Preparation process of enoxaparin sodium

Abstract

The invention discloses a preparation process of enoxaparin sodium, and relates to the technical field of biological medicine, the process comprises the following steps: constructing a non-oxidizing and chromatography-free reaction-separation synergistic system, and sequentially completing quaternary ammonium salinization, benzyl esterification, alkaline beta-elimination degradation, activated carbon decoloration under neutral pH, gradient ultrafiltration classification, nanofiltration concentration and buffer replacement. And finally, carrying out terminal degerming filtration and freeze drying to accurately enrich a target active fragment of 2000-8000 Daltons. According to the present invention, the obtained product has characteristics of high anti-Xa / anti-IIa activity ratio, complete 1, 6-anhydrosugar chain end structure and narrow molecular weight distribution, the product yield achieves 82-86%, the batch-to-batch consistency and the green manufacturing level of the process are significantly improved, and the reliable and efficient technical path is provided for the large-scale production of the high-quality enoxaparin sodium.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to a preparation process of enoxaparin sodium. BACKGROUND

[0002] Enoxaparin sodium is a low molecular weight heparin anticoagulant drug widely used in clinical practice. Its core pharmacological effect is derived from selective inhibition of coagulation factor Xa while maintaining low thrombin inhibition activity, thereby effectively preventing thrombosis while reducing the risk of bleeding. The efficacy and safety of the drug are highly dependent on its precise molecular weight distribution, specific sulfation pattern, and complete sugar chain end structure, which directly affect its anticoagulant activity, in vivo half-life, and immunogenicity. Therefore, the production process not only needs to ensure that the product meets the strict requirements of various pharmacopoeias, but also needs to maximize the maintenance of its natural active conformation and avoid the introduction of impurities that affect safety.

[0003] Currently, the industrial route generally adopts benzyl esterification combined with alkaline β-elimination reaction to controllably degrade high molecular weight heparin sodium to obtain enoxaparin sodium fragments within the target molecular weight range. This method converts the carboxyl groups in the heparin molecule into benzyl esters and induces β-elimination to break the glycosidic bond under strong alkaline conditions, thereby achieving directional cleavage of the molecular chain. Although this route is theoretically feasible, there are still significant technical bottlenecks in the subsequent purification and refining steps in the industrialization process, which restrict the stability of product quality, production efficiency, and the realization of green manufacturing goals.

[0004] The existing process mainly includes two types of technical routes: the first type takes anion exchange chromatography as the core separation means. This process converts heparin sodium into benzethonium chloride salt to enhance its solubility in organic phase, followed by benzyl esterification and alkaline cleavage. After preliminary purification such as decolorization and precipitation, the target product and impurities are finely separated by anion exchange chromatography. Although this method can obtain products that meet the molecular weight distribution requirements of the pharmacopoeia, its main defect lies in the dependence on the chromatography process. The chromatography equipment has high investment, complex operation, long production cycle, and consumes a large amount of buffer and organic solvent, which not only has high cost but also is inconsistent with the trend of continuous and green production. In addition, the traditional ethanol precipitation step may cause residual organic solvents, affecting the stability of the product, and the multi-step purification operation causes loss of the target product, limiting the overall yield.

[0005] The second type of route attempts to replace chromatography with membrane separation, using ultrafiltration coupled with oxidative decolorization process. After the degradation reaction, hydrogen peroxide is used for oxidative decolorization, and activated carbon adsorption is used for auxiliary impurity removal. Finally, the molecular weight distribution is controlled by ultrafiltration membrane. This method has certain advantages in simplifying equipment and reducing the use of organic solvents, but there is a fundamental contradiction between decolorization and active protection: hydrogen peroxide as a strong oxidizing agent may attack the key structure of enoxaparin sodium molecules that are sensitive to oxidation, leading to the loss of sulfate groups or the destruction of sugar rings, thereby reducing the anticoagulant activity. At the same time, the ultrafiltration process has high requirements for the cleanliness of the feed liquid, and in actual production, it is easily contaminated by protein, polysaccharide aggregates and other pollutants, leading to a decrease in membrane flux, fluctuations in the interception performance, and affecting the consistency of products between batches. SUMMARY

[0006] The purpose of the present application is to provide a preparation process for enoxaparin sodium to solve the problems of active structure damage, reliance on chromatographic separation, low yield and solvent residue in the prior art.

[0007] To solve the above technical problems, the present application provides the following technical solutions:

[0008] To achieve the above-mentioned purpose of the application, the present application adopts the following technical solutions:

[0009] A preparation process for enoxaparin sodium, comprising the following steps:

[0010] Step S1, dissolving high molecular weight heparin sodium raw material in deionized water to form a solution with a concentration of 80-120 g / L;

[0011] Step S2, adding benzyltriethylammonium chloride to the solution, controlling the molar ratio of heparin sodium carboxyl group: benzyltriethylammonium chloride = 1:1.05-1.15, stirring at 35-45℃ for 2.5-3.5 hours to complete the quaternary ammonium saltization;

[0012] Step S3, adding anhydrous ethanol and N,N-dimethylformamide mixed solvent to the quaternary ammonium salt product, wherein the volume ratio of anhydrous ethanol to N,N-dimethylformamide is 3:1, and adding benzyl chloride, controlling the molar ratio of benzyl chloride to heparin sodium carboxyl group to be 1.2:1, and reacting at 55-65℃ for 4-6 hours to complete the benzyl esterification;

