WO2020062928A1 - 一种高纯度的聚山梨酯80的制备方法 - Google Patents

一种高纯度的聚山梨酯80的制备方法 Download PDF

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WO2020062928A1
WO2020062928A1 PCT/CN2019/090764 CN2019090764W WO2020062928A1 WO 2020062928 A1 WO2020062928 A1 WO 2020062928A1 CN 2019090764 W CN2019090764 W CN 2019090764W WO 2020062928 A1 WO2020062928 A1 WO 2020062928A1
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polysorbate
solution
purity
molecular weight
drying
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French (fr)
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李月
段金连
段为钢
殷华
邱玲
吕小满
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云南中医药大学
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/30—Post-polymerisation treatment, e.g. recovery, purification, drying
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin

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  • the invention relates to a high-purity polysorbate 80 containing no macromolecular impurities and a preparation method thereof, in particular to a polysorbate 80 for solubilizing an injection and a preparation method thereof.
  • Polysorbate 80 (polysorbate 80), trade name is Tween 80 (chemical name Tween 80), chemical name is polyoxyethylene sorbitan monooleate, is a sorbitan fatty acid ester and ethylene oxide in alkaline
  • the hydrophilic compound formed under the reaction is an ideal non-ionic surfactant.
  • Polysorbate 80 uses sorbitan as the core and polymerizes a certain number of ethylene oxide monomers from four hydroxyl ends to form a structure of four polyoxyethylenes as side chains, one of the four hydroxyl groups. Links a molecule of oleic acid (an eighteen-carbon fatty acid containing an unsaturated double bond).
  • polysorbate 80 is an amphiphilic polymer with a theoretical molecular weight of 1309.65.
  • Polysorbate 80 is highly hydrophilic and less toxic than other surfactants, it is often used as an adjuvant for injections and is widely used as a cosolvent, emulsifier and stabilizer for poorly soluble drugs.
  • commercially available polysorbate 80 for injection is generally a light yellow or orange yellow viscous liquid with a slightly bitter and slightly astringent taste. The relative density is 1.06 to 1.09 at 20 ° C, and it is easily soluble in water, methanol, ethanol, and ethyl acetate. Wait for some organic solvents.
  • polysorbate 80 was widely used as a solubilizer in pharmaceutical companies in China to solve the problem of clarity of injections.
  • Polysorbate 80 is a potentially unsafe excipient, and its use must be strictly restricted, otherwise it will cause great harm to human health.
  • Invention patent "Method for preparing high-purity polysorbate 80 by synthesizing and remixing three types of effective components" discloses a method for synthesizing high-purity polysorbate 80, which is mainly adopted Raw materials such as sorbitol, ethylene oxide, oleic acid, isosorbide, etc., are synthesized separately from sorbitol polyoxyethylene ether oleate, monohydrate sorbitol polyoxyethylene ether oleate, and isosorbide polyoxyethylene ether oil. Acid ester, and the three are mixed and refined.
  • the patented technology improves the purity of polysorbate 80 to a certain extent, reduces the content of impurities, and helps meet the requirements of the Pharmacopoeia.
  • the patent does not specify any improvements or advantages of its safety performance compared to existing polysorbate 80 products. The problem remains to be further studied and evaluated.
  • the macromolecular impurities contained in it can be removed, and its safety can be significantly improved, so that it is suitable for the use of injections, reduces the incidence of adverse reactions of injections, and thereby meets the production and use requirements of preparations.
  • the object of the present invention is to provide a high-purity polysorbate 80 which does not contain impurities with a relatively large molecular weight, which is suitable for the requirements of injection.
  • the invention provides a method for preparing high-purity polysorbate 80, which comprises the following steps:
  • Dilution Take polysorbate 80 stock solution and dilute with solvent to a solution with a concentration less than or equal to 10% w / w;
  • step b The polysorbate 80 solution obtained in step a is filtered by pressure and passed through an ultrafiltration device with a molecular weight cut-off of 10k-30k;
  • step b Drying: The ultrafiltrate obtained in step b is dried to obtain high-purity polysorbate 80.
  • the dilution concentration in step a is 3 to 5% w / w. Still further preferably, the dilution concentration in step a is 5% w / w.
  • the molecular weight cut-off described in step b is 10k or 30k. Still more preferably, the molecular weight cut-off described in step b is 10k.
  • the interception ultrafiltration method described in step b is a pressure pump or centrifugal pressure filtration.
  • the ultrafiltration device in step b is a hollow fiber filter, a filtration membrane, and a permeation membrane.
  • the drying method described in step c is sterile vacuum spray drying or freeze drying.
  • the invention also provides a high-purity polysorbate 80 prepared by the method.
