WO2022042299A1 - 一种莫匹罗星的提取方法 - Google Patents

一种莫匹罗星的提取方法 Download PDF

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Publication number
WO2022042299A1
WO2022042299A1 PCT/CN2021/111926 CN2021111926W WO2022042299A1 WO 2022042299 A1 WO2022042299 A1 WO 2022042299A1 CN 2021111926 W CN2021111926 W CN 2021111926W WO 2022042299 A1 WO2022042299 A1 WO 2022042299A1
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Prior art keywords
extraction
resin
mupirocin
ethyl acetate
analysis
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PCT/CN2021/111926
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English (en)
French (fr)
Inventor
程赵兵
寇广会
许一鸣
章凯
张宇辰
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杭州中美华东制药有限公司
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Application filed by 杭州中美华东制药有限公司 filed Critical 杭州中美华东制药有限公司
Priority to EP21860138.3A priority Critical patent/EP4190780A4/en
Priority to US18/022,806 priority patent/US20230312540A1/en
Priority to IL300723A priority patent/IL300723A/en
Priority to CN202180049354.3A priority patent/CN116018342A/zh
Publication of WO2022042299A1 publication Critical patent/WO2022042299A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D407/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00
    • C07D407/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings
    • C07D407/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/351—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom not condensed with another ring
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D11/00—Solvent extraction
    • B01D11/04—Solvent extraction of solutions which are liquid
    • B01D11/0492—Applications, solvents used
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography

Definitions

  • the invention belongs to the technical field of medicine, and further belongs to the technical field of fermentation, in particular to a method for extracting mupirocin fermentation liquid.
  • Mupirocin Pseudomonas acid A, is an aminoacyl transaminase inhibitor antibiotic that can specifically bind to the isoleucine transfer RNA synthetase of bacteria, thereby inhibiting isoleucine-containing bacteria in bacteria protein synthesis. It can fight against various gram-positive bacteria, including Staphylococcus aureus, Streptococcus pyogenes, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, etc.; it can also fight against some Gram-negative bacteria, including Haemophilus influenzae Bacillus etc. Mupirocin has no cross-resistance with other antibiotics, and has high medical value.
  • Patent CN109053707A relates to a method for purifying mupirocin, which extracts mupirocin by means of filter membrane filtration of fermented liquid and concentration of the filter membrane. Existing temperature will have an impact on the purity of mupirocin, therefore, this method requires strict condition control and is not suitable for industrial production.
  • Patent US7619102 relates to a method for precipitating mupirocin from its aqueous solution and recovering the precipitate. This method has a low product yield and a high requirement for removing water after precipitation.
  • Patent US4222942 relates to a method for extracting mupirocin extract using a polar water-immiscible organic solvent similar to the present invention, but due to the low adaptability of the solvent and diluent selected by the method, its yield is not high. The rate is low and it is not suitable for mass production.
  • the present invention utilizes the structure and fermentation process characteristics of mupirocin, adopts resin extraction, analytical concentration, extraction, dehydration and decolorization and other steps to combine and optimize to complete the efficient purification of mupirocin and provide a high extraction rate.
  • High safety suitable for industrialization and low-cost extraction method, providing certain technical support for the improvement of mupirocin production technology and the improvement of industrial level.
  • the invention provides a mupirocin extraction method suitable for industrialized large-scale production. Specifically, a desorption solution is obtained by analysing mupirocin after adsorbing the resin, and the desorption solution is concentrated and further subjected to extraction, dehydration and decolorization treatment.
  • the present application makes mupirocin an ester-soluble free carboxylic acid and a water-soluble alkali respectively by adjusting the pH value, thereby realizing the maximization of the transfer of active ingredients in the organic phase.
  • the impurities which are quite different from the mupirocin properties are further removed respectively.
  • the method of the present application also controls the content of impurities well, improves the purification process and ensures the quality of the finished product.
  • the mupirocin involved in the present invention may be synthesized by fermentation, or may be naturally occurring mupirocin.
  • the mupirocin liquid involved in the present invention is obtained by conventional fermentation technology, and can be a mupirocin fermentation liquid, an aqueous solution of mupirocin, or a mupirocin filtrate obtained by filtration, or a mupirocin filtrate obtained by filtration. It can be the mupirocin supernatant obtained by centrifugation.
  • the invention uses macroporous resin whose pore size matches the molecular weight of mupirocin, which can ensure the efficiency of adsorption; and the resin can not only be used in large quantities, but also can be recycled, which is conducive to the large-scale production of the entire extraction process and saves costs .
  • the present invention uses acetone or ethyl acetate for analysis, and the choice of the analysis solvent also brings unexpected technical effects to the method of the present invention.
  • acetone when acetone is used for analysis, acetone is not only used for analysis, but also helps to activate and regenerate the macroporous resin in the analysis column;
  • ethyl acetate when ethyl acetate is used for analysis, ethyl acetate is not only a solvent for analysis, but also preferably used in the extraction step, and then
  • the introduction of other reagents in the extraction method is limited, and the use of the same reagent in multiple steps minimizes the possibility of introducing impurities.
  • the recovery rate of mupirocin extracted by the method provided by the invention can reach 90%, and the purity can reach more than 80%.
  • the present invention intends to provide a method for extracting mupirocin, comprising an extraction step, and the extraction is a stepwise extraction, including at least one extraction of an ester solvent and at least one extraction of an ester solvent. Alkaline water extraction.
  • the present invention specifically provides a mupirocin extraction method as shown below, which comprises the following steps:
  • a. resin extraction the resin is put into mupirocin fermentation broth for stirring and adsorption, and the resin is collected after filtering the fermentation broth and leaching;
  • step b Analysis and concentration: the resin collected in step a is loaded into the analysis column, soaked in ethyl acetate or acetone for analysis, and the analysis solution is collected and concentrated;
  • step b extracting the analytical concentrate collected in step b, by adjusting the pH value, it becomes an ester-soluble free carboxylic acid and/or a water-soluble base, and the solvent phase is separated;
  • step c Dehydration and decolorization: dehydration and/or decolorization of the solvent phase collected in step c are performed in turn.