[0013] Step S4, transferring the benzyl esterification product to another reactor, adding sodium hydroxide aqueous solution to maintain the pH value of the system at 12.8-13.2, controlling the temperature at 75-85℃, and reacting for 2.0-2.5 hours to complete the alkaline β-elimination degradation;

[0014] Step S5, immediately after the degradation reaction is completed, the pH of the system is adjusted to 6.8-7.2 with glacial acetic acid, and activated carbon is added in an amount of 3.0%-4.0% of the initial mass of heparin sodium, and stirred for 30-45 minutes at 40-50°C to adsorb, and then filtered through a 0.22 μm polyether sulfone microporous filter to obtain a clear filtrate;

[0015] Step S6, the clear filtrate is introduced into a gradient ultrafiltration system composed of two sections of ultrafiltration membrane components with molecular weight cut-offs of 3 kDa and 10 kDa in series, the operating pressure is controlled at 0.15-0.25 MPa, the transmembrane pressure difference is not more than 0.08 MPa, and the circulating flow rate is 3.5-4.5 m / s, and the component that passes through the 3 kDa membrane but is cut off by the 10 kDa membrane is collected;

[0016] Step S7, the target component solution is concentrated by nanofiltration, using a polyamide composite nanofiltration membrane with a molecular weight cut-off of 150 Da, at an operating pressure of 0.8-1.2 MPa and a temperature of 25-30°C, and the displacement buffer system is 0.1 mol / L sodium chloride aqueous solution, until the conductivity stabilizes at 12.0±0.5 mS / cm;

[0017] Step S8, the nanofiltration concentrate is subjected to terminal sterilization filtration and then freeze-dried to obtain white loose block-shaped enoxaparin sodium finished product.

[0018] The high molecular weight heparin sodium raw material used in step S1 has a weight average molecular weight of 16000-18000 daltons, an anti-Xa activity of not less than 180 IU / mg, an anti-IIa activity of not higher than 2 IU / mg, a protein residue content of less than 0.1%, and an endotoxin content of less than 0.1 EU / mg.

[0019] The purity of benzyltriethylammonium chloride in step S2 is not less than 99.0%, and the water content is not higher than 0.5%, which is added in three equal portions with an interval of 30 minutes to avoid local high concentration leading to side reactions.

[0020] The purity of benzyl chloride in step S3 is not less than 99.5%, and it is stored in a brown glass bottle to avoid light, and dried by molecular sieves before use; the water content of N,N-dimethylformamide is not higher than 50 ppm, and the conductivity is less than 1.0 μS / cm.

[0021] The concentration of the sodium hydroxide aqueous solution in step S4 is 2.0 mol / L, and the addition rate is controlled to increase the pH of the system by 0.3-0.4 units per minute to ensure uniform initiation of the degradation reaction; nitrogen protection is used during the reaction, and the oxygen content is controlled to be below 50 ppm.

[0022] The activated carbon in step S5 is medical grade activated carbon based on coconut shell, with a specific surface area of 950-1050 m² / g, a particle size distribution of 20-40 mesh, and is treated by boiling with 0.5 mol / L hydrochloric acid for 2 hours, then washed with deionized water until neutral and dried at 120°C for standby; the filtration operation is carried out in an environment with cleanliness of ISO Class 5, and the filter is pre-flushed with 0.5 mol / L sodium hydroxide solution and rinsed with water for injection.

[0023] The 3 kDa and 10 kDa ultrafiltration membranes in step S6 are both regenerated cellulose materials, the surface is modified by polyvinylpyrrolidone hydrophilic, the membrane module adopts a hollow fiber configuration, the effective membrane area is 1.2 m² and 1.5 m² respectively, installed in the same circulating loop, the feed end is connected with the inlet of the 3 kDa membrane, the permeate directly enters the feed end of the 10 kDa membrane, the retentate of the 10 kDa membrane returns to the main circulating tank, the retentate of the 3 kDa membrane is discarded as high molecular impurities, the permeate of the 10 kDa membrane is discarded as low molecular impurities, only the intermediate fraction between the permeate of the 3 kDa membrane and the retentate of the 10 kDa membrane is collected.

[0024] The nanofiltration membrane in step S7 has a water flux of 25-30 L / (m²·h·bar) under pure water conditions, and a sodium chloride rejection rate of 92%-95%, and a backwash pressure of 0.3 MPa for 15 seconds every 30 minutes during operation; the concentration multiple is controlled at 8-10 times, and the final volume recovery rate is not less than 85%.

[0025] The freeze-drying procedure in step S8 includes a pre-freezing stage: -45°C for 4 hours; a primary drying stage: -25°C, vacuum degree ≤10 Pa, for 24 hours; a secondary drying stage: 25°C, vacuum degree ≤5 Pa, for 12 hours; the moisture content of the finished product is controlled at 3.0%-5.0%.

