A method for removing impurities from an enolate reaction solution

Abstract

This invention provides a method for removing impurities from an enoxylation reaction solution, comprising the following steps: (1) adding methanol and anhydrous sodium acetate solution to the enoxylation reaction solution, mixing and precipitating to obtain a solid-liquid suspension; (2) adding purified water to the solid-liquid suspension while stirring, then stopping the stirring and allowing it to stand, removing the supernatant, taking the wet solid, washing with methanol, and drying to obtain dried enoxylated heparin. This method reduces the amount of methanol used, optimizes the impurity removal effect of the enoxylated heparin process, reduces material transfer operations between equipment, optimizes the process flow, improves the yield and production efficiency of enoxylated heparin, and is convenient and safe to operate, suitable for large-scale production.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceuticals, specifically relating to a method for removing impurities from an enoxin esterification reaction solution. Background Technology

[0002] Enoxaparin sodium is derived from conventional heparin sodium. Its production involves modifying the structure of heparin sodium followed by a β-elimination reaction. The β-elimination reaction forms unsaturated bonds at the Δ4,5 position of the non-reducing end. The processes from heparin sodium to enoxaparin sodium include heparin quaternization, esterification, and hydrolysis.

[0003] In the enochrome esterification process, the esterification reaction solution is first precipitated to obtain a solid-liquid suspension. Then, it undergoes multiple methanol washes to remove organic impurities, such as benzyl chloride from the salting step. The collected wet solids are then dried to obtain enochrome-esterified heparin. However, this production process requires a large amount of methanol. Taking a feed rate of 60 kg heparin quaternary ammonium salt and 700 L of mixed precipitate as an example, the impurity removal operation after the esterification reaction requires approximately 350 L of methanol per batch for washing. Furthermore, the solid-liquid mixture from the methanol wash needs to be transferred from the tank to a centrifuge for centrifugation to remove impurities, and then the wet solids from the centrifuge are transferred back to the tank for the next step. This multi-step material transfer process is inconvenient for personnel and affects overall production efficiency, making it unsuitable for large-scale production. Summary of the Invention

[0004] The purpose of this invention is to provide a convenient and efficient method for removing impurities from the enocarpy esterification reaction solution, based on existing technologies.

[0005] The technical solution of the present invention is as follows:

[0006] A method for removing impurities from an enoxin esterification reaction solution, comprising the following steps:

[0007] (1) Add methanol and anhydrous sodium acetate solution to the enoyl esterification reaction solution, mix and precipitate to obtain a solid-liquid suspension;

[0008] (2) Add purified water to the solid-liquid suspension while stirring, then stop stirring and let it stand, then remove the supernatant.

[0009] The wet solid was collected, washed with methanol, and dried to obtain the dried enoxaparin.

[0010] Because enoxaparin is highly soluble in water, existing methods for removing impurities from the enoxaparin esterification reaction solution involve precipitation followed by multiple methanol washes to remove organic impurities, all conducted under anhydrous conditions. This application addresses this issue by, through extensive experimentation, utilizing the solubility differences between enoxaparin, methanol, organic impurities, and water during the enoxaparin sodium production process. A specific amount of purified water is added to the mixed precipitate solution, and water addition is stopped when the solid-liquid separation critical point is reached. After settling, the supernatant methanol-water solution is discarded, effectively removing impurities. In this method, the amount of purified water must be strictly controlled. Insufficient purified water will affect impurity removal, while excessive purified water will cause the wet solid enoxaparin to dissolve in water, ultimately leading to yield loss.

[0011] The raw material processed by this method is the enoxaparin esterification reaction solution. This solution is obtained during the preparation of enoxaparin sodium by reacting heparin sodium with benzyl chloride via a salting reaction, followed by an esterification reaction with benzyl chloride. Specifically, heparin sodium undergoes a salting reaction with benzyl chloride to obtain enoxaparin quaternary ammonium salt. Then, the enoxaparin quaternary ammonium salt is dissolved in dichloromethane and esterified with benzyl chloride to prepare the esterification product, esterified heparin. The resulting enoxaparin esterification reaction solution is then used.