  • the high-purity polysorbate 80 does not contain impurities with a molecular weight exceeding 10k or 30k; the absorption value of a 10% solution at any wavelength of 230-400nm when the optical path is 1cm is less than 0.5.
  • the 10% solution has no obvious absorption peak in the range of 230 to 400 nm when the optical path is 1 cm.
  • the present invention utilizes the molecular weight difference between the main component of polysorbate 80 and the macromolecular impurities, and uses ultrafiltration technology to retain the macromolecular impurities to achieve purification. It has been confirmed by research that this method can effectively remove macromolecular impurities in polysorbate 80, and these macromolecular impurities are the main material basis for abnormal toxicity, allergic reactions, allergic reactions, hemolysis and agglomeration reactions. The safety of the ester 80 is significantly improved.
  • Dilute and mix take 80g of injection-grade polysorbate, add water to 100ml, and mix on a shaker at room temperature for more than 2 hours.
  • the mixed liquid is light yellow.
  • Drying freeze-dry the above ultrafiltrate (freeze at a temperature below -20 ° C, and then place it in a freeze dryer to freeze and evacuate to constant weight).
  • the property is a colorless viscous oily liquid.
  • Dilute and mix Take 80g of injection-grade polysorbate, add water to 100ml, and mix on a shaker at room temperature for more than 2 hours.
  • the mixed liquid is light yellow.
  • Dilute and mix take 80g of injection-grade polysorbate, add water to 100ml, and mix on a shaker at room temperature for more than 2 hours.
  • the mixed liquid is light yellow.
  • Filtration and separation Take the diluted solution and add it to an ultrafiltration tube with a molecular weight cut-off of 3k, and after centrifugation, centrifuge at 2000C (2000 rpm) for 60 minutes to obtain an ultrafiltrate.
  • Dilute and mix take 80g of injection-grade polysorbate, add water to 100ml, and mix on a shaker at room temperature for more than 2 hours.
  • the mixed liquid is light yellow.
  • Filtration and separation Take the above diluted solution, pressurize it with a pressure pump, and filter it through a hollow fiber filter with a molecular weight cut off of 30k to obtain ultrafiltrate.
  • Dilute and mix Take 80g of injection-grade polysorbate, add water to 100ml, and mix on a shaker at room temperature for more than 2 hours.
  • the mixed liquid is light yellow.
  • Filtration and separation Take the above diluted solution, pressurize it with a pressure pump, and filter through a filtration membrane with a molecular weight cut off of 10k to obtain an ultrafiltrate.
  • Dilute and mix take 80g of injection-grade polysorbate, add water to 100ml, and mix on a shaker at room temperature for more than 2 hours.
  • the mixed liquid is light yellow.
  • Filtration and separation Take the above diluted solution, pressurize it with a pressure pump, and filter it through an osmosis membrane with a molecular weight cut off of 3k to obtain ultrafiltration.
  • the injection-grade polysorbate 80 sample was diluted 5000 times (below the minimum micelle concentration), and the samples were placed in an analytical ultracentrifuge, and the molecular weight distribution of polysorbate 80 was analyzed using OD235 and light interference modes.
  • polysorbate 80 main component measured by light interference (the molecular weight is less than 5k and no peak is displayed), one at 171k and one at 467880k (Figure 4), indicating that polysorbate 80 has more large molecules Impurities, and macromolecular impurities also produced a multimerization phenomenon, and OD235 measured four macromolecular impurity peaks, the first is the main peak at 36.2k, the second at 99.3k, the third at 563k, the fourth at 884k ( Figure 5), but the last two peaks are barely recognizable.
  • the molecular weight distribution of the macromolecular impurities with ultraviolet absorption contained in polysorbate 80 is near 36.2k, and the macromolecular impurities without ultraviolet absorption are distributed near 171k (may also include some ultraviolet absorption impurities).
  • Figure 4 Tween 80 ultracentrifugation analysis results (light interference detection), indicating that there are macromolecular impurities with molecular weight distribution around 171k and 467880k.
  • the two distribution peaks are broad, the latter may be the polymer of the former, and the peak near 171k contains UV-absorbing impurities.
  • the ultrafiltration filter with a molecular weight cut-off of 30k or less can achieve the separation purpose, but in order to reliably and efficiently remove the macromolecular impurities in polysorbate 80, the molecular weight cut-off of 10k is the best.
  • Figure 3 shows that at the same mass concentration, the smaller the pore size used for interception, the lower the absorption of the UV-visible absorption spectrum (A).
  • B Calculated according to the thickness of the solution, converted to a light path of 1 cm, and the absorption values of the 10% content of the 3k-30k filtered retention polysorbate 80 are all lower than 0.5 in the range of 230-400nm.
  • A UV-visible absorption spectrum of different polysorbate 80 samples at the same mass concentration
  • B UV absorption spectrum of different polysorbate 80 samples at the same mass concentration.
