A quality control method for the production process of nadroparin calcium

Abstract

The application discloses a quality control processing method for a nadroparin calcium production process, and belongs to the technical field of biochemical pharmacy, and comprises the following steps: (1) taking a nadroparin calcium ultrafiltration concentrated solution as a sample for central control detection; (2) detecting the sodium content of the sample obtained in the step (1) by using an inductively coupled plasma atomic emission spectrometer; (3) when the sodium content is not greater than 6.7 mg / mL, the quality control is qualified; when the sodium content is greater than 6.7 mg / mL, the quality control is unqualified, and a calcium conversion treatment needs to be continuously performed until the detection is qualified. The application can directly perform sampling detection on the sodium content in the production process, efficiently monitors the calcium conversion degree, and then guides the production work, thereby avoiding the problems of long time consumption and difficult remediation in the traditional way, and greatly reducing the production cost.

Description

Technical Field

[0001] This invention belongs to the field of biochemical pharmaceutical technology, specifically relating to a quality control method for the production process of nadroparin calcium. Background Technology

[0002] Heparin is a sulfated glycosaminoglycan compound extracted from mammalian tissues and was officially used in clinical treatment in 1935. As a highly effective anticoagulant, heparin products have long been the first-line drugs for treating thrombotic diseases. Initially, unfractionated heparin was used clinically. In the mid-1980s, Europe first developed low molecular weight heparin (LMWH), which is mainly obtained from unfractionated heparin through physical separation, chemical cleavage, and enzymatic cleavage. The European Pharmacopoeia (EP) further classifies LMWH into: nadroparin calcium, enoxaparin sodium, dalteparin sodium, parhexaparin sodium, and tinhexaparin sodium, based on differences in production methods and quality standards. LMWH has a higher anti-FXa / anti-FIIa potency ratio than unfractionated heparin, thus greatly reducing the risk of bleeding during antithrombotic processes, and has been more widely used in clinical practice.

[0003] Nadroparin calcium is one of the most commonly used LMWHs, existing in the form of a calcium salt. It is typically prepared from heparin sodium through steps including nitrite cracking, sodium borohydride reduction, calcium conversion, purification, alcohol precipitation, and drying. The calcium conversion step, in particular, requires a relatively long time to complete due to the equilibrium of sodium and calcium ions in the solution and steric hindrance. Furthermore, because heparin sodium has a complex structure, multiple components, and non-uniform sugar chain lengths and substituents, the time required for calcium conversion varies depending on the source and batch of heparin sodium used in the preparation of nadroparin calcium.

[0004] Currently, calcium content testing is generally only performed on the finished product. If samples are taken from the feed solution during production, subjected to alcohol precipitation, drying, or direct freeze-drying, and then tested for calcium content, the process is time-consuming and cannot achieve rapid, intermediate-level control. Furthermore, the presence of excessive calcium chloride in the feed solution significantly interferes with the calcium content results. Therefore, a rapid and accurate detection method has been developed to monitor the calcium conversion effect in real time during production, addressing the industry pain point of the inability to achieve intermediate-level control of calcium content during nadroparin calcium production. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies that cannot monitor the degree of calcium conversion during production, and to establish a method for detecting intermediate solutions during the production process, and to formulate intermediate control qualification standards. When intermediate control fails to meet the standards, corresponding process operations can be taken in a timely manner to ensure that the calcium content of the final product meets the requirements.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution: A quality control method for the production process of nadroparin calcium includes the following steps: (1) Take the nadroparin calcium ultrafiltration concentrate as the sample for central control testing; (2) The sodium content of the sample obtained in step (1) was detected by inductively coupled plasma atomic emission spectrometry. (3) When the sodium content is not greater than 6.7 mg / mL, the quality control is qualified. When the sodium content is greater than 6.7 mg / mL, the quality control is unqualified and the calcium conversion process needs to be continued until the test is qualified.

[0007] Furthermore, the nadroparin calcium ultrafiltration concentrate mentioned in step (1) is the concentrate that is concentrated to 5 times the amount of heparin sodium fed during production.

[0008] Furthermore, in step (2), the flow rate of the nebulizing gas is controlled to be 0.60-0.90 L / min, the flow rate of the plasma gas is 10.0-15.0 L / min, and the flow rate of the auxiliary gas is 0.80-1.30 L / min during the inductively coupled plasma atomic emission spectrometer test.

[0009] Furthermore, in step (2), the pump speed is controlled at 10-13 rpm during the inductively coupled plasma atomic emission spectrometer test.