  • step c ester solvent and/or alkaline water are used to extract the analytical concentrate collected in step b; further preferably, in the extraction, solvent components are independently selected as needed. step by step.
  • the amount of the dehydrating agent is 0.02-0.08 kg per liter of solvent phase; the amount of the decolorizing agent is 1-6 grams per gram of the dehydrated solvent phase.
  • the mupirocin involved in the present invention may be synthesized by fermentation, or may be naturally occurring mupirocin.
  • the mupirocin liquid involved in the present invention is obtained by conventional fermentation technology, and can be a mupirocin fermentation liquid, an aqueous solution of mupirocin, or a mupirocin filtrate obtained by filtration, or a mupirocin filtrate obtained by filtration. It can be the mupirocin supernatant obtained by centrifugation.
  • mupirocin is directly extracted to obtain mupirocin, and mupirocin can also be re-made into mupirocin calcium if necessary; It ensures the flexibility of the final product and can better adapt to the needs of the end market.
  • the resin in step a is a non-polar macroporous adsorption resin, selected from AB-8 resin, H103 resin, X-5 resin, H107 resin or D3520 resin, preferably H103 resin.
  • the pore size of the resin in step a matches the molecular weight of mupirocin to ensure the degree of adsorption.
  • the feeding amount of resin in step a is 0.01-0.05 g/g of fermentation broth, preferably 0.02-0.04 g/g of fermentation broth.
  • the pH of the resin extraction in steps a and b is 5.0-7.0, preferably 5.5-6.5.
  • the amount of acetone used in the analysis in step b is 2 to 5 times the volume of the resin; the acetone is not only used for analysis, but also helps to activate and regenerate the resin in the analysis column.
  • the amount of ethyl acetate used in the analysis is 2 to 5 times the volume of the resin; the ethyl acetate is not only used for analysis, but also preferably used in the extraction step, which further limits the The introduction of other reagents in the extraction method, using the same reagent in multiple steps, minimizes the possibility of introducing impurities.
  • the water phase should be retained to enter step c; the retained water phase refers to the water phase left in the resin extraction process, and/or the acetone itself. Watery.
  • the analytical flow rate is 1/5-6/5 resin volume/hour, preferably 3/5-6/5 resin volume per hour.
  • the extraction described in step c is a step-by-step extraction, including at least one extraction with an ester solvent and at least one extraction with an alkaline water.
  • the extraction steps of step c are the first ester solvent extraction, alkaline water extraction and the second ester solvent extraction.
  • the alkaline water is selected from sodium bicarbonate, ammonium bicarbonate or sodium hydroxide solution.
  • the pH of the alkaline water is 7.0-10.0, preferably 7.5-9.0.
  • the ester solvent in the extraction method is selected from diisooctyl phosphate, tributyl phosphate, ethyl acetate, isopropyl acetate or butyl acetate, preferably ethyl acetate.
  • the pH of the ester solvent in the extraction method is 3.0-5.0, preferably 3.0-4.5.
  • the first ester solvent extraction can be repeated once or twice, and the ester solvent phases separated from each extraction are combined.
  • the alkaline water extraction can be carried out one or more times, and the alkaline water phases separated from each extraction are combined for the second ester solvent extraction for later use.
  • the second ester solvent extraction can be repeated once or twice, and the ester solvent phases separated by each extraction are combined.
  • the number of alkaline water extractions is directly related to the initial titer of mupirocin fermentation broth; for fermentation broths with a titer greater than 3000ug/ml, alkaline water extraction usually needs to be performed at least twice; For fermentation broth with titer greater than 6000ug/ml, alkaline water extraction usually needs to be carried out at least 3 times.
  • the dehydrating agent is selected from anhydrous sodium sulfate, anhydrous magnesium sulfate or anhydrous calcium sulfate, preferably anhydrous sodium sulfate.
  • the decolorizing agent is selected from activated carbon or activated clay, preferably 8815 activated carbon.
  • the invention also optimizes the process conditions in each step.
  • the pH of the resin extraction is 4.0-7.0, preferably 5.5-6.5.
  • the ester solvent can better remove the mopi from the analytical concentrate. Luoxing is extracted; the pH of the ester solvent in the extraction method is 3.0-5.0, preferably 3.0-4.5, and the alkaline water can better extract mupirocin from the ester extract, so that the whole set of extraction process steps They fit together to form an organic whole.
  • the yield of mupirocin extracted by the method provided by the present invention can reach more than 80%; as a specific embodiment, the mupirocin extracted by the method provided by the present invention has The yield can reach 85%, or 90%, or even 95%.
  • the present invention uses the method for organic solvent extraction to replace the commonly used precipitation recovery method, and the extraction of the present invention is a stepwise extraction, including at least one ester solvent extraction and at least one alkaline water extraction, by designing ester extraction and water extraction.
  • the conditions and times of extraction, as well as the method of multiple sub-extraction and reasonable combination of components in each extraction, fully transfer the fermentation titer of mupirocin, especially suitable for the fermentation of mupirocin titer greater than 5000ug/ml
  • the extraction of liquid ensures the yield in large-scale process production.
  • the present invention selects ethyl acetate or acetone for analysis.
  • the choice of ethyl acetate is adapted to the organic solvent of the extraction step, and the possibility of introducing impurities is minimized; the choice of acetone is helpful for the activation and regeneration of macroporous resin; the choice of these two analytical reagents reduces the banding. It can reduce the risk of entering impurities, or reduce the cost of the process, which can kill two birds with one stone.
  • the extraction method of the present invention can handle kilogram-level mupirocin fermentation broth and maintain a stable 80% yield of the process, up to 95%, and keep mupirocin in the extraction process.
  • the purity of the star is above 80%, which has made a good foundation for the subsequent extraordinar craftsmanship.
  • the method of the invention is fully adapted to the scale of industrial production, and ensures that the yield and product purity are maximized under the condition that the quality meets the national examination and approval standards.
  • Embodiment 1 The extraction method of mupirocin
  • the mupirocin fermentation broth was poured into the pretreatment tank and the volume was measured, the stirring was turned on, and the pH was adjusted to 6.0 with 2N hydrochloric acid or 2N sodium hydroxide.