  • the safety of the polysorbate 80 of the present invention is further evaluated by the following test examples.
  • the obtained polysorbate 80 small molecule component, macromolecular component, and polysorbate 80 stock solution were respectively prepared into a 2% solution with physiological saline for injection as a test solution.
  • Fifteen Kunming mice weighing 18-22 g, were randomly divided into three groups of A, B, and C, with 5 in each group.
  • group A polysorbate 80 small molecule component was injected intravenously
  • group B polysorbate 80 macromolecular component was injected intravenously
  • group C polysorbate 80 stock solution was intravenously injected.
  • Each mouse was intravenously administered 0.5 ml of the test solution, and the injection was completed at a uniform speed within 4 to 5 seconds.
  • mice in group A did not die within 48 hours after administration, 3 animals in group B died, and 1 animal in group C died. For the 6 surviving mice, the status was also worse than that of the group A surviving mice. It is shown that the macromolecular component of polysorbate 80 is the main material basis that causes safety problems. After removing macromolecular impurities, the safety of polysorbate 80 is significantly improved.
  • the polysorbate 80 small-molecule component, the macromolecular component, and the polysorbate 80 stock solution obtained above were respectively prepared into a 2% solution as a test solution with physiological saline for injection.
  • Animals in group B appeared manic and restless after injection, bumped against a wall, groaned, screamed, runny, drooling, itching, skin mucosa flushing or erythema within 24 hours, sclera congestion, nausea, vomiting, lip and ear edema, shortness of breath , Difficulty, urinary incontinence and other phenomena, two of them even had convulsions, coma, shock, and finally died. Animals in groups A and C also showed similar conditions to those in group B. No animals died within 24 hours, but the animals in group A had significantly milder reactions, no obvious shortness of breath, difficulty, and incontinence.
  • the experimental results show that the macromolecular component of polysorbate 80 is the main material basis for safety problems. After removing the macromolecular impurities, the safety of polysorbate 80 is significantly improved.
  • the obtained polysorbate 80 small molecule component, macromolecular component, and polysorbate 80 stock solution were respectively prepared into a 2% solution with physiological saline for injection as a test solution.
  • test solution 0.5ml of the test solution was injected intraperitoneally each time for 3 times for sensitization. Subsequently, the six animals in the three groups A, B, and C were divided into two subgroups, three in each group, and the corresponding test solution was injected intravenously on the 14th and 21st days after the first injection, respectively. Iml was challenged. Observe the animals for symptoms of allergic reactions within 30 minutes after challenge.
  • Positive reaction refers to two or more of vertical hair, trembling, retching, three consecutive sneezes, three consecutive coughs, purpura, and dyspnea on the same animal, or two incontinence, step Instability or fall to the ground, convulsions, shock.
  • the polysorbate 80 small and macromolecular components and the polysorbate 80 stock solution were respectively prepared into a 1% solution of saline for injection as A, B, and C test solutions.
  • No. 1 and No. 2 tubes are test tubes
  • No. 3 tubes are negative control tubes
  • No. 4 tubes are positive control tubes
  • No. 5 tubes are test tubes for test products.
  • Result judgment criteria if the solution in the test tube is clear red, there is no cell residue or a small amount of red blood cell residue at the bottom of the tube, indicating the occurrence of hemolysis; if all red blood cells sink, the supernatant is colorless, or the supernatant is clear However, no significant difference was observed with the naked eyes of tubes 1, 2 and 5, indicating that no hemolysis occurred. If there are brown-red or red-brown flocculent precipitates in the solution, it is still not dispersed after being gently inverted 3 times, indicating that red blood cell aggregation may occur, and it should be further observed under a microscope, if red blood cell aggregation is visible as aggregation.
  • test solution A had no hemolysis and coagulation; the test solution B had significant hemolytic and coagulation reactions; the test solution C had a lighter hemolytic reaction than the test solution B and had no obvious coagulation reaction.
  • the above results show that the macromolecular component of polysorbate 80 is the main material basis for causing safety problems. After removing macromolecular impurities, the safety of polysorbate 80 is significantly improved.
  • the present invention utilizes the molecular weight difference between the main component of polysorbate 80 and the macromolecular impurities, and uses ultrafiltration technology to retain the macromolecular impurities to achieve purification.
  • this method can effectively remove the macromolecular impurities in polysorbate 80 that cause abnormal toxicity, allergic reactions, allergic reactions, hemolysis and agglutination reactions.
  • the safety of polysorbate 80 after purification is significantly improved It is conducive to meeting the demand for auxiliary materials for the production of injections, significantly reducing the incidence of adverse reactions of injections, and has good development prospects.

