Polysorbate 20 with high biological safety and preparation method thereof

Abstract

The invention provides a preparation method of high-biosafety polysorbate 20, which comprises the following steps: by taking sorbitol, ethylene oxide and coconut oil acid as initial raw materials, synthesizing polyoxyethylene-sorbitan coconut oil acid ester through the processes of dehydration, purification, polymerization, esterification and the like; and blending the polysorbate 20 with low-esterification-degree polyoxyethylene sorbitan coconut oleate prepared by adopting a specific esterification process according to a ratio to prepare a polysorbate 20 product which meets the pharmacopeia index requirements and has high biological safety at the same time. The production method disclosed by the invention is simple, efficient, high in yield and suitable for industrial production.

Description

Technical Field

[0001] The invention belongs to the technical field of preparation of medical supplies, and specifically relates to polysorbate 20 with high biological safety and a preparation method thereof. Background Art

[0002] Polysorbate 20 (PS20), also known as Tween 20, is an amphiphilic nonionic surfactant with a chemical name of polyoxyethylene (20) sorbitan monolaurate. Polysorbate 20 is often used as a stabilizer, solubilizer, emulsifier, and wetting agent, and is widely used in the pharmaceutical, food, and cosmetic industries. Due to the complex composition of polysorbate 20, and the fact that it will inevitably degrade to varying degrees during storage, this will lead to a decrease in the concentration of polysorbate 20 and weaken its functionality. On the other hand, the degradation products may also affect the stability of biological preparations. The latter will not only cause protein aggregation, but also some degradation products will react chemically with proteins to produce new compounds and increase the immunogenicity of the preparation. In addition, there are literature reports that the presence of polyoxyethylene isosorbide fatty acid esters in polysorbate series products will affect the biosafety of the product. However, a significant reduction in polyoxyethylene isosorbide fatty acid esters will not only affect the product's application performance in preparations such as solubility and stability, but will also cause the product indicators to deviate from the range specified in the pharmacopoeia.

[0003] Zebrafish is an internationally recognized new model organism, which is used to evaluate the toxicity and safety of drugs. Its characteristics are that it covers a wide range of toxicological endpoints, has high sensitivity of the test method, uses a small amount of samples, has a short test cycle, and has low reagent and instrument costs. Zebrafish experiments are whole animal studies that can reflect the absorption, distribution, metabolism and excretion of compounds. At the same time, the similarity between zebrafish and human genomes is close to 90%, and they have similar toxicity characteristics and signal transduction pathways to humans, making them a highly comparable model animal. Zebrafish experiments can be used to test and compare the acute toxicity and allergic reactions of different polysorbate 20. Summary of the invention

[0004] In response to the above problems, the applicant conducted a biosafety comparison of polyoxyethylene isosorbide fatty acid esters with different esterification degrees and found that the safety of polyoxyethylene isosorbide fatty acid monoester esterified at one end is better than that of polyoxyethylene isosorbide fatty acid diester esterified at both ends. Based on this finding, the applicant reduced the proportion of polyoxyethylene isosorbide fatty acid diester while maintaining the performance of polysorbate 20 by keeping the content of polyoxyethylene isosorbide fatty acid ester basically unchanged, thereby improving the biosafety of polysorbate 20.