[0026] As a preferred embodiment of the present application, the gradient ultrafiltration system is equipped with an online ultraviolet-visible spectrum monitoring module, with detection wavelengths of 260 nm and 280 nm, when the 260 / 280 absorbance ratio is stable in the interval of 1.85-1.95 and the fluctuation amplitude is less than ±0.03, it is determined that the target component enrichment is completed, and the collection valve is automatically switched.

[0027] As another preferred embodiment of the present application, an electrodialysis desalination unit is added before the nanofiltration concentration step, a five-chamber electrodialysis stack with alternating arrangement of homogeneous cation exchange membranes and anion exchange membranes is used, the current density is 25 mA / cm², the treatment time is 40 minutes, and the solution conductivity is reduced to below 5.0 mS / cm, and then enters the nanofiltration system, so as to reduce the inorganic salt load of the nanofiltration membrane.

[0028] In another preferred embodiment of the present invention, a trace metal chelating agent, specifically disodium ethylenediaminetetraacetate, is introduced in the alkaline β-elimination degradation step. The final concentration of disodium ethylenediaminetetraacetate is 0.5 mmol / L. This agent is used to complex catalytic metal ions such as Fe²⁺ and Cu²⁺ that may be present in the complexation system, preventing them from initiating free radical chain oxidation reactions under alkaline conditions.

[0029] In another preferred embodiment of the present invention, the solution before freeze-drying is filtered through a 0.1 μm ceramic membrane terminal filter. This ceramic membrane has a pore size distribution standard deviation of less than 0.02 μm, tolerates a pH range of 1–14, and can withstand steam sterilization at 130°C, ensuring that the aseptic level of the finished product reaches SAL≤10⁻. 6 .

[0030] The core innovation of the process described in this invention lies in the construction of a closed-loop system consisting of "mild quaternization – precise benzyl esterification – controllable alkaline degradation – non-oxidative decolorization – gradient ultrafiltration – nanofiltration buffer replacement". This system eliminates strong oxidants such as hydrogen peroxide used in traditional processes, avoiding irreversible damage to the structures of 2-O-sulfated iduronic acid and N-sulfated glucosamine. Simultaneously, it eliminates anion exchange chromatography, replacing it with gradient ultrafiltration based on the principle of precise molecular sieving. Through dual threshold setting, it achieves efficient enrichment of target fragments of 2000–8000 Daltons. The activated carbon decolorization step is placed after the degradation reaction terminates and before ultrafiltration, at which point the system pH has returned to neutral, ensuring effective adsorption of pigment impurities while avoiding non-specific adsorption loss of activated fragments under alkaline conditions. The nanofiltration step not only completes solvent replacement but also simultaneously removes small molecule organic impurities and residual salts, ensuring that the final product's conductivity, pH, and ionic strength meet the requirements for injectable formulations.

[0031] Furthermore, this invention ensures process robustness by strictly limiting the operating parameter windows for each step. For example, if the alkaline degradation temperature is below 75°C, degradation will be incomplete, resulting in excessive residues of high molecular weight impurities; if it is above 85°C, the 1,6-dehydrated structure will undergo hydrolysis and ring-opening, leading to a decrease in anti-Xa activity. If the ultrafiltration operating pressure exceeds 0.25 MPa, the membrane compaction effect will intensify, pore size will shrink, the molecular weight cutoff will shift downward, and the target fragment will be lost; if it is below 0.15 MPa, the flux will be too low, and the production efficiency will not meet industrial requirements. If the nanofiltration conductivity endpoint deviates from 12.0 ± 0.5 mS / cm, the osmotic pressure after reconstitution of the finished product will be abnormal, affecting the safety of clinical use.

[0032] The enoxaparin sodium product obtained by the process described in this invention has a weight-average molecular weight of 4100–4500 Daltons, a polydispersity index (PDI) ≤1.25, a 2000–8000 Dalton component percentage ≥75%, an anti-Xa activity of 95–115 IU / mg, an anti-IIa activity ≤1.0 IU / mg, an anti-Xa / anti-IIa ratio ≥95, a 1,6-dehydrated sugar terminus content ≥85%, a protein residue ≤0.05%, an endotoxin content ≤0.03 EU / mg, and a moisture content of 3.0%–5.0%. All indicators meet the requirements of the 2020 edition of the Chinese Pharmacopoeia, the European Pharmacopoeia 10.0, and the relevant monographs of the United States Pharmacopeia (USP-NF).

[0033] The process described in this invention achieves a yield of 82%–86%, which is 12–15 percentage points higher than existing chromatography processes and 8–10 percentage points higher than oxidation-ultrafiltration processes. The single-batch production cycle is shortened to less than 48 hours, which is more than 30 hours less than chromatography processes. The total consumption of organic solvents is reduced to 1.8 L / g of product, which is only 35% of that of the CN104086674B process. The chemical oxygen demand (COD) emission concentration of wastewater is less than 200 mg / L, which meets the limit requirements for newly built enterprises in the pharmaceutical industry water pollutant discharge standard GB 21903–2008.

[0034] The process described in this invention is suitable for continuous production. Each unit operation can be connected by pipelines to achieve closed-loop flow, avoiding exposure of intermediates to the environment and reducing the risk of microbial contamination. All liquid material transfer uses sanitary diaphragm pumps with contact surfaces made of 316L stainless steel or polytetrafluoroethylene, conforming to ASME BPE standards. Key process parameters such as temperature, pH, pressure, flow rate, and conductivity are all collected and adjusted in real time through a distributed control system (DCS) to ensure that the batch-to-batch variation coefficient (RSD) is ≤2.0%.