[0012] The enoxacid reaction solution is subsequently precipitated using a sodium acetate methanol solution. The resulting solid-liquid suspension is also referred to in this invention as: solid-liquid suspension solution after precipitation of the enoxacid reaction solution, or simply as solid-liquid suspension solution, solid-liquid suspension, or solid-liquid suspension material.

[0013] In the impurity removal method of the present invention, in step (1), preferably, the composition of the methanol anhydrous sodium acetate solution is an anhydrous sodium acetate to methanol mass-volume ratio of 1:8-12 g / ml, more preferably 1:9-11 g / ml, and more preferably 1:10 g / ml.

[0014] In the impurity removal method of the present invention, in step (1), preferably, the volume ratio of methanol anhydrous sodium acetate solution to enolate reaction solution is 1-1.5:1, and the mixing precipitation condition is mixing precipitation at 20℃-30℃ for ≥0.5 hours. To ensure the reproducibility of subsequent experiments, the volume ratio of methanol anhydrous sodium acetate solution to enolate reaction solution is selected as 1:1, and the mixing precipitation condition is mixing precipitation at room temperature for 1 hour.

[0015] In the impurity removal method of the present invention, in step (2), preferably, the amount of purified water added is 8% to 25% of the volume of the solid-liquid suspension, more preferably 10% to 22%. The purified water is added until precipitation occurs, and the precipitation phenomenon includes one or more of the following: solid precipitate, no floating matter in the supernatant, and clear supernatant without yellowing.

[0016] In the impurity removal method of the present invention, in step (2), preferably, the stirring speed is 5-150 rpm, more preferably 10-100 rpm.

[0017] In the impurity removal method of the present invention, in step (2), preferably, the stirring time is 5-150 min, more preferably 10 min-120 min.

[0018] In the impurity removal method of the present invention, in step (2), preferably, the standing time is 0.5 to 10 hours, more preferably 0.5 hours to 8 hours.

[0019] In the impurity removal method of the present invention, in step (2), preferably, the supernatant is removed by centrifugation or extraction after standing, and more preferably by extraction.

[0020] In the impurity removal method of the present invention, in step (2), a specific methanol washing method is as follows: add water to dissolve the wet solid, the volume of the solution after dissolution is 3-6 times the weight of the heparin quaternary ammonium salt, add 2-4 times the volume of methanol to precipitate according to the solution volume, repeat at least 2 times, and collect the wet solid.

[0021] The present invention further includes a method for reducing the impurity content in enoxaparin sodium, the method comprising the following steps:

[0022] S1 salt formation: Sodium heparin reacts with benzyl chloride in a salting reaction to obtain enoxaparin quaternary ammonium salt;

[0023] S2 Esterification: The enoxaparin quaternary ammonium salt is dissolved in dichloromethane and esterified with benzyl chloride to obtain the enoxaparin esterification reaction solution.

[0024] S3 Impurity Removal: The enoxin esterification reaction solution is treated with the method of claim 1 to obtain enoxin esterified heparin;

[0025] S4 Hydrolysis: The enoxaparin esterified is hydrolyzed to obtain enoxaparin sodium.

[0026] The present invention also includes a method for removing impurities from the solid-liquid suspension after mixing and precipitation of the enoxetine esterification reaction solution, which includes the following steps: adding purified water to the solid-liquid suspension after mixing and precipitation of the enoxetine esterification reaction solution under stirring, then stopping stirring and letting it stand, then removing the supernatant, taking the wet solid, then washing with methanol and drying to obtain the dried enoxetine esterification heparin.

[0027] The solid-liquid suspension of this invention includes: esterified heparin, a product of esterification reaction, as well as sodium acetate, methanol, dichloromethane, and benzyl chloride. This invention utilizes the differences in solubility of different materials in water: anhydrous sodium acetate, methanol > benzyl chloride > esterified heparin, and the properties that dichloromethane is readily soluble in methanol while esterified heparin is insoluble in dichloromethane, to remove various impurities.