[0005] To achieve the above object, the present invention provides the following technical solutions: A method for preparing polysorbate 20 with high biosafety comprises the following steps: (1) Using sorbitol as a raw material, dehydrating under an acidic catalyst and vacuum conditions to obtain a crude dehydrated sorbitol mixture; then separating and purifying to obtain a dehydrated sorbitol mixture; (2) using sorbitol as a raw material, dehydrating under an acidic catalyst and vacuum conditions to obtain a crude disorbitol mixture; or using the crude disorbitol mixture described in step (1) as a raw material, heating under an acidic catalyst and vacuum conditions, and further dehydrating to obtain a crude disorbitol mixture; separating and purifying the crude disorbitol mixture to obtain a disorbitol mixture; the amount of catalyst added is 0.1% to 1%; (3) mixing the anhydrous sorbitol mixture and sorbitol, and subjecting the mixture to a polymerization reaction catalyzed by ethylene oxide under the action of an alkaline catalyst to obtain an anhydrous sorbitol ether; (4) subjecting the monosorbitan ether to an esterification reaction with coconut oil acid in the presence of an esterification catalyst to obtain polyoxyethylene monosorbitan coconut oil ester; (5) subjecting the disorbitol mixture to a catalytic polymerization reaction with ethylene oxide under the action of an alkaline catalyst to obtain disorbitol ether; (6) subjecting the disorbitan ether to an esterification reaction with coconut oil acid in the presence of an esterification catalyst to obtain polyoxyethylene disorbitan coconut oil ester; (7) The polyoxyethylene monosorbitan cocoate obtained in step (4) and the polyoxyethylene disorbitan cocoate obtained in step (6) are blended to obtain the polysorbate 20.

[0006] Preferably, the mass ratio of the polyoxyethylene monosorbitan cocoate to the polyoxyethylene disorbitan cocoate in step (7) is 8-20:1.

[0007] Preferably, the sorbitol in step (1) or step (2) is a sorbitol aqueous solution with a mass fraction of 50% to 80%.

[0008] Preferably, the acidic catalyst in step (1) or step (2) is a protonic acid, and more preferably, the protonic acid is sulfuric acid, phosphoric acid or phosphorous acid.

[0009] Preferably, the dehydration in step (1) or step (2) includes a step of neutralizing with a base to obtain a product after the reaction is completed.

[0010] Preferably, the separation and purification in step (1) or step (2) comprises the steps of decolorization, filtration, drying and distillation. Preferably, the distillation is vacuum distillation and the fraction at 175-185° C. is collected.

[0011] Preferably, the amount of the acidic catalyst added in step (1) or step (2) is 0.1% to 1% by mass of the total reaction materials.

[0012] Preferably, step (1) is to use sorbitol as a raw material, vacuum dehydrate at 100-120°C, then add an acidic catalyst and continue vacuum dehydration at 105-120°C to obtain a crude dehydrated sorbitol mixture; then neutralize with alkali, decolorize, filter and dry to obtain the dehydrated sorbitol mixture.

[0013] Preferably, the temperature in step (2) is increased to 150-170°C.

[0014] Preferably, the alkaline catalyst in step (3) or step (5) is one or more of potassium hydroxide, sodium hydroxide, potassium alcoholate or sodium alcoholate.

[0015] Preferably, the amount of the alkaline catalyst added in step (3) or step (5) is 0.01% to 0.5% by mass of the total reaction materials.

[0016] Preferably, the reaction conditions in step (3) or step (5) are: 100-120° C., 0.4-0.6 MPa.

[0017] Preferably, step (3) or step (5) further comprises the steps of neutralizing, adsorbing, vacuum dehydrating, and filtering the reaction product.

[0018] Preferably, the esterification catalyst in step (4) or step (6) is one or more of phosphate, tin salt or solid super acid.

[0019] Preferably, the temperature of the esterification reaction in step (4) or step (6) is 200-240°C.

[0020] Preferably, step (4) or step (6) further comprises the steps of adsorption and filtration after the esterification reaction.

[0021] Preferably, in step (6), the mass ratio of the disorbitol ether to the coconut oil acid is greater than 1:0.3 and the molar ratio is greater than 1:1.

[0022] The present invention also provides polysorbate 20 prepared by the method.

[0023] Compared with the prior art, the present invention has the following beneficial effects: The method of the present invention prepares polyoxyethylene disorbitol coconut ester monomer with low esterification degree through a special esterification process, and then by increasing the degree of polymerization of ethylene oxide, or improving the degree of esterification of polyoxyethylene sorbitol / monosorbitol coconut ester, the mixture is blended according to a specific ratio, and the acute toxicity and sensitization problems existing in the existing products are solved under the premise of ensuring that the product index meets the requirements of the pharmacopoeia and the application performance is consistent with the conventional process product. Through a more refined process, polyoxyethylene isosorbide difatty acid ester is controlled, and the biosafety of polysorbate 20 products is significantly improved. Compared with the prior art, the method of the present invention is more targeted, and the overall change of polysorbate 20 products is smaller, thereby reducing the impact on product indexes and application performance. Under the premise of ensuring that the application effects such as solubilization remain basically unchanged, the safety is improved. The product prepared by the method of the present invention can be directly used in injections, and provides an excellent injection-grade pharmaceutical excipient for the development of new injection dosage forms in the pharmaceutical industry.

