Quality control method for low molecular weight heparin

Abstract

This invention belongs to the field of low molecular weight heparin production technology, specifically relating to a method for determining the concentration of low molecular weight heparin. The method provided by this invention involves dissolving and diluting a sample in purified water to prepare gradient solutions of different concentrations. The TOC values ​​of samples at different concentrations are measured using a TOC analyzer, and a calibration curve between TOC and sample concentration is plotted to obtain a linear equation. Then, the TOC value of the intermediate product solution during the production process is measured using a TOC analyzer, and the product concentration is calculated using the linear equation. The method for determining the concentration of low molecular weight heparin provided by this invention features simple operation, short detection time, and high accuracy. It is suitable for rapid detection of product concentration during the preparation of dalteparin sodium or nadroparin calcium, facilitating concentration control and ensuring that the final product quality meets EP standard requirements.

Description

Technical Field

[0001] This invention belongs to the field of low molecular weight heparin production technology, and specifically relates to a quality control method for low molecular weight heparin. Background Technology

[0002] Heparin is a sulfated glycosaminoglycan compound, primarily produced by mast cells and basophils. Its molecular structure is extremely complex, consisting of alternating components of glucosamine, L-iduronide, N-acetylglucosamine, and D-glucuronic acid. Currently, unfractionated heparin is extracted and purified from porcine small intestinal mucosa and is mainly used for the prevention and treatment of thromboembolic diseases and as an anticoagulant in hemodialysis.

[0003] Low molecular weight heparin is prepared by chemical or enzymatic cleavage of unfractionated heparin. It has a higher anti-FXa / anti-FIIa potency ratio and fewer bleeding side effects, and is more widely used in clinical practice. The European Pharmacopoeia (EP) classifies low molecular weight heparin into nadroparin calcium, dalteparin sodium, enoxaparin sodium, parheparin sodium, and tinhexaparin sodium according to different manufacturing processes. Low molecular weight heparin prepared by different methods has different molecular weight distributions and terminal structures, resulting in different physicochemical properties.

[0004] Dalteparin sodium and nadroparin calcium are prepared by nitrous acid pyrolysis. The products prepared by this method do not contain carbon-carbon double bonds and therefore have no ultraviolet absorption, making it difficult to directly determine the product concentration in the intermediate solution. Currently, traditional gel size exclusion chromatography, combined with GPC software calculations, can only determine the molecular weight and molecular weight distribution of dalteparin sodium and nadroparin calcium, but cannot determine their concentration.

[0005] Patent document CN103149170A discloses a method for determining the solution concentration of heparin calcium in nagqu using o-phenanthroline-zinc sulfate ultraviolet spectroscopy, which involves measuring Zn... 2+ The concentration of the product was determined by the ultraviolet absorbance of a mixed solution of ions, o-phenanthroline, and nadroparin calcium. Zhang Yun et al., in their article "Study on the Determination of Heparin Content by Ultraviolet Spectrophotometry," pointed out that methylene blue can undergo a complexation reaction with heparin, producing a characteristic peak at 664 nm, which can then be used to determine the heparin concentration spectrophotometrically. Si Wenhui et al., in their article "Determination of Heparin Sodium by Night Blue Decolorization Spectrophotometric Method," indicated that in an acetate-sodium acetate buffer solution at pH 4.5, the interaction between heparin sodium and night blue causes the night blue to decolorize, allowing the concentration of heparin sodium to be determined spectrophotometrically at 618 nm.