[0028] We attempted to analyze the principles behind the use of sodium acetate solution for precipitation and the subsequent use of purified water for impurity removal in this invention. We believe the possible reasons and mechanisms are as follows:

[0029] (1) After adding methanol and anhydrous sodium acetate solution to the enoxylation reaction solution, methanol and anhydrous sodium acetate together remove water from the dissolved enoxylated heparin and benzyl chloride. Therefore, enoxylated heparin can be precipitated from the reaction solution. Methanol will also remove some dichloromethane from the reaction solution, which can achieve the effect of removing some impurities (dichloromethane and anhydrous sodium acetate).

[0030] (2) Continue to add water to the solid-liquid suspension. Since the solubility of benzyl chloride is greater than that of enoxaparin, benzyl chloride will dissolve as the amount of water added slowly increases. When enough water has been dissolved in methanol, anhydrous sodium acetate, and benzyl chloride, if more water is added, enoxaparin will begin to dissolve. Therefore, there is a critical point. At this point, the aqueous phase is methanol, anhydrous sodium acetate, and benzyl chloride, the oil phase is dichloromethane, and the solid phase is enoxaparin. By removing the aqueous phase and the oil phase, the impurities in enoxaparin (benzyl chloride, anhydrous sodium acetate, methanol, and dichloromethane) can be removed.

[0031] (3) This is a relatively ideal state. When the water volume is slightly insufficient, the impurities that may exist in enoxaparin include benzyl chloride and incomplete collection of enoxaparin, which will affect the yield. When the water volume is excessive, enoxaparin will dissolve, resulting in a decrease in product yield. Therefore, the amount of water added for precipitation is a critical control point. At the same time, considering that the solution will turn yellow after enoxaparin dissolves in water, the upper limit of water addition can be determined. The endpoint of water addition can be observed by the precipitation phenomenon (solid phase precipitate is firm, there are no floating objects in the supernatant and the supernatant is clear and does not turn yellow).

[0032] This invention includes a specific method for reducing the impurity content in enoxaparin sodium, characterized in that the method comprises the following steps:

[0033] 1) Salt formation: Sodium heparin reacts with benzyl chloride to form enoxaparin quaternary ammonium salt;

[0034] 2) Esterification: The enoxaparin quaternary ammonium salt obtained in step 1) above is dissolved in dichloromethane and reacted with benzyl chloride to obtain an esterification solution.

[0035] 3) Mixed precipitation: Add methanol and anhydrous sodium acetate solution to the esterification solution obtained in step 2) above to obtain a solid-liquid suspension after esterification mixed precipitation;

[0036] 4) Impurity removal: The solid-liquid suspension obtained in step 3) above is purified by adding purified water and then allowed to settle.

[0037] 5) Enoxetine: After the above step 4) is settled, remove the supernatant of the solid-liquid suspension after impurity removal, take the wet solid, purify it with methanol, and then dry it to obtain dried enoxetine.

[0038] 6) Hydrolysis: Enoxaparin is hydrolyzed to obtain enoxaparin sodium.

[0039] In a preferred embodiment, in step 4), while adding purified water to the solid-liquid suspension, stirring needs to be maintained. The stirrer speed is 5-150 rpm, more preferably 5-100 rpm, and even more preferably 10-100 rpm. During the stirring process, the stirring time is 5-150 min, preferably 5-120 min, and even more preferably 10-120 min. The added purified water is 8%-25% of the volume of the solid-liquid suspension, preferably 10%-22%. After adding purified water, the sedimentation time is 0.5-10 h, preferably 0.5-8 h.

[0040] This patented method reduces methanol consumption while ensuring effective impurity removal and improving production efficiency. Based on a feed rate of 60 kg heparin quaternary ammonium salt and 700 L of mixed precipitate, approximately 350 L of methanol can be reduced per batch. The solid-liquid mixture washed with purified water and methanol exhibits solid-liquid stratification; the supernatant is discarded, retaining the wet solid, reducing material transfer and centrifuge usage. This optimizes operator procedures and improves production efficiency.