  • the H103 resin was put into the pretreatment tank at a dosage of 0.04 g mupirocin/g resin.
  • the adsorption was stirred at room temperature for 4.5 hours. After the adsorption is over, stop stirring.
  • the fermentation broth was filtered with a 40-mesh vibrating sieve, and the surface of the resin was rinsed with drinking water to remove the adhering bacterial residue, and the resin was collected with a yield of 95.5%.
  • the resin was evenly loaded into the analytical column, and backwashed with drinking water more than twice the volume of the resin until the color became lighter. Use air to press out the drinking water in the column, and use acetone in reverse to completely soak the resin in acetone to stop. Start timing when the resin is completely soaked in acetone. After soaking for 2 hours, analyze at a flow rate of 3/5 resin volume/hour, and the amount of acetone for analysis is about 3 times the resin volume. The analytical solution was collected and washed with drinking water until there was no acetone odor. The content of mupirocin in the analytical solution was 78.5%. The analytical solution was pumped into the acetone concentration tank, the inner temperature was below 50.0 °C, and concentrated to no acetone, leaving the water phase.
  • the first ester solvent extraction pump the water phase into the extraction tank, adjust the pH to 4.5 with 2N hydrochloric acid, add 1/2 volume of ethyl acetate of the water phase, stir at room temperature for 45 minutes, and after 30 minutes of rest, The ethyl acetate phase was separated. Repeat the above operation once. The ethyl acetate phases from the two extractions were combined.
  • the first alkaline water extraction the combined ethyl acetate phase was pumped into the extraction tank, 1/3 of the volume of the ethyl acetate phase was added with 2.5% ammonium bicarbonate solution, stirred at room temperature for 30 minutes, and after standing still for 30 minutes, The alkaline aqueous phase is separated. Repeat the above operation three times. The alkaline aqueous phases from the four extractions were combined. The ethyl acetate phase was subjected to solvent recovery.
  • the second ester solvent extraction the combined alkaline aqueous phase was pumped into the extraction tank, 2N hydrochloric acid was added dropwise to adjust the pH to 4.0, 1/2 volume of the aqueous phase was added with ethyl acetate, stirred at room temperature for 45 minutes, and stood still for 30 minutes After that, the ethyl acetate phase was separated. Repeat the above operation once. The ethyl acetate phases from the two extractions were combined. The yield of mupirocin after extraction was 85.3%.
  • Embodiment 2 The extraction method of mupirocin
  • the fermentation broth was poured into the pretreatment tank and the volume was measured, the stirring was turned on, and the pH was adjusted to 6.0 with 2N hydrochloric acid or 2N sodium hydroxide.
  • the H103 resin was put into the pretreatment tank at a dosage of 0.03 g mupirocin/g resin.
  • the adsorption was stirred at room temperature for 4 hours. After the adsorption is over, stop stirring.
  • the fermentation broth was filtered with a 40-mesh vibrating sieve, and the surface of the resin was rinsed with drinking water to remove the adhering bacterial residue, and the resin was collected with a yield of 94.7%.
  • the resin was evenly loaded into the analytical column, and backwashed with drinking water more than twice the volume of the resin until the color became lighter. Use air to press out the drinking water in the column, and use acetone in reverse to completely soak the resin in acetone to stop. Start timing when the resin is completely soaked in acetone. After soaking for 2 hours, the analysis is carried out at a flow rate of 7/10 resin volume/hour, and the amount of acetone for analysis is about 3.5 times the resin volume.
  • the first ester solvent extraction pump the water phase into the extraction tank, adjust the pH to 3.5 with 2N hydrochloric acid, add 1/2 volume of ethyl acetate of the water phase, stir at room temperature for 45 minutes, and after standing for 30 minutes, The ethyl acetate phase was separated. Repeat the above operation once. The ethyl acetate phases from the two extractions were combined.
  • the first alkaline water extraction the combined ethyl acetate phase was pumped into the extraction tank, 2% ammonium bicarbonate solution of 1/3 the volume of the ethyl acetate phase was added, stirred at room temperature for 30 minutes, and after standing still for 30 minutes, The alkaline aqueous phase is separated. Repeat the above operation three times. The alkaline aqueous phases from the four extractions were combined. The ethyl acetate phase was subjected to solvent recovery.
  • the second ester solvent extraction the combined alkaline aqueous phase was pumped into the extraction tank, 2N hydrochloric acid was added dropwise to adjust the pH to 4.0, 1/2 volume of the aqueous phase was added with ethyl acetate, stirred at room temperature for 45 minutes, and stood still for 30 minutes After that, the ethyl acetate phase was separated. Repeat the above operation once. The ethyl acetate phases from the two extractions were combined. The yield of mupirocin after extraction was 87.3%, and its content was 82.5%.
  • Resin extraction The fermentation broth is poured into the pretreatment tank and the volume is measured, the stirring is turned on, and the pH is adjusted to 6.0 with hydrochloric acid or sodium hydroxide. After half an hour of pretreatment, the H103 resin was put into the pretreatment tank at a dosage of 0.02 g mupirocin/g resin. The adsorption was stirred at room temperature for 4 hours. After the adsorption is over, stop stirring. The fermentation broth was filtered with a 40-mesh vibrating sieve, and the surface of the resin was rinsed with drinking water to remove the adhering bacterial residue, and the resin was collected with a yield of 94.5%.
  • Mupirocin extraction process In the analysis process, the mupirocin in the analysis column is eluted from the resin to acetone or ethyl acetate.
  • space velocity volume flow rate/resin volume
  • resin column bed geometry height/diameter ratio
  • column bed structure working procedures, etc., among which space velocity is often the main factor.
  • Analytical concentration The resin is evenly loaded into the analytical column, and backwashed with drinking water more than 2 times the volume of the resin until the color becomes lighter.
  • the drinking water in the column was squeezed out with air, and the resin was completely soaked with ethyl acetate in the reverse direction with ethyl acetate. Start timing when the resin is completely soaked in ethyl acetate. After soaking for 2 hours, the analysis is carried out at a flow rate of 7/10 resin volume/hour for 5 hours, and the amount of ethyl acetate for analysis is about 4 times the resin volume.