[0006] While the above methods can all determine heparin concentration, they all suffer from drawbacks such as cumbersome operation, long detection time, small linear range, and susceptibility to interference. In the preparation of dalteparin sodium and nadroparin calcium, it is necessary to promptly measure the product concentration of the ultrafiltration permeate and the feed solution before alcohol precipitation. This is because some small-molecule heparin will pass through the filter membrane and be removed during ultrafiltration. The ultrafiltration endpoint must be properly controlled. If the ultrafiltration time is insufficient, the permeate product concentration will be high, potentially resulting in a higher content of small molecules in the final product. If the ultrafiltration time is too long, small molecules will be removed too thoroughly, resulting in a very low permeate concentration, potentially leading to a low content of small molecules in the final product, a high weight-average molecular weight, and a lower product yield. Furthermore, based on past experience, the product concentration of the feed solution before alcohol precipitation needs to be controlled within a certain range to ensure that the precipitate particle size is within an appropriate range, facilitating ethanol removal during drying. If the concentration is too low, the particles will be too fine, resulting in a sticky product after centrifugation, a high wet weight, and significantly increased ethanol and water content, which is detrimental to drying. If the concentration is too high, the precipitate particles will be large, hindering the diffusion of ethanol during drying. Therefore, researching and developing a rapid and accurate method for determining the concentration of low molecular weight heparin products, for subsequent process control, and to ensure that the final product quality meets the requirements of EP standards remains an unsolved problem. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a quality control method for low molecular weight heparin. This method calculates the product concentration by determining the TOC value of the intermediate solution from the ultrafiltration or alcohol precipitation step in the low molecular weight heparin preparation process. Specifically, it involves selecting multiple batches of dalteparin sodium (molecular structure formula as shown)... Figure 1 (as shown) or nadroneparin calcium (molecular structure formula as shown) Figure 2 The self-made product (shown) was prepared into solutions with different concentration gradients using purified water. The TOC values ​​of heparin sodium or nadroparin calcium solutions at different concentrations were measured. After subtracting the blank, a concentration-TOC calibration curve was plotted to obtain a linear equation. Then, by measuring the TOC value of the intermediate solution during the production process, the product concentration was calculated using the linear equation, and then subsequent process control was carried out.

[0008] This invention provides a quality control method for low molecular weight heparin, comprising the following steps:

[0009] Step S1: Take the sample, dissolve and dilute it with purified water to prepare gradient solutions of different concentrations, and obtain the sample solution;

[0010] Step S2: Determine the TOC value of the sample solution prepared in step S1 using a TOC analyzer. Each sample solution is measured twice in parallel. Plot the TOC-sample concentration calibration curve and obtain the linear equation.

[0011] Step S3: During the preparation of low molecular weight heparin, the TOC value of the test solution is measured. The concentration of low molecular weight heparin in the test solution is calculated using the linear equation in step S2. If necessary, ultrafiltration or water is added to control the concentration of the test solution within a certain range so that the final product quality meets the EP standard requirements.

[0012] Furthermore, the sample in step S1 refers to dalteparin sodium or nadroparin calcium finished product, and the test solution in step S3 refers to the ultrafiltration permeate and the product solution before alcohol precipitation during the preparation process of dalteparin sodium or nadroparin calcium.

[0013] Further, the sample solution in step S1 refers to a gradient solution with concentrations of 5 mg / L, 10 mg / L, 15 mg / L, 20 mg / L, and 25 mg / L.

[0014] Furthermore, the TOC analyzer in step S2 is a GE Sievers M9 TOC analyzer.

[0015] Furthermore, the determination conditions of the TOC analyzer in steps S2 and S3 are as follows: acid solution is set to 0.3~0.6 mL, oxidant solution is set to 0.3~0.6 mL, rinsing time is >300s, and the system suitability check response efficiency is in the range of 85%~115%.

[0016] Furthermore, the acid is a 5-8 mol / L phosphoric acid solution.

[0017] Furthermore, the oxidant is an ammonium persulfate solution with a mass percentage concentration of 10-20%.

[0018] Furthermore, in step S3, controlling the concentration of the test solution within a certain range means that when the product concentration in the permeate drops to 0.2–1.0 mg / mL in the later stage of ultrafiltration, water replenishment is stopped and concentration begins. At this point, small molecule fragments have been removed to a reasonable range, ensuring that the molecular weight distribution of the final product is qualified.

[0019] Furthermore, in step S3, controlling the concentration of the test solution within a certain range means controlling the product concentration within the range of 0.12 to 0.20 g / mL before alcohol precipitation, followed by alcohol precipitation, centrifugation, and drying, so that the ethanol content of the final product is qualified.

[0020] Dalteparin sodium or nadroparin calcium, produced from heparin derived from porcine intestinal mucosa through processes such as nitrite pyrolysis, both possess relatively stable proportions of elements such as C, H, O, and N. The method established in this invention for determining the concentration of dalteparin sodium and nadroparin calcium products by measuring their TOC values ​​is based on the principle that the carbon proportion of the same type of heparin drug remains relatively constant across different batches. By establishing a concentration-TOC linear equation and measuring the TOC value of the intermediate solution, the product concentration can be calculated.