[0041] The advantages of using the technical solution of this invention are as follows:

[0042] 1. While significantly reducing methanol usage, optimize the impurity removal effect of the enoxaparin esterification process.

[0043] 2. Reducing material transfer operations between equipment and optimizing process flow improves the yield and production efficiency of esterified heparin. Attached Figure Description

[0044] Figure 1 This is a comparison chart of conditions 1 and 3 in Example 2.

[0045] Figure 2 This is a comparison chart of conditions 2 and 4 in Example 2.

[0046] Figure 3This is a comparison chart of conditions 1 and 2 in Example 2.

[0047] Figure 4 This is a comparison chart of conditions 3 and 4 in Example 2.

[0048] Figure 5 This is a comparison chart of conditions 6 and 8 in Example 2.

[0049] Figure 6 This is a comparison chart of conditions 7 and 9 in Example 2.

[0050] Figure 7 This is a comparison chart of conditions 1 and 5 in Example 2.

[0051] Figure 8 This is a comparison chart of conditions 6 and 7 in Example 2.

[0052] Figure 9 This is a comparison chart of conditions D1 and D6 in Example 2;

[0053] Figure 10 This is a comparison chart of conditions 5 and 7 in Example 2.

[0054] Figure 11 This is a comparison chart of benzyl chloride removal in Example 3;

[0055] Figure 12 This is a comparison chart of acetate removal in Example 3;

[0056] Figure 13 This is a comparison chart of methanol removal in Example 3;

[0057] Figure 14 This is a comparison chart of dichloromethane removal in Example 3;

[0058] Figure 15 This is a yield comparison chart from Example 3. Detailed Implementation

[0059] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

[0060] In this invention, "water addition ratio" refers to the percentage of purified water added to the volume of the solid-liquid suspension when adding purified water to the solid-liquid suspension for impurity removal, i.e., the volume of purified water ÷ the volume of the solid-liquid suspension after esterification and precipitation × 100%.

[0061] Enoxaparin esterification reaction solution: The starting material is sodium heparin. Then, according to the approved enoxaparin sodium production process, sodium heparin reacts with benzyl chloride in a salting reaction to obtain enoxaparin quaternary ammonium salt. The heparin quaternary ammonium salt is dissolved in dichloromethane and then reacted with benzyl chloride in an esterification reaction to obtain an esterification solution.

[0062] Example 1: Preparation of solid-liquid suspension material after inocyanate-based mixed precipitation

[0063] Take 250 mL of enoxaparin esterification reaction solution and add 250 mL of methanol-anhydrous sodium acetate solution to the esterification solution while stirring. The methanol-anhydrous sodium acetate solution contains 250 mL of methanol and 25 g of anhydrous sodium acetate. Stir for 60 min to obtain a solid-liquid suspension after esterification and precipitation. The composition of other materials except esterified heparin is shown in Table 1 below.

[0064] Table 1. Composition of main materials in solid-liquid suspensions excluding heparin esterification

[0065] Material composition Percentage (subject to batch-to-batch variation; the following percentages are approximate). Benzyl chloride 13％ Anhydrous sodium acetate 5％ methanol 53％ dichloromethane 25％

[0066] Simultaneously, solid-liquid suspensions produced in the workshop were collected for various impurity removal examples or comparative examples. Using existing impurity removal methods, the solid-liquid suspensions were washed with methanol, with the methanol volume being approximately 50% of the suspension volume. For example, 500 ml of solid-liquid suspension yielded 12 g of dried esterified heparin, corresponding to 45 g of enoxaparin quaternary ammonium salt, resulting in a heparin production yield of 27%.

[0067] Example 2: Small-scale trials under various conditions

[0068] In the same batch of enoxaparin production, 27L of solid-liquid suspension material after enoxaparin mixed precipitation prepared according to the method of Example 1 was taken. Each group consisted of 500ml of solid-liquid suspension material. Purified water precipitation was carried out under different conditions in Table 3, using different water addition ratios and experimentally preset stirring and settling conditions. The precipitation phenomenon was observed. After settling, the supernatant was removed, the wet solid was taken, and then washed with methanol and dried to obtain dried enoxaparin.