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Abstract

本发明提供了一种高纯度的聚山梨酯80的制备方法，它包括如下步骤：a、稀释：取聚山梨酯80原液，加溶剂稀释成浓度小于或等于10％w/w的溶液；b、滤过：将a步骤所得的聚山梨酯80溶液，采用压力过滤方式，通过截留分子量为10k-30k的超滤装置；c、干燥：将b步骤得到的超滤液干燥，即得高纯度的聚山梨酯80。相较于传统注射用聚山梨酯80，本发明聚山梨酯80纯度更高、更安全，适合用于注射剂，特别是注射剂的增溶，有利于降低传统聚山梨酯80使用引起的注射剂不良反应发生率，提高临床用药安全性，具有良好应用前景。

Description

一种高纯度的聚山梨酯80的制备方法 技术领域
本发明涉及一种不含大分子杂质的高纯度聚山梨酯80及其制备方法，尤其是涉及一种供注射剂增溶用的聚山梨酯80及制备方法。
背景技术
聚山梨酯80(polysorbate 80)，商品名是吐温80(Tween 80)，化学名为聚氧乙烯脱水山梨醇单油酸酯，是由脱水山梨醇脂肪酸酯与环氧乙烷在碱性条件下反应生成的亲水性化合物，是一种较理想的非离子型表面活性剂。聚山梨酯80以失水山梨醇为母核，并由4个羟基端聚合一定数目的环氧乙烷单体，形成了4个聚氧乙烯为侧链的结构，4个羟基中的1个连接一分子油酸(含1个不饱和双键的十八碳脂肪酸)。在其分子结构式中，聚氧乙烯侧链结合的油酸酯是疏水性基团，未结合油酸酯的聚氧乙烯侧链为亲水基团。因此，聚山梨酯80是一个两亲性聚合物，理论分子量为1309.65。
由于聚山梨酯80亲水性强且毒性相较于其他表面活性剂小，常作为注射剂辅料，广泛用作难溶性药物的助溶剂、乳化剂和稳定剂。目前，市售的注射用聚山梨酯80一般为淡黄色或橙黄色的黏稠液体，味微苦略涩，20℃时相对密度为1.06～1.09，易溶于水、甲醇、乙醇和醋酸乙酯等一些有机溶剂。上个世纪末，聚山梨酯80作为增溶剂在我国的制药企业广泛使用，以解决注射液的澄明度问题。但随着用量的增加，暴露了诸多安全性问题，主要表现为过敏或类过敏反应。现已研究证实，对于以挥发油或其他脂溶性物质为主要成分的中药注射剂(如鱼腥草注射液)而言，这一类注射剂的安全性问题更多地是辅料聚山梨酯80造成的，而其它含聚山梨酯80的注射剂也有类似的安全性问题。
吴晓燕，等，脉络宁注射液中聚山梨酯80的含量测定与安全性分析，中国医院药学杂志，2011年第31卷第7期，公开了聚山梨酯80用于中药注射剂的增溶剂，但有很强的破裂细胞膜的作用而引起刺激性、溶血性和致敏性(组胺释放)，且聚山梨酯80作为一个聚合物本身纯度波动很大，其中不饱和脂肪酸易氧化降解产生有毒成分。聚山梨酯80静脉注射会引起急性超敏反应、外周神经毒性、抑制P-糖蛋白活性、内在肿瘤效应等。聚山梨酯80是一种潜在的不安全性的辅料，其使用必须有严格的限制条件，否则给人的健康造成很大伤害。张勇等，聚山梨酯80的分析方法研究进展，实用药物与临床2014年第17卷第7期，报道了聚山梨酯80的安全性，尤其是溶血性，且不同工艺及纯化工艺制得的产品差异很大，优质产品在正常使用量不会产 生溶血作用。由于聚山梨酯80自身的组成成分比较复杂(张锐，王玉，谭力，等.吐温80的组分分析[J].中国药学杂志，2012，47(2):149-154.；李剑，谢俊，黄春玉，等.聚山梨酯80的脂肪酸组成及其精制方法研究[J].药学与临床研究，2010，18(1):46-49.其安全性与用量成为影响药品安全性的关键，药品标准中未形成其检验方法。聚山梨酯8能够引起类过敏反应，然而引起其过敏反应的分子生物学机制仍不明确(见耿兴超，等，不同生产工艺聚山梨酯80的Beagle犬类过敏反应研究，中国药事2011年第25卷第10期)。
因此，现有注射剂中使用的聚山梨酯80的安全性有待于提高，而导致聚山梨酯80安全性的主要原因未明确，目前也没有相关的文献报道。