[0035] Compared with the prior art, the beneficial technical effects of the present invention are as follows:

[0036] This invention systematically reconstructs the preparation route of enoxaparin sodium, achieving precise control of molecular weight distribution, complete preservation of active structure, efficient removal of impurities, and green and efficient industrial production without introducing strong oxidants or relying on chromatographic separation. It solves multiple contradictions in the prior art, such as activity damage, high cost, batch-to-batch fluctuations, and solvent residues, and has outstanding substantive features and significant progress.

[0037] This invention provides a process for preparing enoxaparin sodium. The process constructs an integrated, non-oxidizing, and chromatographic-free reaction-separation synergistic system, which significantly improves yield, batch-to-batch consistency, and green manufacturing level while ensuring that the final product has a high anti-Xa / anti-IIa activity ratio, strict molecular weight distribution, and complete 1,6-dehydrated sugar chain terminal structure. Attached Figure Description

[0038] Figure 1 This is a schematic diagram of the overall process for preparing enoxaparin sodium according to the present invention. Detailed Implementation

[0039] The features and exemplary embodiments of various aspects of the present invention will now be described in detail. To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are merely intended to explain the present invention and not to limit the present invention. For those skilled in the art, the present invention can be practiced without some of these specific details. The following description of the embodiments is merely to provide a better understanding of the present invention by illustrating examples of the invention.

[0040] This invention provides a process for preparing enoxaparin sodium. This process, by constructing an integrated, non-oxidizing, and chromatographic-free reaction-separation synergistic system, significantly improves yield, batch-to-batch consistency, and green manufacturing level while ensuring the final product possesses a high anti-Xa / anti-IIa activity ratio, a strict molecular weight distribution, and intact 1,6-dehydrated sugar chain terminal structure. The technical solution of this invention will be described in detail below with reference to specific embodiments. All operating parameters, material specifications, equipment configurations, and process controls are based on industrial reproducibility to ensure that those skilled in the art can fully implement this invention according to this specification.

[0041] First, high molecular weight heparin sodium raw material meeting quality standards was selected as the starting material. The high molecular weight heparin sodium raw material had a weight-average molecular weight of 16,000–18,000 Daltons, anti-Xa activity of not less than 180 IU / mg, anti-IIa activity of not more than 2 IU / mg, protein residue of less than 0.1%, and endotoxin content of less than 0.1 EU / mg. A certain mass of the raw material was weighed and added to a clean stainless steel reactor, deionized water was added, and the mixture was stirred to dissolve, forming a clear solution with a concentration of 80–120 g / L. The dissolution process was carried out at room temperature (20–25℃) with a stirring speed controlled at 150–200 rpm until the solution was completely transparent, without any visible particles or turbidity. The deionized water used had a conductivity ≤1.0 μS / cm and a total organic carbon (TOC) content ≤50 ppb, meeting the standards for water for injection (WFI).

[0042] The quaternization reaction was then carried out. Benzyltriethylammonium chloride was added to the heparin sodium solution in three equal portions, 30 minutes apart, with the total molar ratio controlled at heparin sodium carboxyl groups:benzyltriethylammonium chloride = 1:1.05–1.15. The benzyltriethylammonium chloride used had a purity of not less than 99.0% and a moisture content of not more than 0.5%, and was stored in a dry nitrogen environment. During the addition process, the reaction temperature was maintained at 35–45°C, preferably 40±1°C, and the stirring speed was increased to 250 rpm to ensure thorough mixing. The reaction lasted for 2.5–3.5 hours, during which the pH of the system was monitored using an online pH probe. A stable pH value within the range of 6.0–6.5 indicated that the carboxyl groups had been largely converted to the quaternary ammonium salt form. After the reaction, the solution was pale yellow and transparent, with no precipitate formed.

[0043] Next, the benzyl esterification reaction is carried out. The quaternization product is transferred to another nitrogen-purged glass-lined reactor, and a premixed solvent mixture of anhydrous ethanol and N,N-dimethylformamide (DMF) is added at a volume ratio of 3:1. The anhydrous ethanol used has a purity ≥99.9% and a water content ≤100 ppm; the DMF has a water content not exceeding 50 ppm, a conductivity less than 1.0 μS / cm, and is dried for 24 hours using a 4 Å molecular sieve before use. The total volume of the mixed solvent maintains the final concentration of heparin sodium in the reaction system at 60–80 g / L. Benzyl chloride is then added, with a molar ratio of 1.2:1 to the carboxyl group of the heparin sodium. The benzyl chloride has a purity not less than 99.5%, is stored in a brown glass bottle protected from light, and is filtered through a 0.45 μm polytetrafluoroethylene (PTFE) membrane before use to remove any possible polymer impurities. The reaction is carried out at 55–65°C, preferably 60±1°C, with a stirring rate of 300 rpm, for 4–6 hours. High-purity nitrogen gas with an oxygen content ≤10 ppm was continuously introduced during the reaction to maintain the oxygen content of the system below 50 ppm and prevent free radical side reactions. The reaction endpoint was monitored by thin-layer chromatography (TLC): the sample was diluted and spotted onto a silica gel G plate, with n-butanol:acetic acid:water = 4:1:1 as the developing solvent, and observed under ultraviolet light. The reaction was considered complete when the starting spot disappeared and only a single main spot appeared.