[0024] The method of the invention adopts sorbitol aqueous solution, ethylene oxide and coconut oil acid as initial reaction raw materials, the raw materials have wide sources, the overall synthesis and refining process is simple and feasible, the yield is high, and it is suitable for large-scale industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The figure is a flow chart of the method of the present invention. DETAILED DESCRIPTION

[0026] The following will be combined with the specific embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] On the basis of ensuring that the polysorbate 20 index meets the requirements of the pharmacopoeia, and the structural adjustment does not affect the application performance such as solubility and stability, in order to improve its biosafety, the present invention provides a preparation method of polysorbate 20 with high biosafety, which uses sorbitol, ethylene oxide and coconut oil acid as initial raw materials, synthesizes polyoxyethylene sorbitan coconut oil ester II through dehydration, purification, polymerization, esterification and other processes, and then blends it with low esterification degree polyoxyethylene sorbitan coconut oil ester III prepared by a specific esterification process according to a specific ratio to prepare a polysorbate 20 product that meets the requirements of the pharmacopoeia index and has high biosafety. The method is simple and efficient, has a high yield, is environmentally friendly, and is suitable for industrial production.

[0028] The method for acute toxicity and anaphylactoid reaction testing by zebrafish experiment is as follows: (1) Acute toxicity reaction: Different batches of polysorbate 20 were prepared into a 2.00 mg / mL test solution and introduced into zebrafish 2 hours post fertilization (2 hpf) by microinjection. The zebrafish were placed in a standard environment for development. Their development was observed and recorded daily, and the number of zebrafish deaths was counted. The acute toxicity of different samples was compared by comparing the mortality rate.

[0029] (2) Allergic reaction: Different batches of polysorbate 20 were prepared into a 200 mg / mL test solution and introduced into 72 hpf zebrafish by microinjection. The trypsin-specific substrate BAPNA was added and the reaction was allowed to proceed to 120 hpf. The OD 405 value was read using a microplate reader. The OD 405 value was used to compare the sensitizing effect of different samples.

[0030] The present invention is further described below through specific embodiments.

[0031] Example 1

[0032] (1) Add sorbitol aqueous solution to a vacuum dehydration reaction device, heat to 100-120°C for vacuum dehydration for 3 hours, cool to 80°C, add sulfuric acid, heat to 105-120°C, and vacuum dehydrate for 5 hours to obtain a crude product of anhydrous sorbitol mixture I; neutralize with sodium hydroxide aqueous solution to neutrality, add activated carbon for decolorization, filter, dry, and distill under reduced pressure to collect a fraction of 175-185°C, which is an anhydrous sorbitol mixture II. The content of anhydrous sorbitol in the product is 98.9% as determined by liquid chromatography.

[0033] (2) The crude sorbitol mixture I obtained in step (1) was added to a vacuum dehydration reaction device, the temperature was raised to 150-170°C, the dehydration reaction was continued for 6 hours, the mixture was neutralized with an aqueous sodium hydroxide solution until neutral, activated carbon was added for decolorization, the mixture was filtered, and the mixture was dried. The fraction at 175-185°C was collected by vacuum distillation, which was the disorbitol mixture III. The disorbitol content was 99.1% as determined by liquid chromatography.

[0034] (3) A total of 167 g of anhydrous sorbitol mixture II and solid sorbitol were added to the polymerization reaction device, potassium hydroxide catalyst was added, the temperature was raised to 90°C after nitrogen replacement, 1232 g of ethylene oxide was slowly introduced, and the reaction was carried out at 110°C and 0.4 MPa until the pressure no longer decreased, and the temperature was lowered and the material was discharged. Neutralization, adsorption, vacuum dehydration, and filtration were performed to obtain anhydrous sorbitol ether II with a hydroxyl value of 176 mgKOH / g.