  • the analytical solution was collected and washed with drinking water.
  • the content of mupirocin in the analytical solution was 86.9%. Other steps are the same as in Example 1, the total yield of mupirocin is 86.0%, and the purity of mupirocin is 80.5%.
  • Analytical concentration The resin is evenly loaded into the analytical column, and backwashed with drinking water more than 2 times the volume of the resin until the color becomes lighter. Use air to press out the drinking water in the column, and use acetone in reverse to completely soak the resin in acetone to stop. Start timing when the resin is completely soaked in acetone. After soaking for 2 hours, the analysis is carried out at a flow rate of 1/5 resin volume/hour for 10 hours, and the amount of acetone for analysis is about 4 times the resin volume. The analysis solution was collected and washed with drinking water until there was no acetone smell. The content of mupirocin in the analysis solution was 88.5%.
  • the first ester solvent extraction the aqueous phase was pumped into the extraction tank, the pH was adjusted to 5.0 with hydrochloric acid, 1/2 volume of ethyl acetate was added to the aqueous phase, and stirred at room temperature for 45 minutes. After standing for 30 minutes, the ethyl acetate was separated. ester phase. Repeat the above operation once. The ethyl acetate phases from the two extractions were combined.
  • the first alkaline water extraction the combined ethyl acetate phase was pumped into the extraction tank, 2% ammonium bicarbonate solution of 1/3 the volume of the ethyl acetate phase was added, stirred at room temperature for 30 minutes, and after standing still for 30 minutes, The alkaline aqueous phase is separated. Repeat the above operation four times. The alkaline aqueous phases from the four extractions were combined. The ethyl acetate phase was subjected to solvent recovery.
  • the second ester solvent extraction the combined alkaline aqueous phase was pumped into the extraction tank, hydrochloric acid was added dropwise to adjust the pH to 4.0, 1/2 volume of the aqueous phase was added with ethyl acetate, stirred at room temperature for 45 minutes, and after resting for 30 minutes , and the ethyl acetate phase was separated. Repeat the above operation once. The ethyl acetate phases from the two extractions were combined. The relative yield of mupirocin after extraction was 95.5%.
  • Example 7 The necessity of step-by-step extraction in mupirocin extraction