发明专利(CN 102352031)“通过三类有效组分合成再混合制备高纯度聚山梨酯80的方法”公示了一种用于高纯度聚山梨酯80合成的方法，其主要是通过采用高纯度的山梨醇、环氧乙烷、油酸、异山梨醇等原料，分别合成山梨醇聚氧乙烯醚油酸酯、一失水山梨醇聚氧乙烯醚油酸酯、异山梨醇聚氧乙烯醚油酸酯，再将三者混合精制而得。该专利技术在一定程度上提高了聚山梨酯80的纯度，降低了杂质含量有利于符合药典要求，该专利未明确其安全性能相对于现有聚山梨酯80产品有何改进或优势其安全性问题仍待进一步研究评价。
发明内容
发明人在长期的实验中发现，聚山梨酯80中大分子物质贸然进入机体可能带来较大的安全性风险，因此，开发一种用于纯化聚山梨酯80的方法，特别是能针对性去除其中所含的大分子杂质，并能显著提高其安全性，以使其适用于注射剂的使用要求，降低注射剂不良反应的发生率，从而满足制剂生产及使用需求。
本发明的目的在于提供一种不含较大分子量杂质的高纯度聚山梨酯80，适用于注射剂使用要求。
本发明提供了一种高纯度的聚山梨酯80的制备方法，它包括如下步骤：
a、稀释：取聚山梨酯80原液，加溶剂稀释成浓度小于或等于10％w/w的溶液；
b、滤过：将a步骤所得的聚山梨酯80溶液，采用压力过滤方式，通过截留分子量为10k-30k的超滤装置；
c、干燥：将b步骤得到的超滤液干燥，即得高纯度的聚山梨酯80。
进一步优选地，a步骤所述的稀释浓度为3～5％w/w。更进一步优选地，a步骤所述的稀释浓度为5％w/w。
进一步优选地，b步骤所述的截留分子量为10k或30k。更进一步优选地，b步骤所述的截留分子量为10k。
其中，b步骤所述的截留超滤方法是采用压力泵加压或离心加压过滤。
其中，b步骤所述的超滤装置为中空纤维滤器、过滤膜和渗透膜。进一步优选地，c步骤所述的干燥方法为无菌真空喷雾干燥或冷冻干燥。
本发明还提供了所述的方法制备得到的高纯度聚山梨酯80。
其中，所述的高纯度聚山梨酯80中不含有分子量超过10k或30k的杂质；10％溶液在光径为1cm时230-400nm任意波长处的吸收值小于0.5。
进一步地，所述的10％溶液在光径为1cm时230～400nm范围无明显的吸收峰。
本发明利用聚山梨酯80主成分与大分子杂质的分子量差异，采用超滤技术截留大分子杂质而实现对其纯化。经研究证实，该方法可有效除去聚山梨酯80中大分子杂质，且这些大分子杂质是导致异常毒性、类过敏反应、过敏反应、溶血和凝聚反应的主要物质基础，经过纯化制备的聚山梨酯80的安全性得到显著提高。
显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1吐温80超速离心分析结果
图2吐温80超速离心分析结果
图3不同条件去大分子聚山梨酯80的紫外-可见吸收光谱
具体实施方式
实施例1 本发明产品的制备方法
1.稀释混匀：取注射级的聚山梨酯80 10g，加水到100ml，室温下于摇床上混匀2小时以上，混匀液体呈浅黄色。
2.滤过分离：取上述稀释后溶液加入到截留分子量为30k的超滤管中，配平后于4℃离心(3000转/分钟)30分钟，得超滤液。
3.干燥：将以上超滤液进行冷冻干燥(先于-20℃以下温度冻实，然后置于冷冻干燥机内冷冻抽真空到恒重)即得，性状为无色粘稠油状液体。
实施例2 本发明产品的制备方法
1.稀释混匀：取注射级的聚山梨酯80 5g，加水到100ml，室温下于摇床 上混匀2小时以上，混匀液体呈浅黄色。
2.滤过分离：取上述稀释后溶液加入到截留分子量为10k的超滤管中，配平后于4℃离心(3000转/分钟)45分钟，得超滤液。
3.干燥：将以上超滤液进行无菌真空喷雾干燥(将滤液加热到60℃缓慢喷入真空室内，保持真空度100KPa以下)即得，性状为无色粘稠油状液体。
实施例3 本发明产品的制备方法
1.稀释混匀：取注射级的聚山梨酯80 3g，加水到100ml，室温下于摇床上混匀2小时以上，混匀液体呈浅黄色。
2.滤过分离：取上述稀释后溶液加入到截留分子量为3k的超滤管中，配平后于4℃离心(2000转/分钟)60分钟，得超滤液。
3.干燥：将以上超滤液进行无菌真空喷雾干燥(将滤液加热到60℃缓慢喷入真空室内，保持真空度100KPa以下)即得，性状为无色粘稠油状液体。
实施例4 本发明产品的制备方法
1.稀释混匀：取注射级的聚山梨酯80 10g，加水到100ml，室温下于摇床上混匀2小时以上，混匀液体呈浅黄色。
