 by NaOH; after filtering through a 0.45um filter membrane, 20ml of lincomycin concentrated solution with the concentration of 110.2g/L is obtained. Pumping into a macroporous resin column with the loading capacity of 47g/L, washing with a sodium hydroxide solution with the pH value of 10.5 for 3BV, then washing with water for 4BV, then eluting with an ethanol aqueous solution of 80% (V/V) for 2BV, and collecting 90mL of an eluent containing lincomycin; adjusting the pH value to 4.5 by using 1mol/L HCl; finally, the lincomycin hydrochloride is prepared into 2.05g of white powder by rotary evaporation at the temperature of 45 ℃ and under the condition of 100 mbar. Through liquid chromatography detection and calculation, the purity of the lincomycin hydrochloride product reaches 99.74%, and the total process yield reaches 90.6%.
Example 2
The solution was prepared as follows:
mobile phase A: diluting 34g of phosphoric acid with 900ml of water, adjusting the pH to 7 with ammonia water, and metering to 1L;
mobile phase B: 940mL of mobile phase A is added with 60mL of ethanol, and the mixture is uniformly mixed to prepare 6% (V/V) of phosphate-ethanol solution with the constant volume of 1L;
mobile phase C: adding 250mL of ethanol into 750mL of mobile phase A, uniformly mixing to prepare a 25% (V/V) phosphate-ethanol solution, and fixing the volume to 1L;
dissolving 2.35g of lincomycin crude product into a crude product solution by using the mobile phase A, metering the volume to 10mL, and filtering by using a 0.45um filter membrane to obtain a filtered solution with the concentration of 235g/L and the volume of 10 mL.
LX-161 (the average particle size of polymer microspheres is 50-75um) is used as a polymer reversed-phase filler, the flow rate in the whole chromatographic purification process is 4.7ml/min, and a mobile phase A is used for balancing a chromatographic column for 40 min; pumping 10mL of the filtered lincomycin crude product solution into the chromatographic column, wherein the loading capacity is 50g/L (the pure lincomycin mass/the volume of the polymer reverse phase filler column), eluting with a mobile phase B for 80min, eluting with a mobile phase C for 120min, and collecting the eluent; regenerating with 80% ethanol water solution for 60 min; 564mL of the collected eluate was concentrated to 25mL by rotary evaporation at 45 ℃ and 100 mbar;
the flow rate of the whole macroporous resin desalting process is 0.75ml/min, purified water is balanced for 120min, and the concentrated solution obtained in the previous step is adjusted to pH10.5 by NaOH; after filtering through a 0.45um filter membrane, 25ml of lincomycin concentrated solution with the concentration of 85.1g/L is obtained. Pumping into a macroporous resin column, loading 80mg/mL, washing with a sodium hydroxide solution with pH of 10.5 for 3BV, washing with water for 4BV, eluting with an ethanol aqueous solution of 80% (V/V) for 2BV, and collecting 90mL of an eluent containing lincomycin; adjusting the pH value to 4.5 by using 1mol/L HCl; finally, 2.04g of lincomycin hydrochloride powder is obtained by rotary evaporation at 45 ℃ and 100 mbar. The purity of the lincomycin hydrochloride product reaches 99.68%, and the total process yield reaches 89.6%.
Example 3
The solution was prepared as follows:
mobile phase A: diluting 34g of phosphoric acid with 900ml of water, adjusting the pH value to 7 by using ammonia water, and then fixing the volume to 1L;
mobile phase B: adding 950mL of mobile phase A into 50mL of ethanol, uniformly mixing to prepare 5% (V/V) phosphate-ethanol solution, and fixing the volume to 1L;
mobile phase C: adding 250mL of ethanol into 750mL of mobile phase A, uniformly mixing to prepare a 25% (V/V) phosphate-ethanol solution, and fixing the volume to 1L;
dissolving 2.2g of lincomycin crude product into a crude product solution by using the mobile phase A, metering the volume to 10mL, and filtering by using a 0.45um filter membrane to obtain a filtered solution with the concentration of 220g/L and the volume of 10 mL.