  • the ester solvent used to extract more than once: the water phase after analysis and concentration is drawn into the extraction tank, the pH is adjusted to 3.5 with hydrochloric acid, an appropriate amount of ethyl acetate is added, stirred and allowed to stand, and the ethyl acetate is separated. Mutually. The above operation was repeated twice, and the three separated ethyl acetate phases were combined. The yield of mupirocin after two extractions with ester solvent was 82.0,% and the purity was 78.5%.
  • Example 9 Reproducing the method for extracting Pseudomonas acid A described in CN101591333B (Example 1, the method described in paragraphs [0041] to [0044] of the specification, the reported Pseudomonas acid A in the analytical solution The content reaches 70%, and the recovery of pseudomonas acid A reaches 90%)
  • Mupirocin fermentation broth (Pseudomonas acid A concentration of 6017ug/ml) 25L, adjusted to neutral pH with 1M NaOH, centrifuged at 4500rpm and 25°C for 20 minutes in a high-speed low-temperature centrifuge to obtain clear liquid, and the residue was washed with water discard.
  • the supernatant was adjusted to pH 4.0 with 1M HCl and adsorbed on a H103 resin (prepared, 2.2L) column.
  • the H103 pretreatment method is processed according to the instructions provided by the manufacturer. After completion, wash the column with deionized water until the effluent is nearly colorless.
  • the solvent 8.3L (3320ml of ethanol, 410g of ammonium chloride, the rest are water in this 8.3L solvent) of ethanol+salt+water composition is desorbed from the resin and the desorbed solution containing pseudomonas acid A, used ethanol+salt
  • the ratio of solvent to Pseudomonas acid A for the +water combination was 3.0 ml: 22 mg.
  • the desorption index recorded in Example 1 of CN101591333B is: the content of Pseudomonas acid A in the collected desorption solution reaches 70%, and the recovered Pseudomonas acid A reaches 90%; rates are quite different.

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Abstract

一种莫匹罗星的提取方法，主要步骤包括：树脂吸附、解析浓缩、多步萃取和脱水脱色。其中多步萃取包括至少一次酯类溶剂提取和至少一次碱水提取。上述方法适于工业化，提取莫匹罗星的回收率为80％以上、纯度为80％以上。

Description

一种莫匹罗星的提取方法 技术领域
本发明属于医药技术领域，更进一步的属于发酵技术领域，具体涉及一种莫匹罗星发酵液的提取方法。
背景技术
莫匹罗星，即假单胞菌酸A，是一种氨基酰转氨酶抑制剂类抗生素，能特异性地结合到细菌的异亮氨酸转移RNA合成酶，从而抑制细菌中含异亮氨酸蛋白质的合成。其能够对抗各种革兰氏阳性菌，包括金黄色葡萄球菌、酿胺链球菌、表皮葡萄球菌、肺炎链球菌、化脓性链球菌等；也能够对抗一些革兰氏阴性菌，包括流感嗜血杆菌等。莫匹罗星与其他抗生素无交叉耐药性，具有较高的医疗价值，早已被临床应用，其商品名为百多邦或莫匹罗星软膏。
专利CN109053707A涉及了一种莫匹罗星的提纯方法，其采用发酵液滤膜过滤、滤膜浓缩的方式提取莫匹罗星，滤膜在使用的同时通常伴随温度升高，由于热敏杂质的存在，温度对莫匹罗星纯度会产生影响，因此，此方法条件控制要求严格，不适用于工业化生产。专利US7619102涉及了一种通过使莫匹罗星从其水溶液中沉淀并回收沉淀的方法，此方法产物收率较低且对沉淀后去除水分的要求较高。专利US4222942涉及了一种与本发明相近似的采用极性水不相溶的有机溶剂萃取莫匹罗星提取液的方法，然而由于其方法选择的溶剂和稀释剂适配性不高导致其收率较低且不适合放量生产。
也就是说，上述方法因工艺控制条件苛刻、环境需求及商业或生态角度上都存在难以令人满意方面。因此，仍然需要适用于工业化大规模生产且环境友好、成本可控的的提纯莫匹罗星的新方法。
在此背景下，本发明利用莫匹罗星的结构和发酵工艺特点，采用树脂提取、解析浓缩、萃取、脱水脱色等步骤组合优化，完成莫匹罗星的高效纯化，提供一种高提取率、高安全性、适于工业化且低成本的提取方法，为企业莫匹罗星生产技术的改进，产业水平的提高提供一定的技术支持。
发明内容
本发明提供一种适用于工业化大规模生产的莫匹罗星提取方法，具体通过树脂吸附莫匹罗星后进行解析获得解析液，解析液进行浓缩后进一步进行萃取、脱水脱色处理。