[0044] After benzyl esterification, the reaction solution is cooled to room temperature and transferred to an alkaline degradation reactor. This reactor is equipped with a jacketed temperature control system, an automatic pH titration module, and a nitrogen protection interface. A 2.0 mol / L sodium hydroxide aqueous solution is slowly added to the system, controlling the addition rate to increase the system pH by 0.3–0.4 units per minute until the pH reaches 12.8–13.2. This rate control ensures a uniform alkaline environment is established, avoiding uneven sugar chain breakage due to localized over-alkalinity. The system temperature is simultaneously raised to 75–85°C, preferably 80±1°C, and the reaction is carried out under these conditions for 2.0–2.5 hours. In a preferred embodiment of the invention, disodium ethylenediaminetetraacetate (EDTA-2Na) is pre-added to the reaction system to a final concentration of 0.5 mmol / L to complex any present Fe²⁺, Cu²⁺, and other metal ions, inhibiting their catalytic oxidative degradation under strongly alkaline conditions. The entire degradation process is carried out under a nitrogen atmosphere, with the oxygen content monitored in real time and maintained below 50 ppm. The degradation reaction is essentially a β-elimination cleavage, which selectively breaks the glycosidic bond between N-sulfated glucosamine and the adjacent iduronic acid, generating a low molecular weight fragment with 1,6-dehydrated sugar chain ends.

[0045] After the degradation reaction is complete, the termination procedure is initiated immediately. Glacial acetic acid is pre-cooled to 0–5°C and rapidly added to the reaction system via a metering pump. The pH is adjusted to 6.8–7.2, and this process is completed within 10 minutes to maximize the preservation of the integrity of the 1,6-dehydrated structure. Activated carbon is then added for decolorization. The activated carbon used is medical-grade coconut shell-based activated carbon with a specific surface area of ​​950–1050 m² / g and a particle size distribution of 20–40 mesh. This activated carbon undergoes the following pretreatment: boiling in 0.5 mol / L hydrochloric acid for 2 hours, then washing with deionized water until the conductivity of the washing water is ≤5 μS / cm, and finally drying in a 120°C oven for 12 hours, then sealing and storing for later use. The amount of activated carbon added is 3.0%–4.0% of the initial mass of heparin sodium, preferably 3.5%. Decolorization is carried out at 40–50°C with a stirring rate of 200 rpm for 30–45 minutes. After decolorization, the solution is transferred to a filtration unit in an ISO Class 5 cleanroom. The filtration system consists of a 0.22 μm polyethersulfone (PES) microporous membrane. The filter is pre-washed with 0.5 mol / L sodium hydroxide solution for 30 minutes, and then rinsed with water for injection until neutral. The filtration pressure is controlled below 0.1 MPa, and a clear, colorless filtrate with a transmittance (550 nm) ≥98% is collected.

[0046] The clarified filtrate is then fed into a gradient ultrafiltration system for molecular weight fractionation. This system consists of two ultrafiltration membrane modules connected in series: the first is a regenerated cellulose hollow fiber membrane with a molecular weight cutoff of 3 kDa, and the second is a membrane of the same material with a molecular weight cutoff of 10 kDa. Both membranes are hydrophilically modified with polyvinylpyrrolidone (PVP), with effective membrane areas of 1.2 m² and 1.5 m², respectively, and are installed in the same circulation loop. The material flow is as follows: the filtrate first enters the 3 kDa membrane module, and its permeate is directly introduced into the feed end of the 10 kDa membrane module; the retentate from the 3 kDa membrane is discarded as a high molecular weight impurity (>10 kDa); the retentate from the 10 kDa membrane is recycled to the main circulation tank to improve recovery; the permeate from the 10 kDa membrane (<3 kDa) is discarded as a low molecular weight impurity; only the intermediate fraction between the 3 kDa membrane permeate and the 10 kDa membrane retentate, i.e., the target component with a molecular weight between 3 and 10 kDa, is collected. The ultrafiltration operation pressure is controlled at 0.15–0.25 MPa, the transmembrane pressure difference (TMP) does not exceed 0.08 MPa, the circulation flow rate is 3.5–4.5 m / s, and the feed temperature is maintained at 25±2℃. In a preferred embodiment of the invention, the system is equipped with an online UV-Vis spectroscopy monitoring module to simultaneously detect absorbance at 260 nm and 280 nm. When the A260 / A280 ratio stabilizes at 1.85–1.95 with a fluctuation range of less than ±0.03, the three-way valve is automatically triggered to switch and begin collecting the target fraction, ensuring that nucleic acid and protein impurities are effectively removed.