LX-161S is used as a polymer reversed-phase filler, the flow rate of the whole chromatographic purification process is 4.7ml/min, and a chromatographic column is firstly balanced by a mobile phase A for 40 min; pumping 10mL of the filtered lincomycin crude product solution into the chromatographic column, wherein the loading capacity is 45g/L (the pure lincomycin mass/the volume of the polymer reverse phase filler column), eluting with a mobile phase B for 80min, eluting with a mobile phase C for 140min, and collecting the eluent; regenerating with 80% ethanol water solution for 60 min; 658mL of collected eluate was concentrated to 30mL by rotary evaporation at 45 ℃ and 100 mbar;
the flow rate of the whole macroporous resin desalting process is 0.75ml/min, purified water is balanced for 120min, and the concentrated solution obtained in the previous step is adjusted to pH10.5 by NaOH; after filtering through a 0.45um filter membrane, 30mL of lincomycin concentrated solution with the concentration of 64.9g/L is obtained. Pumping into a macroporous resin column, loading 80mg/mL, washing with a sodium hydroxide solution with pH of 10.5 for 3BV, washing with water for 4BV, eluting with an ethanol aqueous solution of 80% (V/V) for 2BV, and collecting 90mL of an eluent containing lincomycin; adjusting the pH value to 4.5 by using 1mol/L HCl; finally, the lincomycin hydrochloride is prepared into 1.94g of white powder by rotary evaporation at the temperature of 45 ℃ and under the condition of 100 mbar. The purity of the lincomycin hydrochloride product reaches 99.81%, and the total process yield reaches 91.2%.
Example 4
The solution was prepared as follows:
mobile phase A: diluting 34g of phosphoric acid with 900ml of water, adjusting the pH value to 7 by using ammonia water, and then fixing the volume to 1L;
mobile phase B: 930mL of mobile phase A is added with 70mL of ethanol, and the mixture is uniformly mixed to prepare a phosphate-ethanol solution with 7% (V/V), and the constant volume is 1L;
mobile phase C: adding 850mL of mobile phase A into 150mL of ethanol, uniformly mixing to prepare 15% (V/V) of phosphate-ethanol solution, and fixing the volume to 1L;
dissolving 2.2g of lincomycin crude product into a crude product solution by using the mobile phase A, metering the volume to 10mL, and filtering by using a 0.45um filter membrane to obtain a filtered solution with the concentration of 220g/L and the volume of 10 mL.
LX-161S is used as a polymer reversed-phase filler, the flow rate of the whole chromatographic purification process is 4.7ml/min, and a chromatographic column is firstly balanced by a mobile phase A for 40 min; pumping 10mL of the filtered lincomycin crude product solution into the chromatographic column, wherein the loading capacity is 45g/L (the pure lincomycin mass/the volume of the polymer reverse phase filler column), eluting with a mobile phase B for 80min, eluting with a mobile phase C for 160min, and collecting the eluent; regenerating with 80% ethanol water solution for 60 min; collecting 752mL eluate, rotary evaporating at 45 deg.C and 100mbar to concentrate to 32 mL;
the flow rate of the whole macroporous resin desalting process is 0.75ml/min, purified water is balanced for 120min, and the concentrated solution obtained in the previous step is adjusted to pH10.5 by NaOH; after filtering through a 0.45um filter membrane, 32mL of lincomycin concentrated solution with the concentration of 60.5g/L is obtained. Pumping into a macroporous resin adsorption column, loading 80mg/mL, washing with a sodium hydroxide solution with pH of 10.5 for 3BV, washing with water for 4BV, eluting with an ethanol aqueous solution of 80% (V/V) for 2BV, and collecting 90mL of an eluent containing lincomycin; adjusting the pH value to 4.5 by using 1mol/L HCl; finally, the lincomycin hydrochloride is prepared into 1.96g of white powder by rotary evaporation at the temperature of 45 ℃ and under the condition of 100 mbar. The purity of the lincomycin hydrochloride product reaches 99.64 percent, and the total process yield reaches 91.8 percent.
Example 5
The solution was prepared as follows:
mobile phase A: diluting 34g of phosphoric acid with 900ml of water, adjusting the pH value to 7 by using ammonia water, and then fixing the volume to 1L;
mobile phase B: adding 950mL of mobile phase A into 50mL of ethanol, uniformly mixing to prepare 5% (V/V) phosphate-ethanol solution, and fixing the volume to 1L;
mobile phase C: adding 250mL of ethanol into 750mL of mobile phase A, uniformly mixing to prepare a 25% (V/V) phosphate-ethanol solution, and fixing the volume to 1L;
dissolving 3.9g of lincomycin crude product into a crude product solution by using the mobile phase A, metering the volume to 10mL, and filtering by using a 0.45um filter membrane to obtain the filtrate with the concentration of 260g/L and the volume of 15 mL.