[0010] Furthermore, in step (2), the RF power is controlled to be 1.10-1.40kW during the inductively coupled plasma atomic emission spectrometer test.

[0011] Furthermore, in step (2), the plasma preheating time is controlled to be 15-25 min and the reading time is 3-6 s during the inductively coupled plasma atomic emission spectrometer test.

[0012] Furthermore, in step (2), the inductively coupled plasma atomic emission spectrometer is used to control the detection wavelength at 589.592 nm and the observation method is axial.

[0013] Furthermore, the concentration of the sodium standard solution used for sodium content detection in step (2) is: 0, 0.1 ppm, 0.2 ppm, 0.4 ppm, 0.6 ppm, 0.8 ppm, 1.2 ppm.

[0014] Furthermore, when testing the sample described in step (2), the sample volume is 0.05 mL, which is then diluted with ultrapure water to 500 mL.

[0015] Furthermore, the calcium conversion treatment method described in step (3) is as follows: first, continuously add an equal volume of 5% calcium chloride solution for ultrafiltration for 1-2 hours, and then continuously add an equal volume of purified water for ultrafiltration for 0.5-1 hours.

[0016] The present invention has the following advantages over the prior art: This invention provides a quality control testing method that enables direct sampling and testing during the production process. It efficiently measures the sodium content of samples, quickly providing feedback on the degree of sodium salt conversion to calcium salt, guiding production operations, and avoiding the time-consuming and difficult-to-correct problems of traditional methods. This significantly reduces production costs. Furthermore, for non-conforming samples, it allows for rapid reprocessing to convert the sodium salt to calcium, completely solving the industry pain point of not being able to quickly control calcium content during nadroparin calcium production. Because during ultrafiltration calcium conversion, the added calcium chloride is often excessive, directly detecting calcium content cannot accurately calculate the true amount of calcium converted in the sample. Therefore, this invention uses sodium content detection to infer the degree of calcium conversion in the product, enabling timely monitoring and control, and has significant application value. Attached Figure Description

[0017] Figure 1 This is a verification linear spectrum of the method corresponding to Experimental Example 1 of the present invention.

[0018] Figure 2 This is the standard curve diagram for the corresponding sample detection in Experiment Example 2 of this invention.

[0019] Figure 3 This is the sodium content detection spectrum of the corresponding sample in Experimental Example 3 of this invention. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of the embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0021] Example 1 A quality control method for the production process of nadroparin calcium includes the following steps: (1) Take the nadroparin calcium ultrafiltration concentrate as the sample for central control testing; the nadroparin calcium ultrafiltration concentrate is the concentrate that is concentrated to 5 times the amount of heparin sodium added during production. The material tanks of ultrafiltration are marked with liquid level. That is, when running, the total volume including pipelines, membranes and material tanks can be reflected on the liquid level mark of the material tank. For example, if 5kg of heparin sodium is added during production, when it is concentrated in the ultrafiltration step, it is concentrated to 25L (5 times v / M) of the liquid level mark of the material tank, and then ultrafiltration can be stopped. (2) The sodium content of the sample obtained in step (1) was detected using an inductively coupled plasma atomic emission spectrometer; the flow rate of the nebulizer gas was controlled at 0.7 L / min, the flow rate of the plasma gas was 12 L / min, the flow rate of the auxiliary gas was 1 L / min, the pump speed was 12 rpm, the RF power was 1.2 kW, the plasma preheating time was 20 min, the reading time was 5 s, the detection wavelength was controlled at 589.592 nm, and the observation mode was axial. The concentrations of the sodium standard solutions used for sodium content detection were: 0, 0.1 ppm, 0.2 ppm, 0.4 ppm, 0.6 ppm, 0.8 ppm, and 1.2 ppm; when the sample was tested, the sample volume was 0.05 mL, and then diluted with ultrapure water to 500 mL.

[0022] (3) When the sodium content is not greater than 6.7 mg / mL, the quality control is qualified. When the sodium content is greater than 6.7 mg / mL, the quality control is unqualified and the calcium conversion treatment needs to be continued. Specifically, first, add an equal volume of 5% calcium chloride solution for ultrafiltration for 1.5 h, then add an equal volume of purified water for ultrafiltration for 0.8 h, until the above test is repeated and qualified.