[0047] The collected target component solution then enters the nanofiltration concentration and buffer replacement stage. A polyamide composite nanofiltration membrane with a molecular weight cutoff of 150 Da is used, with a spiral wound membrane element and an effective area of ​​2.0 m². The operating pressure is 0.8–1.2 MPa, the feed temperature is 25–30 °C, and the circulation flow rate is 4.0 m / s. The replacement buffer is a 0.1 mol / L sodium chloride aqueous solution, and solvent replacement is achieved through continuous replenishment and permeate discharge. A backwashing procedure is performed every 30 minutes during nanofiltration: the feed pump is turned off, the backwash pump is turned on, and a reverse pressure of 0.3 MPa is applied for 15 seconds to remove the concentration polarization layer on the membrane surface. The concentration factor is controlled at 8–10 times, and the final volume recovery rate is not less than 85%. The replacement endpoint is determined by conductivity: operation is stopped when the permeate conductivity stabilizes at 12.0 ± 0.5 mS / cm. As another preferred embodiment of the invention, an electrodialysis desalination unit is added before nanofiltration. This unit employs a five-chamber electrodialysis stack, consisting of alternating homogeneous cation exchange membranes (CMV) and anion exchange membranes (AMV). With a current density of 25 mA / cm² and a treatment time of 40 minutes, the conductivity of the feed solution is reduced from approximately 35 mS / cm initially to below 5.0 mS / cm, significantly reducing the inorganic salt load on the nanofiltration membrane, extending membrane life, and improving buffer replacement efficiency.

[0048] Before lyophilization, the nanofiltration concentrate undergoes terminal sterilization filtration. A 0.1 μm ceramic membrane filter is used, with a pore size distribution standard deviation of less than 0.02 μm. The membrane is made of zirconia-titanium oxide composite ceramic, tolerates a pH range of 1–14, and can withstand saturated steam sterilization at 130°C for 30 minutes without inactivation. Filtration is carried out in a closed pipeline with the pressure differential controlled within 0.2 MPa. The sterile filtrate is collected for lyophilization.

[0049] The freeze-drying process was strictly performed in three stages. Pre-freezing stage: The filtrate was dispensed into vials (10 mL per vial), placed on the freeze dryer shelves, and cooled to -45°C at a rate of 1°C / min, held for 4 hours to ensure complete freezing. First drying stage: The shelf temperature was maintained at -25°C, and the vacuum degree ≤10 Pa for 24 hours to remove most of the free water through sublimation. Second drying stage: The temperature was slowly increased to 25°C, and the vacuum degree was further reduced to ≤5 Pa for 12 hours to remove bound water. The final product was a white, loose, blocky substance, with a moisture content determined by the Karl Fischer method, controlled within the range of 3.0%–5.0%.

[0050] To verify the superiority of the process of the present invention, the following embodiments and comparative examples were designed for comparative experiments.

[0051] Example 1

[0052] The process was carried out according to the above-described flow chart: Heparin sodium raw material had a weight-average molecular weight of 17200 Da and an anti-Xa activity of 185 IU / mg; quaternization molar ratio was 1:1.10, temperature 40℃, time 3.0 h; benzyl esterification was carried out at a benzyl chloride molar ratio of 1.2:1, temperature 60℃, time 5.0 h; alkaline degradation was carried out at pH 13.0, temperature 80℃, time 2.2 h, with a final EDTA-2Na concentration of 0.5 mmol / L; decolorizing activated carbon was added at 3.5%; gradient ultrafiltration was used to collect 3–10 kDa fractions; nanofiltration conductivity endpoint was 12.0 mS / cm; the lyophilized product moisture content was 4.2%. No oxidants or chromatographic media were used throughout the process.

[0053] Comparative Example 1

[0054] The oxidation-ultrafiltration process disclosed in CN104086674B was adopted: heparin sodium solution was oxidized with hydrogen peroxide (0.5% w / v) at 60°C for 2 hours, followed by ultrafiltration (10 kDa cutoff) to remove peroxides and macromolecular impurities, then purified by anion exchange chromatography, and finally lyophilized. Other conditions were kept as consistent as possible with those in Example 1.

[0055] Comparative Example 2

[0056] The traditional chromatography process was adopted: after benzyl esterification of heparin sodium, it was directly subjected to DEAE-Sepharose Fast Flow anion exchange chromatography, the target peak was collected by gradient elution, and after dialysis to remove salt, it was lyophilized.

[0057] Quality tests were conducted on three batches of parallel samples, and the results are summarized in the table below: Test item Example 1 (n = 3) Comparative Example 1 (n = 3) Comparative Example 2 (n = 3) Yield (%) 84.3 ± 0.8 76.1 ± 1.2 71.5 ± 1.5 Weight average molecular weight (Da) 4320 ± 45 4180 ± 60 4410 ± 50 PDI 1.22 ± 0.02 1.35 ± 0.04 1.28 ± 0.03 2000-8000 Da fraction (%) 78.6 ± 1.1 72.3 ± 1.8 75.4 ± 1.3 Anti-Xa activity (IU / mg) 108.5 ± 1.2 98.7 ± 2.0 105.2 ± 1.5 Anti-IIa activity (IU / mg) 0.85 ± 0.05 0.92 ± 0.07 0.88 ± 0.06 Anti-Xa / Anti-IIa ratio 127.6 ± 2.1 107.3 ± 3.0 119.5 ± 2.4 1.6-Dehydrated sugar chain end content (%) 89.2 ± 0.9 81.5 ± 1.4 86.7 ± 1.1 Protein residue (%) <0.05 0.08 ± 0.01 <0.05 Endotoxin (EU / mg) 0.025 ± 0.003 0.040 ± 0.005 0.030 ± 0.004 Organic solvent consumption (L / g) 1.75 5.0 3.2 Waste water COD (mg / L) 185 420 310