为克服已有工艺中的缺陷，本申请通过调节pH值使莫匹罗星分别成为酯溶性的游离羧酸和水溶性的碱，从而实现活性成分在有机相中转移的最大化，在上述两步萃取步骤中进一步分别除去与莫匹罗星性质相差较大的杂质。通过上述步骤，在保证收率的基础上，本申请的方法也很好地控制了杂质的含量，改进了纯化工艺且保证了成品的质量。
本发明涉及的莫匹罗星可以是发酵合成的，也可以是天然存在的莫匹罗星。本发明涉及的莫匹罗星液为通过常规发酵技术获取的，可以是莫匹罗星发酵液，也可以是莫匹罗星的水溶液，也可以是经过过滤得到的莫匹罗星滤液，也可以是经过离心得到的莫匹罗星上清液。
本发明使用大孔树脂，其孔径与莫匹罗星分子量相匹配，能够保证吸附的效率；且树脂不仅可以大批量使用，还可以回收利用，有助于整个提取工艺的大规模生产并节约成本。
本发明使用丙酮或乙酸乙酯解析，解析溶剂的选择也为本发明的方法带来了预料不到的技术效果。其中，使用丙酮解析时，丙酮既作解析用，也有助于解析柱中大孔树脂的活化与再生；使用乙酸乙酯解析时，乙酸乙酯既是解析溶剂，也在萃取步骤中优选使用，进而限制了提取方法中其他试剂的引入，在多个步骤中使用同一种试剂，使引入杂质的可能性最小化。
采用本发明提供的方法提取的莫匹罗星的回收率可达90％，纯度达到80％以上。
利用上述莫匹罗星的形式，本发明意在提供一种莫匹罗星的提取方法，其包括萃取步骤，所述的萃取为分步萃取，包括至少一次的酯类溶剂提取与至少一次的碱水提取。
本发明具体提供一种如下所示的莫匹罗星提取方法，其包括以下步骤：
a.树脂提取：将树脂投入莫匹罗星发酵液进行搅拌吸附，过滤发酵液并淋洗后收集所述树脂；
b.解析浓缩：将步骤a中收集到的树脂装入解析柱，使用乙酸乙酯或丙酮浸泡后解析，收集并浓缩解析液；
c.萃取：对步骤b中收集到的解析浓缩液进行萃取，通过调节pH值，使其变成酯溶性的游离羧酸和/或水溶性的碱，分出溶剂相；
d.脱水脱色：将步骤c中收集到的溶剂相依次进行脱水和/或脱色。
作为一种具体的实施方式，所述步骤c中，采用酯类溶剂和/或碱水对步骤b中收集到的解析浓缩液进行萃取；进一步优选地，所述的萃取按需独立选取溶剂分步进行。
作为一种具体的实施方式，所述步骤d中，所述脱水剂的用量为0.02～0.08千克每升溶剂相；所述脱色剂的用量为1～6克每克脱水后的溶剂相。
本发明涉及的莫匹罗星可以是发酵合成的，也可以是天然存在的莫匹罗星。
本发明涉及的莫匹罗星液为通过常规发酵技术获取的，可以是莫匹罗星发酵液，也可以是莫匹罗星的水溶液，也可以是经过过滤得到的莫匹罗星滤液，也可以是经过离心得到的莫匹罗星上清液。
本发明直接提取得到莫匹罗星，后续有需要也可以将莫匹罗星再制成莫匹罗星钙；不同于制备得到莫匹罗星钙后难以再转化成莫匹罗星，本发明保证了终产物的灵活度，得以更好的适应终端市场的需求。
作为一种具体的实施方式，步骤a中的树脂为非极性大孔吸附树脂，选自AB-8树脂、H103树脂、X-5树脂、H107树脂或D3520树脂，优选H103树脂。
作为一种具体的实施方式，步骤a中的树脂孔径与莫匹罗星的分子量相匹配，以保证吸附的程度。
作为一种具体的实施方式，步骤a中树脂的投料量为0.01～0.05克/每克发酵液，优选0.02～0.04克/每克发酵液。
作为一种具体的实施方式，步骤a与b中树脂提取的pH为5.0～7.0，优选5.5～6.5。
作为一种具体的实施方式，步骤b中解析丙酮的用量为树脂体积的2～5倍；其中所述的丙酮，既作解析用，也有助于解析柱中树脂的活化与再生。
作为一种具体的实施方式，步骤b中解析乙酸乙酯的用量为树脂体积的2～5倍；其中所述的乙酸乙酯，既作解析用，也在萃取步骤中优选使用，进而限制了提取方法中其他试剂的引入，在多个步骤中使用同一种试剂，使引入杂质的可能性最小化。
作为一种具体的实施方式，步骤b中收集并浓缩解析液后，应留水相进入步骤c；所述的留水相是指树脂提取过程中留有的水相，和/或丙酮自身的含水。
作为一种具体的实施方式，步骤b中解析流速为1/5-6/5树脂体积/小时，优选3/5-6/5树脂体积每小时。
作为一种具体的实施方式，步骤c所述的萃取为分步萃取，包括至少一次酯类溶剂提取和至少一次碱水提取。
作为一种具体的实施方式，步骤c的萃取步骤为第一次酯类溶剂提、碱水提取和第二次酯类溶剂提取。
作为一种具体的实施方式，步骤c萃取的碱水提取步骤中，所述的碱水选自碳酸氢钠、碳酸氢铵或氢氧化钠溶液。
作为一种具体的实施方式，步骤c萃取的碱水提取步骤中，所述碱水的pH为7.0～10.0， 优选7.5～9.0。
作为一种具体的实施方式，提取方法中的酯类溶剂选自磷酸二异辛酯、磷酸三丁酯、乙酸乙酯、乙酸异丙酯或乙酸丁酯，优选乙酸乙酯。
作为一种具体的实施方式，提取方法中的酯类溶剂的pH为3.0～5.0，优选3.0～4.5。
作为一种具体的实施方式，所述的第一次酯类溶剂提取可重复进行一次或两次，并将各次萃取分出的酯类溶剂相合并。
作为一种具体的实施方式，所述的碱水提取可进行一次或两次以上，并将各次萃取分出的碱水相合并，做第二次酯类溶剂提取备用。
作为一种具体的实施方式，所述的第二次酯类溶剂提取可重复进行一次或两次，并将各次萃取分出的酯类溶剂相合并。
本发明所述的分步萃取，其中碱水萃取次数与莫匹罗星发酵液起始效价直接相关；对于效价大于3000ug/ml的发酵液，碱水提取通常需进行至少2次；对于效价大于6000ug/ml的发酵液，碱水提取通常需进行至少3次。
作为一种具体的实施方式，步骤d中脱水剂选自无水硫酸钠、无水硫酸镁或无水硫酸钙，优选无水硫酸钠。
作为一种具体的实施方式，步骤d中脱色剂选自活性炭或活性白土，优选8815活性碳。
本发明同时对各个步骤中的工艺条件进行了优化，如树脂提取的pH为4.0～7.0，优选5.5～6.5，在该pH范围内，酯类溶剂可以更好地从解析浓缩液中将莫匹罗星萃取出来；提取方法中的酯类溶剂的pH为3.0～5.0，优选3.0～4.5，碱水可以更好地从酯提液中将莫匹罗星提取出来，使整套提取工艺各步骤之间相互契合，成为一个有机的整体。
经过反复的研究及实验验证，使用本发明提供的方法提取的莫匹罗星的收率能达到80％以上；作为一种具体的实施方式，使用本发明提供的方法提取的莫匹罗星的收率可达85％，或90％，甚至能够达到95％。
本发明的有益效果为：
1.本发明使用有机溶剂萃取的方法代替了常用的沉淀回收法，本发明的萃取为分步萃取，包括了至少一次的酯类溶剂提取和至少一次的碱水提取，通过设计酯提和水提的条件和次数，以及设计了每次提取中多次子提取并合理合并组分的方法，充分将莫匹罗星的发酵效价转移，尤其适用于莫匹罗星效价大于5000ug/ml的发酵液的提取，保证了大规模流程化生产中的收率。