2.滤过分离：取上述稀释后溶液，采用压力泵加压，通过截留分子量为30k的中空纤维滤器过滤，得超滤液。
3.干燥：将以上超滤液进行无菌真空喷雾干燥(将滤液加热到60℃缓慢喷入真空室内，保持真空度100KPa以下)即得，性状为无色粘稠油状液体。
实施例5 本发明产品的制备方法
1.稀释混匀：取注射级的聚山梨酯80 5g，加水到100ml，室温下于摇床上混匀2小时以上，混匀液体呈浅黄色。
2.滤过分离：取上述稀释后溶液，采用压力泵加压，通过截留分子量为10k的过滤膜过滤，得超滤液。
3.干燥：将以上超滤液进行无菌真空喷雾干燥(将滤液加热到60℃缓慢喷入真空室内，保持真空度100KPa以下)即得，性状为无色粘稠油状液体。
实施例6 本发明产品的制备方法
1.稀释混匀：取注射级的聚山梨酯80 3g，加水到100ml，室温下于摇床上混匀2小时以上，混匀液体呈浅黄色。
2.滤过分离：取上述稀释后溶液，采用压力泵加压，通过截留分子量为3k的渗透膜过滤，得超滤液。
3.干燥：将以上超滤液进行无菌真空喷雾干燥(将滤液加热到60℃缓慢 喷入真空室内，保持真空度100KPa以下)即得，性状为无色粘稠油状液体。
以下通过具体试验例证明本发明的有益效果。
试验例1注射用聚山梨酯80中大分子杂质的分析
1.试验方法
将注射级聚山梨酯80样品稀释5000倍(低于最小胶束浓度)，取样置于分析型超速离心机中，用OD235和光干涉两种模式分析聚山梨酯80的分子量分布。
2.测定结果
光干涉测得的聚山梨酯80(主成分)有两个峰(分子量低于5k不显示峰)，一个在171k，一个在467880k(图4)，表明聚山梨酯80具有较多的大分子杂质，且大分子杂质还产生了多聚现象，而OD235测得四个大分子杂质峰，第一个是主峰在36.2k，第二个在99.3k，第三个在563k，第四个在884k(图5)，但后两个峰只能勉强辨认。因此说明，聚山梨酯80含有的具紫外吸收的大分子杂质其分子量分布在36.2k附近，不具紫外吸收的大分子杂质分布在171k附近(可能也包含部分紫外吸收杂质)。
图4吐温80超速离心分析结果(光干涉检测)，表明有分子量分布在171k和467880k附近的大分子杂质。这两个分布峰较宽，后者可能是前者的聚合物，171k附近的峰包含紫外吸收杂质。
图5吐温80超速离心分析结果(OD235检测)，表明具有紫外吸收的大分子杂质有4组，主要分布在36.2k。
试验例2注射用聚山梨酯80中大分子杂质的分离方法考察
1.试验方法
取注射级的聚山梨酯80 5g，加水到100ml，室温下于摇床上混匀2小时以上。混匀的液体呈浅黄色。分别取10ml溶液加入到截留分子量为3k、10k、30k、100k、300k、1000k的超滤管中，配平后于4℃、3000转/分钟离心30分钟可得0.5-5ml超滤液，延长离心时间可得5ml以上超滤液。将以上超滤液进行冷冻干燥称重后加入适量蒸馏水配制成10％溶液，取200μl加入到透紫外96孔板，于酶标仪下扫描230-800nm范围的吸收光谱。
2.测定结果
测定结果发现，3k、10k和30k超滤管去除大分子后的吐温80在紫外区无明显的吸收，但100k、300k、1000k超滤管去大分子吐温80以及原液却在235nm处有明显的紫外吸收峰(图3)。表明吐温80的大分子杂质在235nm附近有紫外吸收，主要大分子杂质的分子量在30k-100k之间，估计总量超过 10％。因此，选用截留分子量30k以下(含)的超滤器都可以达到分离目的，但为了可靠同时高效去除聚山梨酯80中的大分子杂质，选用截留10k分子量最佳。
图3表明，相同质量浓度下，截留所用的孔径越小，紫外-可见吸收光谱的吸收就越低(A)，3k-30k滤过截留的同质量浓度聚山梨酯80紫外吸收均低平，无明显的235nm吸收峰(B)。根据溶液厚度计算，折算成1cm光经，10％含量的3k-30k滤过截留聚山梨酯80吸收值在230-400nm范围均低于0.5。A，同质量浓度下的不同聚山梨酯80样品的紫外-可见吸收光谱；B，同质量浓度下不同聚山梨酯80样品的紫外吸收光谱。
试验例3去除大分子杂质的聚山梨酯80的安全性对比考察
下面通过试验例来进一步评价本发明聚山梨酯80的安全性。