[0023] Example 2 A quality control method for the production process of nadroparin calcium includes the following steps: (1) Take the nadroparin calcium ultrafiltration concentrate as the sample for central control testing; the nadroparin calcium ultrafiltration concentrate is the concentrate that is concentrated to 5 times the amount of heparin sodium added during production. The material tanks of ultrafiltration are marked with liquid level. That is, when running, the total volume including pipelines, membranes and material tanks can be reflected on the liquid level mark of the material tank. For example, if 5kg of heparin sodium is added during production, when it is concentrated in the ultrafiltration step, it is concentrated to 25L (5 times v / M) of the liquid level mark of the material tank, and then ultrafiltration can be stopped. (2) The sodium content of the sample obtained in step (1) was detected using an inductively coupled plasma atomic emission spectrometer; the flow rate of the nebulizer gas was controlled at 0.60 L / min, the flow rate of the plasma gas was 10.0 L / min, the flow rate of the auxiliary gas was 0.80 L / min, the pump speed was 10 rpm, the RF power was 1.10 kW, the plasma preheating time was 15 min, the reading time was 3 s, the detection wavelength was controlled at 589.592 nm, and the observation method was axial. The concentrations of the sodium standard solutions used for sodium content detection were: 0, 0.1 ppm, 0.2 ppm, 0.4 ppm, 0.6 ppm, 0.8 ppm, and 1.2 ppm; when the sample was tested, the sample volume was 0.05 mL, and then diluted with ultrapure water to 500 mL.

[0024] (3) When the sodium content is not greater than 6.7 mg / mL, the quality control is qualified. When the sodium content is greater than 6.7 mg / mL, the quality control is unqualified and the calcium conversion treatment needs to be continued. Specifically, first, add an equal volume of 5% calcium chloride solution for ultrafiltration for 1 hour, then add an equal volume of purified water for ultrafiltration for 0.5 hours, until the above test is repeated and qualified.

[0025] Example 3 A quality control method for the production process of nadroparin calcium includes the following steps: (1) Take the nadroparin calcium ultrafiltration concentrate as the sample for central control testing; the nadroparin calcium ultrafiltration concentrate is the concentrate that is concentrated to 5 times the amount of heparin sodium added during production. The material tanks of ultrafiltration are marked with liquid level. That is, when running, the total volume including pipelines, membranes and material tanks can be reflected on the liquid level mark of the material tank. For example, if 5kg of heparin sodium is added during production, when it is concentrated in the ultrafiltration step, it is concentrated to 25L (5 times v / M) of the liquid level mark of the material tank, and then ultrafiltration can be stopped. (2) The sodium content of the sample obtained in step (1) was detected using an inductively coupled plasma atomic emission spectrometer; the flow rate of the nebulizer gas was controlled at 0.90 L / min, the flow rate of the plasma gas was 15.0 L / min, the flow rate of the auxiliary gas was 1.30 L / min, the pump speed was 13 rpm, the RF power was 1.40 kW, the plasma preheating time was 25 min, the reading time was 6 s, the detection wavelength was controlled at 589.592 nm, and the observation method was axial. The concentrations of the sodium standard solutions used for sodium content detection were: 0, 0.1 ppm, 0.2 ppm, 0.4 ppm, 0.6 ppm, 0.8 ppm, and 1.2 ppm; when the sample was tested, the sample volume was 0.05 mL, and then diluted with ultrapure water to 500 mL.

[0026] (3) When the sodium content is not greater than 6.7 mg / mL, the quality control is qualified. When the sodium content is greater than 6.7 mg / mL, the quality control is unqualified and the calcium conversion treatment needs to be continued. Specifically, first, add an equal volume of 5% calcium chloride solution for ultrafiltration for 2 hours, then add an equal volume of purified water for ultrafiltration for 1 hour, until the above test is repeated and qualified.

[0027] Experimental Example 1 To compare the effects of the present invention, the method of Example 1 was specifically verified: 1. Linearity and Range: Sodium standard solutions of 0, 0.1, 0.2, 0.4, 0.6, 0.8, and 1.2 ppm were prepared and injected sequentially from low to high concentrations. The resulting standard curve and linear regression equation are: y = 325272.2599 * x + 127.2821, where y represents the peak area and x represents the concentration; the correlation coefficient r = 0.99990, indicating good linearity. See attached figure for details. Figure 1 As shown.

[0028] 2. Limit of Detection and Limit of Quantification: The LOD of this method was calculated to be 0.0009 ppm and the LOQ was 0.0029 ppm, based on the concentrations corresponding to 3 times and 10 times the standard deviation of the response value of the blank solution after 7 consecutive determinations.

[0029] 3. Specificity: A sodium standard solution (0.4 ppm), a nadroparin calcium sample solution (1 mg / mL), and a 1:1 mixture of the two were prepared and injected separately. Nitric acid solution (0.002%) and a blank solution (water) were also injected separately. The results showed that the sodium peak wavelength was consistent across the three solutions, with no other absorption peaks within ±0.05 nm. No interfering peaks were observed in the nitric acid solution or the blank solution.