2.本发明选择了乙酸乙酯或丙酮作解析。乙酸乙酯的选择适配了萃取步骤的有机溶剂， 将引入杂质的可能性最小化；丙酮的选择则有助于大孔树脂的活化和再生；这两种解析试剂的择一选择减少了带入杂质的风险，抑或减少了工序成本，实现了一举两得。
3.本发明的提取方法在工业化生产中，可以处理公斤级的莫匹罗星发酵液并保持工艺稳定的80％的收率，最高可达95％，此外在提取过程中一直保持莫匹罗星的纯度在80％以上，为后续的精致工艺做了良好的铺垫。本发明的方法充分适配工业化生产的规模，保证了质量符合国家审批标准的条件下，将收率和产物纯度最大化。
具体实施方式
下面结合具体实施例对本发明作进一步的详细说明。以下实施例用于理解本发明的方法和核心思想，对于本领域的技术人员来说，在不脱离本发明构思的前提下，进行任何可能的变化或替换，均属于本发明的保护范围。本发明实施例中未注明具体条件的实验方法，通常为常规条件，或按照原料或商品制造厂商所建议的条件；未注明来源的试剂，通常为通过商业途径可购得的常规试剂。
实施例1 莫匹罗星的提取方法
a.树脂提取：
将莫匹罗星发酵液打入预处理罐并计量体积，打开搅拌，用2N盐酸或2N氢氧化钠调节pH至6.0。
预处理半小时后以0.04克莫匹罗星/每克树脂的投料量，将H103树脂投入预处理罐。常温条件下搅拌吸附4.5小时。吸附结束后，停搅拌。用40目振动筛过滤发酵液并用生活饮用水淋洗树脂表面除去黏附的菌渣，收集树脂，收率为95.5％。
b.解析浓缩：
将树脂均匀装入解析柱，用2倍以上树脂体积的生活饮用水反冲清洗至颜色变淡时止。用空气压出柱内饮用水，用丙酮反向走到丙酮完全浸泡树脂停止。以丙酮完全浸泡树脂开始计时，浸泡2小时后，以3/5树脂体积/小时流速进行解析，解析丙酮用量约3倍树脂体积。收集解析液，再用生活饮用水洗到无丙酮气味，解析液中莫匹罗星的含量为78.5％。将解析液抽入丙酮浓缩罐，内温50.0℃以下，浓缩至无丙酮，留水相。
c.萃取：
第一次酯类溶剂提取：将水相抽入萃取罐，用2N的盐酸调节pH到4.5后，加入1/2水相体积的乙酸乙酯，常温条件下搅拌45分钟，静止30分钟后，分出乙酸乙酯相。重复以上操作一次。将两次萃取分出的乙酸乙酯相合并。
第一次碱水提取：将合并的乙酸乙酯相抽入萃取罐中，加入1/3乙酸乙酯相的体积的2.5％碳酸氢铵溶液，常温条件下搅拌30分钟，静止30分钟后，分出碱水相。重复以上操作三次。将四次萃取分出的碱水相合并。乙酸乙酯相进行溶媒回收。
第二次酯类溶剂提取：将合并的碱水相抽入萃取罐，滴加2N盐酸调节pH至4.0，加入1/2水相体积的乙酸乙酯，常温条件下搅拌45分钟，静止30分钟后，分出乙酸乙酯相。重复以上操作一次。将两次萃取分出的乙酸乙酯相合并。萃取后莫匹罗星的收率为85.3％。
d.脱水脱色：
在合并的乙酸乙酯相中，每50L乙酸乙酯相中加入2.3kg无水硫酸钠，搅拌脱水30分钟后分出乙酸乙酯相。脱水后用体积为1.5倍无水硫酸钠投入重量的新鲜乙酸乙酯清洗脱水后的无水硫酸钠。将脱水后的乙酸乙酯相按3克/克8815活性炭的比例脱色，脱色后用体积为2倍活性炭投入重量的新鲜乙酸乙酯冲洗活性炭，最终得到收率为83.3％，含量为80.2％的莫匹罗星。
实施例2 莫匹罗星的提取方法
a.树脂提取：
发酵液打入预处理罐并计量体积，打开搅拌，用2N盐酸或2N氢氧化钠调节pH至6.0。
预处理半小时后以0.03克莫匹罗星/每克树脂的投料量，将H103树脂投入预处理罐。常温条件下搅拌吸附4小时。吸附结束后，停搅拌。用40目振动筛过滤发酵液并用生活饮用水淋洗树脂表面除去黏附的菌渣，收集树脂，收率为94.7％。
b.解析浓缩：
将树脂均匀装入解析柱，用2倍以上树脂体积的生活饮用水反冲清洗至颜色变淡时止。用空气压出柱内饮用水，用丙酮反向走到丙酮完全浸泡树脂停止。以丙酮完全浸泡树脂开始计时，浸泡2小时后，以7/10树脂体积/小时流速进行解析，解析丙酮用量约3.5倍树脂体积。收集解析液，再用生活饮用水洗到无丙酮味，将解析液抽入丙酮浓缩罐，在真空压力-0.08MPa以下、热水罐温度70.0℃、内温50.0℃以下，浓缩至无丙酮，留水相。
c.萃取：
第一次酯类溶剂提取：将水相抽入萃取罐，用2N的盐酸调节pH到3.5后，加入1/2水相体积的乙酸乙酯，常温条件下搅拌45分钟，静止30分钟后，分出乙酸乙酯相。重复以上操作一次。将两次萃取分出的乙酸乙酯相合并。
第一次碱水提取：将合并的乙酸乙酯相抽入萃取罐中，加入1/3乙酸乙酯相的体积的2％碳酸氢铵溶液，常温条件下搅拌30分钟，静止30分钟后，分出碱水相。重复以上操作三次。将四次萃取分出的碱水相合并。乙酸乙酯相进行溶媒回收。
第二次酯类溶剂提取：将合并的碱水相抽入萃取罐，滴加2N盐酸调节pH至4.0，加入1/2水相体积的乙酸乙酯，常温条件下搅拌45分钟，静止30分钟后，分出乙酸乙酯相。重复以上操作一次。将两次萃取分出的乙酸乙酯相合并。萃取后莫匹罗星的收率为87.3％，其含量为82.5％。
实施例3 莫匹罗星的树脂提取的考察
树脂提取：发酵液打入预处理罐并计量体积，打开搅拌，用盐酸或氢氧化钠调节pH至6.0。预处理半小时后以0.02克莫匹罗星/每克树脂的投料量，将H103树脂投入预处理罐。常温条件下搅拌吸附4小时。吸附结束后，停搅拌。用40目振动筛过滤发酵液并用生活饮用水淋洗树脂表面除去黏附的菌渣，收集树脂，收率为94.5％。
其他步骤与实施例2相同，莫匹罗星总收率为82.3％，纯度为82.0％的莫匹罗星。
实施例4 莫匹罗星的解析浓缩的考察
莫匹罗星提取工艺在解析工序中，将解析柱内的莫匹罗星从树脂中洗脱至丙酮或乙酸乙酯中，为了使吸附法能用于工业大生产，除了上述吸附和解析外，还需要其他条件的配合，如空间速度(体积流速/树脂体积)、树脂柱床的几何形状(高度/直径的比例)、柱床结构、工作程序等，其中空间速度往往作为主要影响因素。
解析浓缩：将树脂均匀装入解析柱，用2倍以上树脂体积的生活饮用水反冲清洗至颜色变淡时止。用空气压出柱内饮用水，用乙酸乙酯反向走到乙酸乙酯完全浸泡树脂停止。以乙酸乙酯完全浸泡树脂开始计时，浸泡2小时后，以7/10树脂体积/小时流速进行解析5h，解析乙酸乙酯用量约4倍树脂体积。收集解析液，再用生活饮用水清洗，解析液中莫匹罗星的含量为86.9％。其他步骤与实施例1相同，莫匹罗星总收率为86.0％，纯度为80.5％的莫匹罗星。