[0035] (4) The above-mentioned monosorbitan ether II, coconut oil acid and phosphate catalyst were added to the esterification device, and the mixture was stirred and reacted at 220°C for 5 hours under nitrogen protection. The mixture was cooled to room temperature, and then adsorbed and filtered to obtain polyoxyethylene monosorbitan coconut oil ester II, which was numbered 20220601-1, had a hydroxyl value of 105 mgKOH / g, and a saponification value of 45 mgKOH / g.

[0036] (5) Add 146g of di-anhydrous sorbitol mixture III to the polymerization reaction device, add sodium hydroxide catalyst, and heat to 90°C after nitrogen replacement. Slowly introduce 880g of ethylene oxide, react at 110°C and 0.4MPa until the pressure no longer decreases, cool and discharge. Neutralize, adsorb, vacuum dehydrate, and filter to obtain di-anhydrous sorbitol ether III with a hydroxyl value of 109 mgKOH / g.

[0037] (6) Add 1026 g of the above-mentioned sorbitan ether III and a tin salt catalyst to the esterification device, and add 200 g of coconut oil acid dropwise at 220° C. under nitrogen protection while stirring for reaction. The dropping time is controlled to 4 hours. After the dropping is completed, the reaction is continued for 1 hour, and the temperature is cooled to room temperature. Polyoxyethylene sorbitan coconut oil ester III is obtained by adsorption and filtration. The number is 20220601-2, the hydroxyl value is 46.5 mgKOH / g, and the saponification value is 46.5 mgKOH / g.

[0038] (7) 1026 g of the disorbitan ether III obtained in step (5), 300 g of coconut oil acid and a catalyst were added to the esterification device, and the mixture was reacted at 220° C. for 5 hours under nitrogen protection, and then cooled to room temperature. The post-treatment was basically the same as in step (6) to obtain polyoxyethylene sorbitan coconut oil ester III, which was numbered 20220601-3, had a hydroxyl value of 21.6 mgKOH / g, and a saponification value of 64.7 mgKOH / g.

[0039] Example 2

[0040] (1) Add sorbitol aqueous solution to the vacuum dehydration reaction device, heat to 100-120°C for vacuum dehydration for 3 hours, cool to 80°C, add phosphoric acid, heat to 105-120°C, and vacuum dehydrate for 7 hours to obtain a crude dehydrated sorbitol mixture; neutralize with sodium hydroxide aqueous solution to neutrality, add activated carbon for decolorization, filter, dry, and collect the 175-185°C fraction after vacuum distillation, which is a dehydrated sorbitol mixture II. The content of dehydrated sorbitol in the mixture is 89.5% as determined by liquid chromatography.

[0041] (2) The crude sorbitol mixture obtained in step (1) was added to a vacuum dehydration reaction device, the temperature was raised to 150-170° C., the dehydration reaction was continued for 4 hours, the mixture was neutralized with an aqueous sodium hydroxide solution until neutral, activated carbon was added for decolorization, the mixture was filtered, and dried. The fraction at 175-185° C. was collected by vacuum distillation, which was the disorbitol mixture III. The disorbitol content was 88.7% as determined by liquid chromatography.

[0042] (3) A total of 167 g of anhydrous sorbitol mixture II and solid sorbitol were added to the polymerization reaction device, and potassium alcohol catalyst was added. After nitrogen replacement, the temperature was raised to 90°C, and 1276 g of ethylene oxide was slowly introduced. The reaction was carried out at 120°C and 0.6 MPa until the pressure no longer decreased, and the temperature was lowered and the material was discharged. After neutralization, adsorption, vacuum dehydration, and filtration, anhydrous sorbitol ether II was obtained, and the hydroxyl value was 170 mgKOH / g.

[0043] (4) Add the above-mentioned monosorbitan ether II, coconut oil acid and solid superacid catalyst into the esterification device, stir and react at 240°C for 5 hours under nitrogen protection, cool to room temperature, adsorb and filter to obtain polyoxyethylene monosorbitan coconut oil ester II.