[0069] Simultaneously, 500 ml of solid-liquid suspension was taken and washed and purified in batches with 250 ml of methanol, and then dried to obtain enoxaparin dried product (this operation is also referred to as "adjustment pretreatment").

[0070] The factors affecting the sedimentation effect were investigated as follows: stirring conditions (stirring speed, stirring time), sedimentation settling time, and the proportion of purified water added. The experimental parameters for each group are shown in Table 2.

[0071] Table 2

[0072]

[0073] Based on the execution conditions in Table 2, the impurity removal under different purified water ratios was studied under 9 different conditions. The specific conditions are described in Table 3.

[0074] Table 3

[0075]

[0076]

[0077] Complete small-scale tests were conducted according to the conditions described in Table 3. Dried heparin products were collected, and the yield of heparin was calculated. The results are shown in Tables 4 and 5.

[0078] Table 4

[0079]

[0080] Table 5

[0081]

[0082]

[0083] In each group of experiments, the precipitation phenomenon was more obvious for the ratio of purified water to water, as summarized in Table 6.

[0084] Table 6

[0085] Purified water addition ratio Precipitation phenomenon 6％ The liquid was cloudy with a small amount of white flocculent material precipitating out; this material settled to the bottom of the beaker after standing. 10％ The solid precipitate is firm, the supernatant is free of floating matter, and the supernatant is clear and not yellowish. 14％ The solid precipitate is firm, the supernatant is free of floating matter, and the supernatant is clear and not yellowish. 18％ The solid precipitate is firm, the supernatant is free of floating matter, and the supernatant is clear and not yellowish. 22％ The solid precipitate is firm, the supernatant is free of floating matter, and the supernatant is clear and not yellowish. 26％ The solid precipitate is viscous, and the supernatant is clear with no floating matter, but it is yellowish.

[0086] The experimental results under various conditions were studied, compared, and summarized. The relevant classifications, comparisons, and results are shown in Table 7 and... Figure 1-10 .

[0087] Table 7

[0088]

[0089] Results analysis:

[0090] Within the range of 0.5h-8h of standing, 10-100rpm of stirring speed and 10min-120min of stirring time, the yield of esterified heparin did not differ significantly. Among them, when the proportion of purified water added was 10%-22%, the yield of esterified heparin could be consistently above 27%.

[0091] Too short a settling time, too low a stirring speed, or too short a stirring time can all lead to insufficient mixing of purified water and organic solvent, resulting in incomplete collection of esterified heparin and affecting the yield.

[0092] Excessive standing time, excessive stirring speed, and excessive stirring time can all cause the supernatant to become turbid, making it difficult to distinguish the solid-liquid interface and affecting the yield.

[0093] Example 3: Mass Production Comparison

[0094] Based on the results of Example 2, the water precipitation effect of batch production was tracked, and the difference in esterification yield and impurity removal effect before and after adjustment were compared.

[0095] Select a purified water ratio of 16% and proceed with batch production according to the stirring conditions and settling time specified in Table 8. Specifically, control the stirring speed as shown in Table 8, agitate the solid-liquid suspension after the esterified heparin mixture precipitates, and calculate the required amount of purified water to add based on the solid-liquid suspension volume multiplied by 16%. Add water while stirring, and follow the stirring time specified in the table below. Then allow the mixture to stand, observe and record the precipitation phenomenon, and remove the supernatant (by centrifugation or supernatant extraction). To reduce material transfer operations and avoid unnecessary material loss, collect the wet solid esterified heparin by supernatant extraction, purify it with methanol, and then dry it to obtain the dried enoxaparin product (this step is detailed in Table 9 and...). Figure 11-15 (In Chinese, this is simply referred to as "purified water precipitation").

[0096] Simultaneously, the solid-liquid suspension was treated with half the volume of methanol for batch washing and purification, followed by drying to obtain enoxaparin-containing dried product (this operation is described in Table 9 and...). Figure 11-15 (referred to as "methanol washing" in Chinese).