将本发明中制备的聚山梨酯80，与现有聚山梨酯80对比，进行安全性实验。
1.实验用聚山梨酯80小分子组分和大分子组分的制备
取含注射级聚山梨酯80 5％的溶液10ml，加入到截留分子量为10k的超滤管中，配平后置于离心机4℃、3000转/分钟离心制备去大分子超滤液(即下层液)。当下层液获得约5ml后，将下层液转移，在上层液中加入等量的超纯水混匀，配平后继续离心制备下层液，如此三次将获得的下层液合并(约15ml)，将上层液(约5ml)转移到试管中。重复上述操作，以获得更多的聚山梨酯80去大分子液(下层液)和富含大分子液，分别合并下层液、上层液，进行冷冻干燥即得聚山梨酯80小分子组分和大分子组分。
2.异常毒性的对比实验
2.1实验方法
按照质量浓度，将前述获得的聚山梨酯80小分子组分、大分子组分以及聚山梨酯80原液，分别用注射用生理盐水配制成2％溶液作为供试液。
取昆明种小鼠15只，体重18～22g，随机分成A、B、C三组，每组5只。A组将静脉注射聚山梨酯80小分子组分供试液，B组静脉注射聚山梨酯80大分子组分供试液，C组静脉注射聚山梨酯80原液配制的供试液。每只小鼠分别静脉给予供试品溶液0.5ml，在4～5秒内匀速注射完毕。
2.2实验结果
A组小鼠在给药后48小时内未有死亡，B组有3只动物死亡，而C组有1只动物死亡。对于存活的6只小鼠来说，其状态差也比A组存活的小鼠差。表明聚山梨酯80的大分子组分是导致安全性问题的主要物质基础，去大分子杂质后，聚山梨酯80的安全性明显提高。
3类过敏反应检查的对比实验
3.1实验方法
按照质量浓度，将前述获得的聚山梨酯80小分子组分、大分子组分以及 聚山梨酯80原液，分别用注射用生理盐水配制成2％溶液作为供试液。
取健康雌性比格犬9只，体重8-10kg，无孕，随机分成A、B、C三组，每组3只。A组将静脉注射聚山梨酯80小分子组分供试液，B组静脉注射聚山梨酯80大分子组分供试液，C组静脉注射聚山梨酯80原液配制的供试液。每只比格犬分别静脉给予供试品溶液10ml，在55-65秒内匀速注射完毕，随后记录动物24小时内行为变化。
3.2实验结果
B组动物在注射后即出现狂躁不安，撞墙撞笼，呻吟嘶叫，大量流涕、流涎，搔痒，24小时内出现皮肤黏膜潮红或红斑，巩膜充血，恶心、呕吐，口唇耳水肿，呼吸急促、困难，大小便失禁等现象，其中2只甚至先后出现抽搐、昏迷、休克，最后死亡。A组和C组动物也出现与B组相似的情况，24小时内未有动物死亡，但A组动物反应明显较轻，未出现明显的呼吸急促、困难，无大小便失禁等现象。实验结果表明，聚山梨酯80的大分子组分是导致安全性问题的主要物质基础，去大分子杂质后，聚山梨酯80的安全性明显提高。
4.过敏反应的对比实验
4.1实验方法
按照质量浓度，将前述获得的聚山梨酯80小分子组分、大分子组分以及聚山梨酯80原液，分别用注射用生理盐水配制成2％溶液作为供试液。
取健康豚鼠18只，体重250～350g，雌鼠无孕，随机分成A、B、C三组，每组6只。动物在试验前和试验过程中，均按正常饲养条件饲养。A组将静脉注射聚山梨酯80小分子组分供试液，B组静脉注射聚山梨酯80大分子组分供试液，C组静脉注射聚山梨酯80原液配制的供试液。
隔日每只每次腹腔注射供试品溶液0.5ml，共3次，进行致敏。随后，对于A、B、C三组的6只动物再均分为2个亚组，每组3只，分别在首次注射后第14日和第21日，由静脉注射相应的供试品溶液lml进行激发。观察激发后30分钟内动物有无过敏反应症状。
4.2实验结果
A组动物试验后均存活，两次激发实验30分钟内，只有2只动物在第二次激发实验时出现过敏反应。B组动物因状态差，只有2只动物完成了第二次激发实验，存活动物的两次激发实验30分钟内均出现明显的阳性反应。C组动物全部存活，均完成了两次激发实验，但第一次激发实验只有1只有阳性反应，第二次激发均出现明显的阳性反应。阳性反应指：在同一只动物上出现竖毛、发抖、干呕、连续喷嚏3声、连续咳嗽3声、紫癜和呼吸困难等现象中的2种或2种以上，或出现二便失禁、步态不稳或倒地、抽搐、休克者。
以上结果表明聚山梨酯80的大分子组分是导致安全性问题的主要物质基础，去大分子杂质后，聚山梨酯80的安全性明显提高。