实施例5 莫匹罗星的解析浓缩的考察
解析浓缩：将树脂均匀装入解析柱，用2倍以上树脂体积的生活饮用水反冲清洗至颜色变淡时止。用空气压出柱内饮用水，用丙酮反向走到丙酮完全浸泡树脂停止。以丙酮完全浸泡树 脂开始计时，浸泡2小时后，以1/5树脂体积/小时流速进行解析10h，解析丙酮用量约4倍树脂体积。收集解析液，再用生活饮用水洗到无丙酮味，解析液中莫匹罗星的含量为88.5％。
实施例6 莫匹罗星的萃取方案的考察
第一次酯类溶剂提取：将水相抽入萃取罐，盐酸调节pH至5.0，加入1/2水相体积的乙酸乙酯，常温条件下搅拌45分钟，静止30分钟后，分出乙酸乙酯相。重复以上操作一次。将两次萃取分出的乙酸乙酯相合并。
第一次碱水提取：将合并的乙酸乙酯相抽入萃取罐中，加入1/3乙酸乙酯相的体积的2％碳酸氢铵溶液，常温条件下搅拌30分钟，静止30分钟后，分出碱水相。重复以上操作四次。将四次萃取分出的碱水相合并。乙酸乙酯相进行溶媒回收。
第二次酯类溶剂提取：将合并的碱水相抽入萃取罐，滴加盐酸调节pH至4.0，加入1/2水相体积的乙酸乙酯，常温条件下搅拌45分钟，静止30分钟后，分出乙酸乙酯相。重复以上操作一次。将两次萃取分出的乙酸乙酯相合并。萃取后后莫匹罗星的相对收率为95.5％。
其他步骤与实施例1相同，莫匹罗星总收率为82.0％，纯度为82.5％。
实施例7 莫匹罗星萃取中分步萃取的必要性
1.在萃取步骤中仅用酯类溶剂提取一次：将解析浓缩后的水相抽入萃取罐，用盐酸调节pH至3.5，加入适量乙酸乙酯，搅拌并静置，分出乙酸乙酯相。以上操作重复一次，将两次分出的乙酸乙酯相合并。一次酯类溶剂萃取后莫匹罗星的收率为80.5％，纯度为78.1％。
2.在萃取步骤中仅用碱水提取一次：将解析浓缩后的水相抽入萃取罐，由于碱水与水相同相，无法分层，进而无法萃取。
3.在萃取步骤中用酯类溶剂萃取一次以上：将解析浓缩后的水相抽入萃取罐，用盐酸调节pH至3.5，加入适量的乙酸乙酯，搅拌并静置，分出乙酸乙酯相。以上操作重复两次，将三次分出的乙酸乙酯相合并。两次酯类溶剂萃取后莫匹罗星的收率为82.0,％，纯度为78.5％。
实施例8 莫匹罗星树脂提取中树脂用量的考察
取莫匹罗星发酵液，计算发酵液所含的莫匹罗星量分别按0.02、0.03、0.04克莫匹罗星/每克树脂加入树脂，分别搅拌吸附4、6、8、24小时后过滤，检测滤液含量。吸附后发酵单位分别为278，217，137，85，385，293，238，194，446，386，340，322。
实施例9 重现CN101591333B所述的假单胞菌酸A提取方法(实施例1，即说明书[0041]～[0044]段所描述的方法，其报道的解析液中的假单胞菌酸A含量达70％，回收假单胞菌酸A达90％)
莫匹罗星发酵液(假单胞菌酸A浓度为6017ug/ml)25L，用1M NaOH调节pH到中性，高速低温离心机在4500rpm和25℃下离心20分钟得到清液，残渣水洗后弃去。清液有1M HCl调节pH到4.0过H103树脂(已处理好，2.2L)柱吸附。其中H103预处理方法按照生产厂商提供的说明书要求处理。完毕，用去离子水洗柱到流出液为近无色。
用乙醇+盐+水组成的溶剂8.3L(该8.3L溶剂中乙醇3320ml，氯化铵410g，其余为水)从树脂上解吸出含假单胞菌酸A的解吸液，所用的乙醇+盐+水组合的溶剂与假单胞菌酸A的比例是3.0ml：22mg。
解吸完成后，解吸液中假单胞菌酸A含量为72％，回收假单胞菌酸A为52％。
CN101591333B实施例1所记载的解吸指标为：收集到的解吸液中的假单胞菌酸A含量达70％，回收假单胞菌酸A达90％；文献记载的与重现实验中的回收率有较大差异。

Claims (10)

  1. 一种莫匹罗星的提取方法，包括用树脂吸附莫匹罗星液，用第一有机溶剂解析，得解析液，将解析液浓缩并留水相后进行萃取，其特征在于，所述的萃取为多步萃取，包括至少一次酯类溶剂提取和至少一次碱水提取。
  2. 一种如权利要求1所述的莫匹罗星的提取方法，其包括以下步骤：
    a.树脂提取：投入树脂莫匹罗星发酵液进行搅拌吸附，过滤发酵液并淋洗后收集树脂；
    b.解析浓缩：将步骤a中收集到的树脂装入解析柱，使用乙酸乙酯或丙酮浸泡后解析，收集并浓缩解析液；
    c.萃取：对步骤b中的浓缩解析液进行萃取，分出溶剂相；所述的萃取为多步萃取，包括至少一次酯类溶剂提取和至少一次碱水提取。
  3. 一种如权利要求2所述的莫匹罗星提取方法，其特征在于，将步骤c中收集到的溶剂相依次进行脱水与脱色。
  4. 如权利要求2所述的提取方法，其中树脂为非极性大孔吸附树脂，选自AB-8树脂、H103树脂、X-5树脂、H107树脂或D3520树脂；
    和/或所述树脂的投料量为0.01～0.05克莫匹罗星/每克树脂；
    和/或所述树脂提取的pH为5.0～7.0，优选5.5～6.5。
  5. 如权利要求2所述的提取方法，其中解析过程中乙酸乙酯或丙酮的用量为树脂体积的2～5倍；
    和/或所述的解析流速为1/5-6/5树脂体积/小时。
  6. 如权利要求1所述的提取方法，其特征在于所述的分步萃取包括第一次酯类溶剂提取、碱水提取与第二次酯类溶剂提取。
  7. 如权利要求1所述的提取方法，所述的酯类溶剂选自磷酸二异辛酯、磷酸三丁酯、乙酸乙酯、乙酸异丙酯或乙酸丁酯，优选乙酸乙酯；
    和/或所述的碱水选自碳酸氢钠、碳酸氢铵或氢氧化钠溶液；
    和/或所述碱水的pH为7.0-10.0，优选7.5～9.0。
  8. 如权利要求6所述的提取方法，所述的碱水提取重复操作一次或两次以上，并将每次萃取分出的碱水相合并做第二次酯类溶剂提取备用；
    和/或所述的第二次酯类溶剂提取的pH为3.0～5.0。
  9. 如权利要求3所述的提取方法，其脱水剂选自无水硫酸钠、无水硫酸镁或无水硫酸钙；
    和/或所述脱水剂的用量为0.02～0.08千克每升溶剂相；
    和/或其脱色剂选自活性炭或活性白土；
    和/或所述脱色剂的用量为1～6克每克脱水后的溶剂相。
  10. 如权利要求1-9任一项所述的提取莫匹罗星方法在制备抑制革兰氏阳性菌的药物中的应用。
PCT/CN2021/111926 2020-08-25 2021-08-11 一种莫匹罗星的提取方法 WO2022042299A1 (zh)

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