[0097] The impurity content of the obtained heparin was determined, and the impurity removal effect and heparin yield were studied. The results are shown in Table 9. The data in Table 9 were compared according to different impurities, and the results are shown below. Figure 11-15 .

[0098] Table 8

[0099] Batch 1 Batch 2 Batch 3 Stirring speed 10rpm 100rpm 50rpm Stirring time 10min 120min 60min resting time 0.5h 8h 4h

[0100] Table 9

[0101]

[0102]

[0103] The comparison of the impurity removal ratio before and after the adjustment shows that using purified water precipitation, standing for 0.5h-8h, stirring speed of 10-100rpm, and stirring time of 10min-120min, the impurity removal effect is better than that of methanol washing.

[0104] A summary analysis of the yields of esterified heparin before and after adjustment under batch production conditions was conducted, and the results are shown in Table 10 and... Figure 15 .

[0105] Table 10

[0106]

[0107] As shown in Table 10, under batch production conditions, the precipitation phenomenon after adding purified water is the same as that observed in the small-scale test. This means that the results of the small-scale test on the proportion of purified water can be used as a reference. Furthermore, under a wide range of stirring, sedimentation and settling conditions, the yield of the purified water addition precipitation operation is at least 1% higher than that of the methanol washing operation.

[0108] After the mixed precipitation of esterified heparin, the solid-liquid suspension was changed from methanol washing to water precipitation with purified water. This reduces methanol usage, increases product yield, and further optimizes the impurity removal effect of the enoxaparin esterification process.

[0109] Furthermore, by reducing material transfer operations between equipment, production efficiency can be guaranteed while optimizing the process flow and improving the yield of esterified heparin.

[0110] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications may still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions may be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for removing impurities from an enoxin esterification reaction solution, characterized in that... Includes the following steps: (1) Add anhydrous sodium acetate solution to the enocyanate reaction solution and mix to precipitate to obtain a solid-liquid suspension; the anhydrous sodium acetate solution has a mass-volume ratio of anhydrous sodium acetate to methanol of 1:8-12 g / ml, the volume ratio of anhydrous sodium acetate solution to enocyanate reaction solution is 1-1.5:1, and the mixing and precipitation conditions are stirring at 20℃-30℃ for no less than 0.5 hours; (2) Add purified water to the solid-liquid suspension under stirring at 5-150 rpm until precipitation occurs, then stop stirring and let stand, remove the supernatant, take the wet solid, wash with methanol, and dry to obtain enoxaparin dried product. In step (2), the precipitation phenomenon includes one or more of the following: solid precipitate, no floating matter in the supernatant, and clear supernatant without yellowing; the amount of purified water added is 10%-22% of the volume of the solid-liquid suspension; the stirring time is 5-150 min; the standing time is 0.5-10 h; after standing, the supernatant is removed by centrifugation or extraction. The enoxaparin esterification reaction solution is obtained by using heparin sodium as a raw material, first reacting it with benzyl chloride in a salting reaction, and then reacting it with benzyl chloride in an esterification reaction during the preparation of enoxaparin sodium.

2. The method according to claim 1, characterized in that... In step (2), the stirring speed is 10-100 rpm; the stirring time is 10 min-120 min; the settling time is 0.5 h-8 h; after settling, the supernatant is removed by extraction.

3. A method for reducing the impurity content in enoxaparin sodium, characterized in that, The method includes the following steps: S1 salt formation: Sodium heparin reacts with benzyl chloride in a salting reaction to obtain enoxaparin quaternary ammonium salt; S2 Esterification: The enoxaparin quaternary ammonium salt is dissolved in dichloromethane and esterified with benzyl chloride to obtain the enoxaparin esterification reaction solution. S3 Impurity Removal: The enoxin esterification reaction solution is treated with the method of claim 1 to obtain enoxin esterified heparin; S4 Hydrolysis: The enoxaparin esterified is hydrolyzed to obtain enoxaparin sodium.

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