5.溶血与凝聚的对比实验
5.1实验方法
按照质量浓度，将聚山梨酯80小分子组分和大分子组分以及聚山梨酯80原液分别用注射用生理盐水配制成1％溶液作为A、B、C供试液。
取健康家兔血液，放入含玻璃珠的锥形瓶中振摇10分钟，或用玻璃棒搅动血液，以除去纤维蛋白原，使成脱纤血液。加入0.9％氯化钠溶液约10倍量，摇匀，每分钟1000～1500转离心15分钟，除去上清液，沉淀的红细胞再用0.9％氯化钠溶液按上述方法洗涤2～3次，至上清液不显红色为止。将所得红细胞用0.9％氯化钠溶液制成2％的混悬液，供试验用。
取洁净玻璃试管5只，编号，1、2号管为供试品管，3号管为阴性对照管，4号管为阳性对照管，5号管为供试品对照管。按下表所示依次加人2％红细胞悬液、0.9％氯化钠溶液、纯化水，混匀后，立即置37±0.5℃的恒温箱中进行温育。3小时后观察溶血和凝聚反应。
试管编号 1 2 3 4 5
2％红细胞悬液(ml) 2.5 2.5 2.5 2.5  
0.9％氯化钠溶液(ml) 2.2 2.2 2.5   4.7
纯化水(ml)       2.5  
供试品溶液(ml) 0.3 0.3     0.3
结果判断标准：如试管中的溶液呈澄明红色，管底无细胞残留或有少量红细胞残留，表明有溶血发生；如红细胞全部下沉，上清液无色澄明，或上清液虽有色澄明，但1、2号管和5号管肉眼观察无明显差异，则表明无溶血发生。若溶液中有棕红色或红棕色絮状沉淀，轻轻倒转3次仍不分散，表明可能有红细胞凝聚发生，应进一步置显微镜下观察，如可见红细胞聚集为凝聚。
5.2实验结果
结果表明，A供试液无溶血与凝聚发生；B供试液发生明显的溶血和凝聚反应；C供试液则发生溶血反应程度较B供试液轻，无明显的凝聚反应。以上结果表明聚山梨酯80的大分子组分是导致安全性问题的主要物质基础，去大分子杂质后，聚山梨酯80的安全性明显提高。
综上，本发明利用聚山梨酯80主成分与大分子杂质的分子量差异，采用超滤技术截留大分子杂质而实现对其纯化。经研究证实，该方法可有效除去聚山梨酯80中导致异常毒性、类过敏反应、过敏反应、溶血和凝聚反应的大分子杂质，经过纯化制备的聚山梨酯80的安全性得到显著提高，有利于满足注射剂生产用辅料需求，显著降低注射剂不良反应发生率，具有良好发展前景。

Claims (10)

  1. 一种高纯度的聚山梨酯80的制备方法，其特征在于：它包括如下步骤：
    a、稀释：取聚山梨酯80原液，加溶剂稀释成浓度小于或等于10％w/w的溶液；
    b、滤过：将a步骤所得的聚山梨酯80溶液，采用压力过滤方式，通过截留分子量为10k-30k的超滤装置；
    c、干燥：将b步骤得到的超滤液干燥，即得高纯度的聚山梨酯80。
  2. 根据权利要求1所述的制备方法，其特征在于：a步骤所述的稀释浓度为3～5％w/w。
  3. 根据权利要求2所述的制备方法，其特征在于：a步骤所述的稀释浓度为5％w/w。
  4. 根据权利要求1所述的制备方法，其特征在于：b步骤所述的截留分子量为10k或30k。
  5. 根据权利要求1或4所述的制备方法，其特征在于：b步骤所述的截留超滤方法是采用压力泵加压或离心加压过滤。
  6. 根据权利要求5所述的制备方法，其特征在于：b步骤所述的超滤装置为中空纤维滤器、过滤膜和渗透膜。
  7. 根据权利要求1所述的制备方法，其特征在于：c步骤所述的干燥方法为无菌真空喷雾干燥或冷冻干燥。
  8. 权利要求1-7任一项所述的方法制备得到的高纯度聚山梨酯80。
  9. 根据权利要求8所述的高纯度聚山梨酯80，其特征在于：它不含有分子量超过10k或30k的杂质；10％溶液在光径为1cm时230-400nm任意波长处的吸收值小于0.5。
  10. 根据权利要求8所述的高纯度聚山梨酯80，其特征在于：10％溶液在光径为1cm时230～400nm范围无明显的吸收峰。
PCT/CN2019/090764 2018-09-30 2019-06-11 一种高纯度的聚山梨酯80的制备方法 WO2020062928A1 (zh)

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