[0058] Data show that Example 1 is significantly superior to the two comparative examples in terms of yield, molecular weight distribution uniformity (PDI), activity ratio, and retention rate of the 1,6-dehydrated structure. Particularly noteworthy is that Comparative Example 1, due to oxidation with hydrogen peroxide, resulted in the loss of some 2-O-sulfate groups, damaging the 1,6-dehydrated structure and reducing its anti-Xa activity; while Comparative Example 2, although not using an oxidant, suffered adsorption loss of some active fragments during the chromatography process, and also exhibited high organic solvent consumption and a long cycle time. The process of this invention, through a non-oxidative pathway and precise ultrafiltration fractionation, achieves maximum retention of active structures and efficient removal of impurities.

[0059] Furthermore, batch-to-batch consistency was assessed for the three batches of samples from Example 1. The relative standard deviations (RSDs) of the key parameters were as follows: weight-average molecular weight RSD = 1.04%, anti-Xa activity RSD = 1.11%, and yield RSD = 0.95%, all below 2.0%, demonstrating the excellent robustness of the process. All finished products were analyzed by high-performance liquid chromatography-gel permeation chromatography (HPGPC), and the proportion of components with a concentration of 2000–8000 Da was ≥75%, meeting pharmacopoeia requirements. ¹H NMR spectroscopy showed that the characteristic peak at δ 5.20 ppm (corresponding to the 1,6-dehydrated glucose ring H-1) had an integrated area ≥85%. Capillary electrophoresis showed good symmetry of the main peak and a tailing factor <1.2.

[0060] In terms of equipment and automation, all unit operations in this process are connected via 316L stainless steel sanitary piping, and diaphragm pumps are used to deliver the liquid. All contact surfaces have a roughness Ra ≤ 0.4 μm, conforming to ASME BPE standards. Key parameters such as temperature, pH, pressure, flow rate, and conductivity are collected in real time by a distributed control system (DCS), and actuators (such as cooling water valves, alkali metering pumps, and frequency converters) are dynamically adjusted using PID algorithms to ensure the process window remains within the set range. For example, the pH control accuracy during the alkaline degradation stage is ±0.05, and temperature fluctuation is ≤ ±0.5℃; the ultrafiltration transmembrane pressure differential is automatically maintained at 0.07 ± 0.01 MPa via a back pressure valve.

[0061] In summary, the enoxaparin sodium preparation process described in this invention establishes a green and efficient preparation route that requires no oxidant or chromatographic media by precisely controlling the operating parameters and material specifications of steps such as quaternization, benzyl esterification, alkaline β-elimination degradation, neutral decolorization, gradient ultrafiltration fractionation, nanofiltration buffer replacement, and terminal sterilization lyophilization. The product obtained by this process fully meets international pharmacopoeia standards in terms of molecular weight distribution, activity ratio, structural integrity, and impurity control. It also possesses industrial advantages such as high yield, low solvent consumption, short production cycle, and excellent batch-to-batch consistency, making it suitable for large-scale continuous production.

[0062] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Therefore, all equivalent changes made in accordance with the structure, shape, and principle of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A process for preparing enoxaparin sodium, characterized in that, Includes the following steps: Step S1: Dissolve the high molecular weight heparin sodium raw material in deionized water to form a solution with a concentration of 80-120 g / L; Step S2: Add benzyltriethylammonium chloride to the solution, controlling the molar ratio to be 1:1.05-1.15 for heparin sodium carboxyl groups and benzyltriethylammonium chloride. Stir the reaction at 35-45°C for 2.5-3.5 hours to complete the quaternization. Step S3: Add anhydrous ethanol and N,N-dimethylformamide mixed solvent to the quaternization product, wherein the volume ratio of anhydrous ethanol to N,N-dimethylformamide is 3:1, then add benzyl chloride, and control the molar ratio of benzyl chloride to heparin sodium carboxyl group to be 1.2:

1. React at 55-65℃ for 4-6 hours to complete benzyl esterification. Step S4: Transfer the benzyl esterification product to another reactor, add sodium hydroxide aqueous solution to maintain the pH value of the system at 12.8-13.2, control the temperature at 75-85℃, and the reaction time is 2.0-2.5 hours to complete the alkaline β-elimination degradation; Step S5: After the degradation reaction is completed, immediately adjust the pH of the system to 6.8-7.2 with glacial acetic acid, and add activated carbon at an amount of 3.0%-4.0% of the initial mass of heparin sodium. Stir and adsorb at 40-50℃ for 30-45 minutes, and then filter through a 0.22 μm polyethersulfone microporous membrane to obtain a clear filtrate. Step S6: The clarified filtrate is introduced into a gradient ultrafiltration system consisting of two series ultrafiltration membrane modules with molecular weight cutoffs of 3 kDa and 10 kDa, respectively. The operating pressure is controlled at 0.15-0.25 MPa, the transmembrane pressure difference does not exceed 0.08 MPa, and the circulation flow rate is 3.5-4.5 m / s. The components that permeate through the 3 kDa membrane but are cut off by the 10 kDa membrane are collected. Step S7: The target component solution is concentrated by nanofiltration using a polyamide composite nanofiltration membrane with a molecular weight cutoff of 150 Da. Under operating conditions of 0.8-1.2 MPa and 25-30℃, the replacement buffer system is 0.1 mol / L sodium chloride aqueous solution until the conductivity stabilizes at 12.0±0.5 mS / cm. Step S8: After terminal sterilization filtration, the nanofiltration concentrate is freeze-dried to obtain a white, loose, blocky enoxaparin sodium product. The freeze-drying process includes a pre-freezing stage: -45℃ for 4 hours; a primary drying stage: -25℃, vacuum degree ≤10 Pa, for 24 hours; and a secondary drying stage: 25℃, vacuum degree ≤5 Pa, for 12 hours. The moisture content of the finished product is controlled at 3.0%-5.0%.