LX-161M (the average particle size of polymer microspheres is 75-100um) is used as a polymer reversed-phase filler, the flow rate in the whole chromatographic purification process is 4.7ml/min, and a mobile phase A is used for balancing a chromatographic column for 40 min; pumping 15mL of the filtered lincomycin crude product solution into the chromatographic column, wherein the loading capacity is 80g/L (the pure lincomycin mass/the volume of the polymer reverse phase filler column), eluting with a mobile phase B for 80min, eluting with a mobile phase C for 140min, and collecting the eluent; regenerating with 80% ethanol water solution for 60 min; 658mL of collected eluate was concentrated to 35mL by rotary evaporation at 45 ℃ and 100 mbar;
the flow rate of the whole macroporous resin desalting process is 0.75ml/min, purified water is balanced for 120min, and the concentrated solution obtained in the previous step is adjusted to pH10.5 by NaOH; after filtering through a 0.45um filter membrane, 35mL of lincomycin concentrated solution with the concentration of 95.4g/L is obtained. Pumping into a macroporous resin adsorption column, loading 80mg/mL, washing with a sodium hydroxide solution with pH of 10.5 for 3BV, washing with water for 4BV, eluting with an ethanol aqueous solution of 80% (V/V) for 2BV, and collecting 90mL of an eluent containing lincomycin; adjusting the pH value to 4.5 by using 1mol/L HCl; finally, the lincomycin hydrochloride is prepared into 3.31g of white powder by rotary evaporation at the temperature of 45 ℃ and under the condition of 100 mbar. The purity of the lincomycin hydrochloride product reaches 99.31 percent, and the total process yield reaches 86.3 percent.
Example 6
The solution was prepared as follows:
mobile phase A: diluting 34g of phosphoric acid with 900ml of water, adjusting the pH value to 7 by using ammonia water, and then fixing the volume to 1L;
mobile phase B: adding 950mL of mobile phase A into 50mL of ethanol, uniformly mixing to prepare 5% (V/V) phosphate-ethanol solution, and fixing the volume to 1L;
mobile phase C: adding 250mL of ethanol into 750mL of mobile phase A, uniformly mixing to prepare a 25% (V/V) phosphate-ethanol solution, and fixing the volume to 1L;
dissolving 3.9g of lincomycin crude product into a crude product solution by using the mobile phase A, metering the volume to 10mL, and filtering by using a 0.45um filter membrane to obtain the filtrate with the concentration of 260g/L and the volume of 15 mL.
LX-161S is used as a polymer reversed-phase filler, the flow rate of the whole chromatographic purification process is 4.7ml/min, and a chromatographic column is firstly balanced by a mobile phase A for 40 min; pumping 15mL of filtered lincomycin crude product solution into the chromatographic column, wherein the loading capacity is 80g/L (the pure lincomycin mass/the volume of the polymer reverse phase filler column), eluting with a mobile phase B for 80min, eluting with a mobile phase C for 140min, and collecting the eluent; regenerating with 80% ethanol water solution for 60 min; 658mL of collected eluate was concentrated to 35mL by rotary evaporation at 45 ℃ and 100 mbar;
the flow rate of the whole macroporous resin desalting process is 0.75ml/min, purified water is balanced for 120min, and the concentrated solution obtained in the previous step is adjusted to pH10.5 by NaOH; after filtering through a 0.45um filter membrane, 35mL of lincomycin concentrated solution with the concentration of 95.4g/L is obtained. Pumping into a macroporous resin adsorption column, loading 80mg/mL, washing with a sodium hydroxide solution with pH of 10.5 for 3BV, washing with water for 4BV, eluting with an ethanol aqueous solution of 80% (V/V) for 2BV, and collecting 90mL of an eluent containing lincomycin; adjusting the pH value to 4.5 by using 1mol/L HCl; finally, the lincomycin hydrochloride is prepared into 3.33g of white powder by rotary evaporation at the temperature of 45 ℃ and under the condition of 100 mbar. The purity of the lincomycin hydrochloride product reaches 99.62%, and the total process yield reaches 88.2%.