[0021] The method for determining the concentration of low molecular weight heparin provided by this invention has the advantages of reliability, speed, and convenience, and can be used for quality control in the preparation process of dalteparin sodium or nadroparin calcium.

[0022] (1) In the ultrafiltration step of preparing dalteparin sodium or nadroparin calcium, the permeate is sampled, diluted and the TOC value is measured. The product concentration of the permeate is calculated by linear equation to control the endpoint of ultrafiltration and achieve the purpose of purification. Specifically, the TOC method is used to determine the product concentration of dalteparin sodium or nadroparin calcium permeate. This facilitates timely cessation of ultrafiltration (stopping when the product concentration in the dalteparin sodium permeate drops to 0.2–0.6 mg / mL, and when the product concentration in the nadroparin calcium permeate drops to 0.6–1.0 mg / mL). This ensures that the product molecular weight and molecular weight distribution meet the EP standard requirements, namely: for dalteparin sodium, the weight-average molecular weight is 5600–6400, with fractions less than 3000 ≤13.0% and fractions greater than 8000 15.0%–25.0%; for nadroparin calcium, the weight-average molecular weight is 3600–5000, with fractions less than 2000 ≤15.0%, fractions between 2000 and 8000 75.0%–95.0%, and fractions between 2000 and 4000 35.0%–55.0%.

[0023] (2) In the alcohol precipitation step of preparing dalteparin sodium or nadroparin calcium, the TOC value of the feed solution before alcohol precipitation is measured after dilution. The product concentration of the feed solution is calculated by linear equation to control the product concentration before alcohol precipitation (purified water is added if necessary), and to control the proportion and amount of ethanol in the alcohol precipitation system, so as to facilitate the removal of ethanol during the later drying process. Specifically, before alcohol precipitation of dalteparin sodium or nadroparin calcium, the product concentration is determined by the TOC method, and the concentration of dalteparin sodium or nadroparin calcium is controlled at 0.12-0.20 g / mL to establish the optimal product-water-ethanol alcohol precipitation system, which is conducive to the removal of ethanol during the later drying process, so that the residual ethanol content is ≤0.5%.

[0024] Compared with existing technologies, the method for determining the concentration of low molecular weight heparin provided by this invention has the advantages of simple operation, short detection time, and high accuracy. It is fully applicable to the rapid detection of the product concentration of intermediate solutions in the ultrafiltration or alcohol precipitation steps during the preparation of dalteparin sodium or nadroparin calcium, and to process control, so that the final product quality meets the requirements of EP standards. Attached Figure Description

[0025] Figure 1 The molecular structure diagram of heparin sodium;

[0026] Figure 2 The molecular structure diagram of nadroparin calcium;

[0027] Figure 3 This is a standard curve graph of Example 1;

[0028] Figure 4 This is the standard curve graph for Example 2; Detailed Implementation

[0029] This invention applies the TOC determination method to ultrafiltration, alcohol precipitation, and other steps in the production of dalteparin sodium and nadroparin calcium. Other production steps not listed, as well as the preparation of other heparin products, where the TOC method is used for detection and production control, are all within the scope of protection of this patent.

[0030] The present invention will be further described below through specific embodiments, but this is not a limitation of the present invention. Those skilled in the art can make various modifications or improvements based on the basic idea of ​​the present invention, but as long as they do not depart from the basic idea of ​​the present invention, they are all within the scope of the present invention.

[0031] Example 1: System Suitability Test of TOC Analyzer

[0032] Referring to General Chapter 0682 "Determination of Total Organic Carbon in Pharmaceutical Water" in the 2020 edition of the Chinese Pharmacopoeia, water for total organic carbon testing, sucrose reference solution (easily oxidizable), and 1,4-p-benzoquinone reference solution (difficultly oxidizable) were injected separately, and the instrument's total organic carbon response value was recorded sequentially. The response efficiency should be 85%–115% calculated using the following formula.