[0044] (5) Add 146g of di-anhydrous sorbitol mixture III to the polymerization reaction device, add sodium alcohol catalyst, and heat to 90°C after nitrogen replacement. Slowly introduce 924g of ethylene oxide, react at 110°C and 0.4MPa until the pressure no longer decreases, cool and discharge. Neutralize, adsorb, vacuum dehydrate, and filter to obtain di-anhydrous sorbitol ether III with a hydroxyl value of 104 mgKOH / g.

[0045] (6) Add 1026 g of the above-mentioned disodium sorbitol ether III and a solid superacid catalyst to the esterification device, and add 220 g of coconut oil acid dropwise at 200° C. under nitrogen protection while stirring for reaction. The dropping time is controlled to 4 hours. After the dropping is completed, the reaction is continued for 1 hour, and the temperature is cooled to room temperature. Polyoxyethylene disodium sorbitol coconut oil ester III is obtained by adsorption and filtration. The hydroxyl value is 41.8 mgKOH / g and the saponification value is 51.1 mgKOH / g.

[0046] Example 3

[0047] (1) to (5) are the same as in Example 1.

[0048] (6) 1026 g of the sorbitan ether III obtained in step (5), 220 g of coconut oil acid and a catalyst were added to the esterification device, and the mixture was reacted at 220° C. for 5 hours under nitrogen protection, then cooled to room temperature and post-treated in the same manner as in Example 1 to obtain polyoxyethylene sorbitan coconut oil ester III having a hydroxyl value of 41.8 mgKOH / g and a saponification value of 51.1 mgKOH / g.

[0049] Example 4

[0050] The polyoxyethylene monosorbitan cocoate II obtained by the esterification reaction in step (4) of Example 1 and the polyoxyethylene disorbitan cocoate III obtained in step (6) of Example 1 were added to the blending device in a mass ratio of 10:1. The mixture was kept warm and stirred at 80°C for 1 hour. After vacuum dehydration and precision filtration, a polysorbate 20 product was obtained, which was numbered 20220602-1, had a hydroxyl value of 99.5 mgKOH / g, and a saponification value of 45 mgKOH / g.

[0051] The blending mass ratio of polyoxyethylene sorbitan cocoate II and polyoxyethylene sorbitan cocoate III was adjusted to 8:1. The finished product number was 20220602-2. The hydroxyl value was 98.5 mgKOH / g and the saponification value was 45 mgKOH / g.

[0052] The blending mass ratio of polyoxyethylene sorbitan cocoate II and polyoxyethylene sorbitan cocoate III was adjusted to 20:1. The finished product number was 20220602-3. The hydroxyl value was 102.2 mgKOH / g and the saponification value was 45.1 mgKOH / g.

[0053] The blending mass ratio of polyoxyethylene sorbitan cocoate II and polyoxyethylene sorbitan cocoate III was adjusted to 5:1. The finished product number was 20220602-4. The hydroxyl value was 95.2 mgKOH / g and the saponification value was 45.2 mgKOH / g.

[0054] Two commercially available polysorbate 20 products were also selected and numbered 20220701-1 and 20220701-2 respectively.

[0055] Each polysorbate 20 product, commercially available product and specific structural monomer were tested. Uninjected wild-type zebrafish samples were used as the normal control group, and wild-type zebrafish samples injected with 10 nL / tail of normal saline were used as the solvent control group. The acute toxicity test results are shown in Table 1, and the allergic reaction test results are shown in Table 2.

[0056] Table 1 Acute toxicity test results of samples

[0058] Table 2 Sample allergic reaction test results

[0059]

[0060] From the above examples and test results, it can be seen that the polysorbate 20 prepared by the present invention is significantly superior to conventional commercially available polysorbate 20 in terms of important biosafety indicators such as acute toxicity and sensitization. And the key indicators of the products prepared by polyoxyethylene sorbitan cocoate II and polyoxyethylene sorbitan cocoate III in different proportions, such as hydroxyl value and saponification value, all meet the requirements of the pharmacopoeia. In actual operation, the finished polysorbate 20 product that meets the needs of preparation solubilization or other applications can be prepared according to the differences in usage scenarios.