Example 7
The solution was prepared as follows:
mobile phase A: diluting 34g of phosphoric acid with 900ml of water, adjusting the pH value to 7 by using ammonia water, and then fixing the volume to 1L;
mobile phase B: adding 950mL of mobile phase A into 50mL of ethanol, uniformly mixing to prepare 5% (V/V) phosphate-ethanol solution, and fixing the volume to 1L;
mobile phase C: adding 150mL of ethanol into 750mL of mobile phase A, uniformly mixing to prepare 15% (V/V) phosphate-ethanol solution, and fixing the volume to 1L;
dissolving 3.9g of lincomycin crude product into a crude product solution by using the mobile phase A, metering the volume to 10mL, and filtering by using a 0.45um filter membrane to obtain the filtrate with the concentration of 260g/L and the volume of 15 mL.
LX-161S is used as a polymer reversed-phase filler, the flow rate of the whole chromatographic purification process is 4.7ml/min, and a chromatographic column is firstly balanced by a mobile phase A for 40 min; pumping 15mL of filtered lincomycin crude product solution into the chromatographic column, wherein the loading capacity is 80g/L (the pure lincomycin mass/the volume of the polymer reverse phase filler column), eluting with a mobile phase B for 80min, eluting with a mobile phase C for 160min, and collecting the eluent; regenerating with 80% ethanol water solution for 60 min; collecting 752mL eluate, rotary evaporating at 45 deg.C and 100mbar to concentrate to 35 mL;
the flow rate of the whole macroporous resin desalting process is 0.75ml/min, purified water is balanced for 120min, and the concentrated solution obtained in the previous step is adjusted to pH10.5 by NaOH; after filtering through a 0.45um filter membrane, 35mL of lincomycin concentrated solution with the concentration of 95.4g/L is obtained. Pumping into a macroporous resin adsorption column, loading 80mg/mL, washing with a sodium hydroxide solution with pH of 10.5 for 3BV, washing with water for 4BV, eluting with an ethanol aqueous solution of 80% (V/V) for 2BV, and collecting 90mL of an eluent containing lincomycin; adjusting the pH value to 4.5 by using 1mol/L HCl; finally, the lincomycin hydrochloride is prepared into 3.52g of white powder by rotary evaporation at the temperature of 45 ℃ and under the condition of 100 mbar. The purity of the lincomycin hydrochloride product reaches 99.55%, and the total process yield reaches 90.2%.
Example 8
In the reverse phase chromatography step, a DAC 100-1500 mm dynamic axial compression column (filled with blue Xiao technology reverse phase chromatography filler LX-161S, produced by Xian blue Xiao technology New Material Co., Ltd., degree of crosslinking of 80-99% (W/W), polymer microspheres with an average particle size of 30-50 μm) was used, the amount of the polymer reverse phase chromatography filler was 10kg, 5L of 50% ethanol aqueous solution (V/V) was used for homogenization, and wet packing was performed at a pressure of 5 Mbar; in the step of desalting with macroporous resin, 100X 1000mm glass column (packed with blue-day-old technology macroporous resin LX-20, produced by Xian blue-day-old technology New Material Co., Ltd., polymer pellet diameter)0.1-1.5mm, pore diameter of
Specific surface area of not less than 100m2G, degree of crosslinking 15-80% (W/W)), 7.5L of macroporous resin, 15L of pure water, mixing, and filling under normal pressure.
The solution was prepared as follows:
mobile phase A: diluting 6.8kg of phosphoric acid with 180L of water, adjusting the pH value to 7 with ammonia water, and then fixing the volume to 200L;
mobile phase B: adding 10L of ethanol into 190L of the mobile phase A, uniformly mixing to prepare 5% (V/V) of phosphate-ethanol solution, and fixing the volume to 200L;
mobile phase C: adding 30L of ethanol into 170L of the mobile phase A, uniformly mixing to prepare 15% (V/V) of phosphate-ethanol solution, and fixing the volume to 200L;
374.5g of lincomycin crude product is dissolved into a crude product solution by using the mobile phase A liquid, the volume is determined to be 1.7L, and the solution is filtered by using a 0.45um filter membrane, wherein the filtered concentration is 220g/L, and the volume is 1.7L.