[0033]

[0034] Where: Rw is the blank response value of water used for total organic carbon testing;

[0035] Rs is the response value of the sucrose reference solution;

[0036] Rss is the response value of the 1,4-p-benzoquinone reference solution.

[0037] The measurement data are shown in Table 1:

[0038] Table 1

[0039]

[0040] As can be seen from Table 1, the TOC analyzer system meets the suitability requirements and can perform total organic carbon testing.

[0041] Example 2: Determination of Dalteparin Sodium Concentration

[0042] Step S1: Take 100 mg of dalteparin sodium self-made product, add 100 mL of purified water to dissolve it, and obtain a solution A with a product concentration of 1000 mg / L. Take 0.25 mL, 0.50 mL, 0.75 mL, 1.0 mL, and 1.25 mL of the solution respectively, and add purified water to make up to 50 mL, so as to obtain gradient solutions with concentrations of 5 mg / L, 10 mg / L, 15 mg / L, 20 mg / L, and 25 mg / L. Take two more batches of dalteparin sodium self-made product, and prepare two sets of gradient solutions in the same way to obtain sample solutions.

[0043] Step S2: The TOC value of the sample solution from Step S1 was determined using a GE Sievers M9 TOC analyzer. The acid was a 6 mol / L phosphoric acid solution, 0.3 mL in volume; the oxidant was a 15% ammonium persulfate solution, 0.3 mL in volume; the rinsing time was 300 s. During system suitability verification, the response efficiency should be within the range of 85-115% before sample detection. Each concentration of sample solution was measured twice in parallel. Six TOC values ​​were obtained for three batches of samples at different concentrations. The average TOC value and RSD were calculated. The RSD of the TOC values ​​for the three batches of samples at different concentrations were all <8%. After subtracting the blank (purified water TOC: 0.017 ppm) from the average TOC, a concentration-TOC calibration curve was plotted, yielding the linear equation: y = 0.2511x -0.0071; R² = 0.9997 (standard curve graph as shown). Figure 3 (As shown). The measurement data are shown in Table 2:

[0044] Table 2

[0045]

[0046] Example 3: Determination of Nadroparin Calcium Concentration

[0047] Step S1: Take 100 mg of nadroparin calcium self-made product, add 100 mL of purified water to dissolve it, and obtain a solution A with a product concentration of 1000 mg / L. Take 0.25 mL, 0.50 mL, 0.75 mL, 1.0 mL and 1.25 mL of solution A respectively, add purified water to make up to 50 mL, and obtain gradient solutions with concentrations of 5 mg / L, 10 mg / L, 15 mg / L, 20 mg / L and 25 mg / L. Take two more batches of nadroparin calcium self-made product, and prepare two sets of gradient solutions in the same way to obtain sample solutions.

[0048] Step S2: The TOC value of the sample solution from Step S1 was determined using a GE Sievers M9 TOC analyzer. The acid was a 6 mol / L phosphoric acid solution, 0.5 mL in volume; the oxidant was a 15% ammonium persulfate solution, 0.5 mL in volume; the rinsing time was 320 s. During system suitability verification, the response efficiency should be within the range of 85-115% before sample detection. Each concentration of sample solution was measured twice in parallel. Six TOC values ​​were obtained for each of the three batches of samples at different concentrations. The average TOC value and RSD were calculated. The RSD of the TOC values ​​for the three batches of samples at different concentrations were all <8%. After subtracting the blank (purified water TOC: 0.024 ppm) from the average TOC, a concentration-TOC calibration curve was plotted, yielding the linear equation: y = 0.2246x + 0.2414; R² = 0.9979 (standard curve graph as shown). Figure 4 (As shown). The measurement data are shown in Table 3:

[0049] Table 3

[0050]

[0051] Experimental Example 1: Specific Applications of Dalteparin Sodium Preparation

[0052] Step A: Dissolve 1 kg of heparin sodium in 5 kg of purified water, adjust the pH to 2.6, and after sodium nitrite pyrolysis, sodium borohydride reduction, neutralization, and column chromatography, use a 3-5 kDa filter membrane for ultrafiltration. Continuously add purified water for equal-volume washing. The permeate mainly contains components with smaller molecular weights. Take samples periodically to determine the TOC value of the permeate. Using purified water as a blank, calculate the product concentration using the formula from Example 1: y = 0.2511x - 0.0071. When the permeate concentration drops to approximately 0.3 mg / mL, most of the small molecules have been removed, and ultrafiltration is stopped.