[0061] In summary, the method of the present invention prepares polyoxyethylene isosorbide coconut ester monomer with low esterification degree through a special esterification process, and then adjusts the polyoxyethylene sorbitol / monosorbide coconut ester with appropriate ethylene oxide polymerization degree and coconut ester esterification degree according to the index, and blends them in a specific ratio. Under the premise of ensuring that the product index meets the requirements of the pharmacopoeia and the application performance is consistent with the conventional process product, the acute toxicity and sensitization problems existing in the existing products are solved, and the biosafety of polysorbate 20 products is significantly improved. The process has the characteristics of simple production process, environmental friendliness, and easy operation of industrial production.

[0062] The matters not described in detail in the present invention are all known technologies to those skilled in the art.

[0063] Finally, it should be noted that the above specific implementation methods are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified and replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A method for preparing polysorbate 20 with high biological safety, characterized in that: The following steps are involved: (1) Using sorbitol as a raw material, dehydrating under an acidic catalyst and vacuum conditions to obtain a crude dehydrated sorbitol mixture; Then, separation and purification are performed to obtain a dehydrated sorbitol mixture; (2) using sorbitol as a raw material, dehydrating under an acidic catalyst and vacuum conditions to obtain a crude disorbitol mixture; or using the crude disorbitol mixture described in step (1) as a raw material, heating under an acidic catalyst and vacuum conditions, and further dehydrating to obtain a crude disorbitol mixture; separating and purifying the crude disorbitol mixture to obtain a disorbitol mixture; the amount of catalyst added is 0.1% to 1%; (3) mixing the anhydrous sorbitol mixture and sorbitol, and subjecting the mixture to a polymerization reaction catalyzed by ethylene oxide under the action of an alkaline catalyst to obtain an anhydrous sorbitol ether; (4) subjecting the monosorbitan ether to an esterification reaction with coconut oil acid in the presence of an esterification catalyst to obtain polyoxyethylene monosorbitan coconut oil ester; (5) subjecting the disorbitol mixture to a catalytic polymerization reaction with ethylene oxide under the action of an alkaline catalyst to obtain disorbitol ether; (6) subjecting the disorbitan ether to an esterification reaction with coconut oil acid in the presence of an esterification catalyst to obtain polyoxyethylene disorbitan coconut oil ester; (7) The polyoxyethylene monosorbitan cocoate obtained in step (4) and the polyoxyethylene disorbitan cocoate obtained in step (6) are blended to obtain the polysorbate 20.

2. The preparation method according to claim 1, characterized in that: The mass ratio of the polyoxyethylene monosorbitan cocoate to the polyoxyethylene disorbitan cocoate in step (7) is 8-20:

1.

3. The preparation method according to claim 1, characterized in that: The acidic catalyst in step (1) or step (2) is a protonic acid, and preferably, the protonic acid is sulfuric acid, phosphoric acid or phosphorous acid.

4. The preparation method according to claim 1, characterized in that: The heating in step (2) is to heat the temperature to 150-170°C.

5. The preparation method according to claim 1, characterized in that: The alkaline catalyst in step (3) or step (5) is one or more of potassium hydroxide, sodium hydroxide, potassium alcoholate or sodium alcoholate.

6. The preparation method according to claim 1, characterized in that: The reaction conditions in step (3) or step (5) are: 100-120° C., 0.4-0.6 MPa.

7. The preparation method according to claim 1, characterized in that: The esterification catalyst in step (4) or step (6) is one or more of phosphate, tin salt or solid super acid.

8. The preparation method according to claim 1, characterized in that: The temperature of the esterification reaction in step (4) or step (6) is 200-240°C.

9. The preparation method according to claim 1, characterized in that: In step (6), the mass ratio of the disorbitol ether to the coconut oil acid is greater than 1:0.3 and the molar ratio is greater than 1:

1.

10. Polysorbate 20 prepared by the preparation method according to any one of claims 1 to 9.

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