LX-161S is used as a polymer reversed-phase filler, the flow rate of the whole chromatographic purification process is 479mL/min, and a chromatographic column is firstly balanced by a mobile phase A for 40 min; pumping 1.7L of the filtered lincomycin crude product solution into the chromatographic column, wherein the loading capacity is 45g/L (the pure lincomycin mass/the volume of the polymer reverse phase filler column), eluting with a mobile phase B for 80min, eluting with a mobile phase C for 140min, and collecting the eluent; regenerating with 80% ethanol water solution for 60 min; concentrating the collected 67L eluate to 3L at 45 deg.C under 100mbar by rotary evaporation;
the flow rate of the whole macroporous resin desalting process is 125ml/min, purified water is balanced for 120min, and the pH value of the concentrated solution obtained in the previous step is adjusted to 10.5 by NaOH; after being filtered by a 0.45um filter membrane, 3L of lincomycin concentrated solution with the concentration of 65.0g/L is obtained. Pumping into a macroporous resin column with the loading capacity of 80mg/ml, washing with a sodium hydroxide solution with the pH value of 10.5 for 3BV, then washing with water for 4BV, then eluting with an ethanol aqueous solution with the concentration of 80% (V/V) for 2BV, and collecting an eluent containing lincomycin for 15L; adjusting the pH value to 4.5 by using 1mol/L HCl; finally, the lincomycin hydrochloride is prepared into 332g of white powder through rotary evaporation at the temperature of 45 ℃ and under the condition of 100 mbar. The purity of the lincomycin hydrochloride product reaches 99.83 percent, and the total process yield reaches 92.2 percent.
Example 9- -example 14
According to the experimental procedures of examples 1 to 8, LX-161S as a reverse phase packing (particle size of 30 to 50 μm) and LX-20 as a macroporous adsorbent resin were used, and different parameters such as loading amount, elution concentration and the like were adjusted, and the remaining parameters were referred to those of example 8, and the results were as follows (the concentrations of the solutions in the tables were all volume percent concentrations):
example 15- -example 17
According to the experimental procedures of examples 1 to 8, LX-161 as a reverse phase packing (particle size 50 to 75 μm) and LX-20 as a macroporous adsorbent resin were used, and different parameters such as loading, elution concentration and the like were adjusted, and the remaining parameters were as in example 8, and the results were as follows (the concentrations of the solutions in the tables are all volume percent concentrations):
EXAMPLE 18 EXAMPLE 20
According to the experimental procedures of examples 1 to 8, LX-161M as a reverse phase packing (particle size of 75 to 100 μ M) and LX-20 as a macroporous adsorbent resin were used, and different parameters such as loading amount, elution concentration and the like were adjusted, and the remaining parameters were referred to those of example 8, and the results were as follows (the concentrations of the solutions in the tables were all volume percent concentrations):
Claims (14)
1. a method for purifying high-purity lincomycin hydrochloride is characterized by comprising the following steps:
step one, purifying a lincomycin crude product by adopting a reverse phase chromatography method to obtain a lincomycin purified solution;
step two, adopting macroporous resin for desalting to obtain a lincomycin desalting solution;
and step three, obtaining lincomycin hydrochloride through salt conversion, concentration and drying.
2. The method for purifying lincomycin hydrochloride with high purity according to claim 1, wherein the reverse phase chromatography method in the first step comprises the following steps:
(1) column assembling: taking polymer reverse phase filler as stationary phase of chromatographic column, adding 1-3CV 0-100% V/V organic solvent-water solution, homogenizing, wet loading to column height not less than 25cm, and loading pressure 0-10 MPa;
(2) column balancing: equilibrating the chromatographic column with 2-6CV buffered saline solution at a process line flow rate of no more than 10 cm/min;
(3) loading: dissolving the lincomycin crude product in a buffer saline solution to obtain a stock solution with the concentration of 200-300g/L, loading the stock solution to a chromatographic column with the loading capacity of 20-100g/L, wherein the process linear flow rate is not higher than 10 cm/min;
(4) impurity washing: washing impurity of the chromatographic column by using an organic solvent-buffer saline solution with 5-10CV content of 5-15% (V/V), wherein the linear flow velocity in the impurity washing process is not higher than 10 cm/min;
(5) and (3) elution: eluting lincomycin adsorbed on a chromatographic column by using an organic solvent-buffer saline solution with the content of 6-14CV (variable solvent/volume) of 15-40% (V/V), wherein the linear flow rate of an elution process is not higher than 10cm/min, and collecting an eluent component to be a lincomycin purified solution;
(6) regeneration: regenerating the chromatographic column by using an organic solvent-water solution with the content of 6-10CV (80-100 percent (V/V)), wherein the linear flow velocity in the regeneration process is not higher than 10 cm/min; and after the regeneration is finished, the balance step of the purification process in the next period is carried out.