[0053] Step B: After concentrating the liquid and irradiating it with ultraviolet light, a sample was taken. Using purified water as a blank, the product concentration was calculated using the formula from Example 1: y = 0.2511x - 0.0071. If necessary, purified water was added to adjust the concentration of dalteparin sodium to 0.14 g / mL. After alcohol precipitation, centrifugation, and drying, 655 g of dalteparin sodium was obtained. The molecular weight, molecular weight distribution, ethanol residue, and other indicators of the product all met the EP requirements.

[0054] Experimental Example 2: Specific Applications of Nadroparin Calcium Preparation

[0055] Step A: Dissolve 1 kg of sodium heparin in 5 kg of purified water, adjust the pH to 2.6, and after sodium nitrite pyrolysis, sodium borohydride reduction, neutralization, and calcium conversion, use a 2-4 kDa filter membrane for ultrafiltration. Continuously add purified water for equal-volume washing. The permeate mainly contains components with smaller molecular weights. Periodically sample and measure the TOC value of the permeate. Using purified water as a blank, calculate the product concentration using the formula from Example 2: y = 0.2246x + 0.2414. When the permeate concentration drops to approximately 0.8 mg / mL, most of the small molecules have been removed, and ultrafiltration is stopped.

[0056] Step B: After concentrating the liquid and irradiating it with ultraviolet light, a sample was taken. Using purified water as a blank, the product concentration was calculated using the formula in Example 2: y = 0.2246x + 0.2414. If necessary, purified water was added to adjust the concentration of nadroparin calcium to 0.17 g / mL. After alcohol precipitation, centrifugation, and drying, 678 g of nadroparin calcium product was obtained. The molecular weight, molecular weight distribution, ethanol residue, and other indicators of the product all met the EP requirements.

[0057] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A quality control method for low molecular weight heparin, characterized in that, Includes the following steps: Step S1: Take the sample, dissolve and dilute it with purified water to prepare gradient solutions of different concentrations, and obtain the sample solution; Step S2: Determine the TOC value of the sample solution prepared in step S1 using a TOC analyzer. Each sample solution is measured twice in parallel. Plot the TOC-sample concentration calibration curve and obtain the linear equation. Step S3: During the preparation of low molecular weight heparin, the TOC value of the test solution is measured, and the concentration of low molecular weight heparin in the test solution is calculated using the linear equation in step S2. If necessary, ultrafiltration or water is added to control the concentration of the test solution within a certain range so that the final product quality meets the EP standard requirements. The sample in step S1 refers to dalteparin sodium or nadroparin calcium finished product, and the test solution in step S3 refers to the ultrafiltration permeate and the product solution before alcohol precipitation during the preparation process of dalteparin sodium or nadroparin calcium. The TOC analyzer used in step S2 is a GE Sievers M9 TOC analyzer. The TOC analyzer was used under the following conditions: acid solution 0.3–0.6 mL, oxidant solution 0.3–0.6 mL, rinsing time > 300 s, and system suitability check response efficiency within the range of 85%–115%. In step S3, controlling the concentration of the test solution within a certain range means that when the product concentration in the sodium dalteparin permeate drops to 0.2–0.6 mg / mL or the product concentration in the calcium nadroparin permeate drops to 0.6–1.0 mg / mL, ultrafiltration is stopped and concentration begins. At this point, small molecule fragments have been removed to a reasonable range, which can make the molecular weight distribution of the final product qualified. In step S3, controlling the concentration of the test solution within a certain range means controlling the concentration of dalteparin sodium or nadroparin calcium within the range of 0.12 to 0.20 g / mL, and then performing alcohol precipitation, centrifugation, and drying to ensure that the ethanol content of the final product is qualified. The sample solution in step S1 refers to a gradient solution with concentrations of 5 mg / L, 10 mg / L, 15 mg / L, 20 mg / L, and 25 mg / L. The acid is a 5-8 mol / L phosphoric acid solution; The oxidant is an ammonium persulfate solution with a mass percentage concentration of 10-20%.

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