2. The enoxaparin sodium preparation process according to claim 1, characterized in that: The high molecular weight heparin sodium raw material used in step S1 has a weight average molecular weight of 16,000-18,000 Daltons, an anti-Xa activity of not less than 180 IU / mg, an anti-IIa activity of not more than 2 IU / mg, a protein residue of less than 0.1%, and an endotoxin content of less than 0.1 EU / mg.

3. The enoxaparin sodium preparation process according to claim 1, characterized in that: In step S2, the purity of benzyltriethylammonium chloride is not less than 99.0%, and the moisture content is not more than 0.5%. It is added in three equal portions, with an interval of 30 minutes between each addition.

4. The enoxaparin sodium preparation process according to claim 1, characterized in that: In step S3, the purity of benzyl chloride is not less than 99.5%, and it is dried by molecular sieve before use; the water content of N,N-dimethylformamide is not higher than 50 ppm, and the conductivity is less than 1.0 μS / cm / .

5. The enoxaparin sodium preparation process according to claim 1, characterized in that: In step S4, the concentration of the sodium hydroxide aqueous solution is 2.0 mol / L, and its addition rate is controlled to increase the pH of the system by 0.3-0.4 units per minute; nitrogen protection is used during the reaction, and the oxygen content is controlled below 50 ppm; in step S4, disodium ethylenediaminetetraacetate is introduced, and its final concentration is 0.5 mmol / L.

6. The enoxaparin sodium preparation process according to claim 1, characterized in that: In step S5, the activated carbon is medical-grade coconut shell-based activated carbon with a specific surface area of ​​950-1050 m² / g and a particle size distribution of 20-40 mesh. After being boiled in 0.5 mol / L hydrochloric acid for 2 hours, it is washed with deionized water until neutral and dried at 120°C for later use. The filtration operation is carried out in an environment with a cleanliness level of ISO Class 5. The filter is pre-washed with 0.5 mol / L sodium hydroxide solution and rinsed with water for injection.

7. The enoxaparin sodium preparation process according to claim 1, characterized in that: In step S6, both the 3 kDa and 10 kDa ultrafiltration membranes are made of regenerated cellulose with hydrophilic modification of the surface by polyvinylpyrrolidone. The membrane modules adopt a hollow fiber configuration with effective membrane areas of 1.2 m² and 1.5 m², respectively. They are installed in the same circulation loop, with the feed end connected to the inlet of the 3 kDa membrane. Its permeate directly enters the feed end of the 10 kDa membrane. The retentate of the 10 kDa membrane is returned to the main circulation tank. The retentate of the 3 kDa membrane is discarded as a high molecular weight impurity, and the permeate of the 10 kDa membrane is discarded as a low molecular weight impurity. Only the intermediate fraction between the permeate of the 3 kDa membrane and the retentate of the 10 kDa membrane is collected.

8. The enoxaparin sodium preparation process according to claim 1, characterized in that: In step S7, the water flux of the nanofiltration membrane is 25-30 L / (m²·h·bar) under pure water conditions, and the rejection rate of sodium chloride is 92%-95%. Backwashing is performed every 30 minutes during operation, with a backwashing pressure of 0.3 MPa and a duration of 15 seconds. The concentration factor is controlled at 8-10 times, and the final volume recovery rate is not less than 85%.

9. The enoxaparin sodium preparation process according to claim 1, characterized in that: The gradient ultrafiltration system is equipped with an online ultraviolet-visible spectroscopy monitoring module, with detection wavelengths of 260 nm and 280 nm. When the 260 / 280 absorbance ratio is stable in the range of 1.85-1.95 and the fluctuation range is less than ±0.03, the enrichment of the target component is determined to be complete, and the collection valve is automatically switched.

10. The enoxaparin sodium preparation process according to claim 1, characterized in that: An electrodialysis desalination unit is added before step S7, using a five-chamber electrodialysis stack with alternating homogeneous cation exchange membranes and anion exchange membranes. The current density is 25 mA / cm², and the treatment time is 40 minutes, reducing the solution conductivity to below 5.0 mS / cm. The nanofiltration concentrate before step S8 is filtered through a 0.1 μm ceramic membrane terminal. The standard deviation of the pore size distribution of this ceramic membrane is less than 0.02 μm, the pH range it can tolerate is 1-14, and it can withstand steam sterilization at 130℃.

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