3. The method for purifying lincomycin hydrochloride with high purity according to claim 1,
the macroporous resin desalting method in the second step comprises the following steps:
(A) concentration: concentrating the lincomycin purified solution collected in the first reverse phase chromatography step, reducing the content of the organic solvent to 1-10% (V/V), and adding alkali to adjust the pH to 10-12;
(B) loading: loading the concentrated solution to a macroporous resin column with a loading capacity of 20-150g/L and a linear flow velocity of not higher than 5 cm/min;
(C) washing: respectively washing the macroporous resin column with 2-6BV of aqueous alkali with pH value of 10-12 and pure water, wherein the linear flow rate is not higher than 5 cm/min;
(D) and (3) elution: eluting lincomycin with 2-6BV (V/V) organic solvent-water solution with the content of 50-100% (V/V), wherein the linear flow rate is not higher than 5cm/min, and collecting eluent, namely lincomycin desalting solution;
(E) column balancing: and (3) rebalancing the macroporous resin column by using 2-6BV of pure water, wherein the linear flow velocity is not higher than 5cm/min, and desalting in the next period.
4. The method for purifying lincomycin hydrochloride with high purity according to claim 1,
the step three is a salt transferring process, namely adding the lincomycin desalted solution in the step two into a hydrochloric acid solution with the concentration of 0.1-4mol/L, adjusting the pH value to 3.5-4.5, wherein the reaction temperature is not more than 20 ℃; the concentration process is to concentrate the collected liquid by 1 to 20 times by using a nanofiltration, distillation or reduced pressure distillation mode; the drying process refers to vacuum drying to obtain powdery lincomycin hydrochloride.
5. The method for purifying lincomycin hydrochloride with high purity according to claim 2,
the reverse phase chromatographic packing is polymer microsphere copolymerized with styrene and divinylbenzene, and has crosslinking degree of 80-99% (W/W), average polymer microsphere grain size of 30-150 micron and pore size of
The specific surface area is 800-1500m 2/g.
6. The method for purifying lincomycin hydrochloride with high purity as claimed in claim 2, wherein the lincomycin crude product is lincomycin with a lincomycin content of 90-97% (W/W) after the lincomycin fermentation broth is subjected to centrifugation, extraction, precipitation and crystallization.
7. The method for purifying lincomycin hydrochloride according to claim 2, wherein the buffered saline solution is phosphate buffered saline solution with pH5-9 and concentration of 0.5-5.0% (W/W).
8. The method for purifying lincomycin hydrochloride according to claim 2, wherein the organic solvent is a solvent miscible with water in any ratio, and includes but is not limited to one or more of methanol, ethanol, acetonitrile, isopropanol and acetone.
9. The method for purifying lincomycin hydrochloride with high purity as claimed in claim 3, wherein the concentration process is to concentrate the collected solution 1-20 times by using nanofiltration, distillation or reduced pressure distillation.
10. The method for purifying lincomycin hydrochloride with high purity as claimed in claim 3, wherein the macroporous resin is polymer beads copolymerized from styrene and divinylbenzene, the polymer beads have a particle size of 0.1-1.5mm and a pore diameter of 0.1-1.5mm
The specific surface area is not less than 100m2/g, and the crosslinking degree is 15-80% (W/W).
11. The method for purifying lincomycin hydrochloride according to claim 3, wherein the alkali is organic alkali with alkalinity, inorganic alkali and organic and inorganic salt which is alkaline in aqueous solution, and the alkali includes but is not limited to one or a mixture of several of methylamine, pyridine, ethylenediamine, triethylamine, tetramethylammonium, tetraethylammonium, sodium hydroxide, potassium hydroxide, sodium phosphate, ammonium phosphate, sodium acetate and ammonium acetate.
12. The method according to claim 3, wherein the organic solvent is a solvent that can be dissolved with water at any ratio, and includes but is not limited to one or more of methanol, ethanol, acetonitrile, isopropanol, and acetone.
13. The method as claimed in claim 5, wherein the reverse phase chromatography packing comprises, but is not limited to, model LX-161 series reverse phase polymer chromatography packing manufactured by seian blue dawn new materials ltd.
14. The method according to claim 10, wherein the macroporous resin comprises, but is not limited to, LX-20 type macroporous adsorbent resin manufactured by seian blue advanced technology materials ltd.
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