[0030] 4. Precision: The test solution (containing 0.4 ppm standard solution) was injected 6 times consecutively, and the RSD at this concentration was 0.2%. Another experimenter prepared the test solution (0.4 ppm standard solution) on different dates and injected it 6 times consecutively, obtaining an RSD of 0.5% at this concentration. The combined RSD of 12 tests by both experimenters was 2.7%.

[0031] 5. Accuracy: 0.1 g of nadroparin calcium sample was dissolved in ultrapure water and diluted to 100 mL. The known sodium content in the solution was determined to be 0.1779 ppm. 5.0 mL of this solution was then added to 5.0 mL of sodium standard solutions with concentrations of 0.1 ppm, 0.2 ppm, and 0.4 ppm, respectively, and injected twice in ascending order of concentration. The recovery rate was calculated. The recovery rates for all six experiments were between 80.0% and 115.0%, with an RSD of 3.4%.

[0032] 6. Durability: By changing the atomizing gas flow rate to 0.68, 0.70, and 0.72 L / min, respectively, the standard solution with a sodium content of 0.4 ppm was measured twice under each condition, for a total of 6 measurements. The RSD of the peak area was 1.6%.

[0033] The verification results of the above method show that the method has the characteristics of anti-interference, high accuracy and strong applicability.

[0034] Experimental Example 2 The method of Example 1 was used for the intermediate control detection of nadroparin calcium sodium content: The first batch of nadroparin calcium ultrafiltration solution was concentrated to 5 times the amount of heparin sodium feed (v / m), and 0.05 mL was taken as a sample and diluted with ultrapure water to 500 mL to obtain the test solution. Sodium standard solutions of 0, 0.1, 0.2, 0.4, 0.6, 0.8, and 1.2 ppm were injected sequentially according to the conditions of Example 1, from low to high, to obtain the standard curve and linear regression equation: y = 334216.3057 * x - 93.0689, where: y refers to the peak area; x refers to the concentration; the correlation coefficient r = 0.99992, as detailed in the attached figure. Figure 2 As shown. The test solution was tested, and the sodium content was calculated to be 0.7042 ppm according to the standard curve. Therefore, the sodium content in the ultrafiltration concentrate was approximately 7.0 mg / mL, exceeding the standard limit of 6.7 mg / mL, and thus unqualified. Ultrafiltration was stopped, and subsequent steps including UV irradiation, alcohol precipitation, centrifugation, and drying were performed. The final product had a calcium content of 9.3% (the standard requirement is 9.5%-11.5%), therefore the calcium content was unqualified. This demonstrates that a high residual sodium ion content can lead to incomplete calcium conversion.

[0035] Experimental Example 3 2. Controlled detection of nadroparin calcium sodium content using the method of Example 1: The second batch of nadroparin calcium ultrafiltration solution was concentrated to 5 times the amount of heparin sodium added (v / m). An initial sample of 0.05 mL was taken and diluted to 500 mL with ultrapure water to obtain test solution 1. The solution was injected under the conditions of Example 1, and the standard curve was calculated according to Example 2. The sodium content of the test solution was 0.83 ppm, therefore the sodium content in the ultrafiltration concentrate was 8.3 mg / mL, exceeding the control limit of 6.7 mg / mL. Therefore, calcium conversion needed to continue. First, 5% calcium chloride solution was continuously added to maintain an equal volume of ultrafiltration in the tank for 1.5 h, then purified water was added to maintain an equal volume of ultrafiltration in the tank for another 0.5 h. Another 0.05 mL sample was taken and diluted to 500 mL with ultrapure water to obtain test solution 2. The solution was injected under the conditions of Example 1, and the standard curve was calculated according to Example 2. The sodium content of the test solution was 0.6437 ppm, therefore the sodium content in the ultrafiltration concentrate was approximately 6.4 mg / mL, which met the intermediate control requirements. Ultrafiltration was then stopped, and subsequent steps including UV irradiation, alcohol precipitation, centrifugation, and drying were performed. The final product had a calcium content of 9.9%, which meets the standard requirements. The above test results are attached. Figure 3 As shown.

[0036] 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. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention are included within the scope of protection of the present invention.

Claims

1. A quality control method for the production process of nadroparin calcium, characterized in that, Includes the following steps: (1) Take the nadroparin calcium ultrafiltration concentrate as the sample for central control testing; (2) The sodium content of the sample obtained in step (1) was detected by inductively coupled plasma atomic emission spectrometry. (3) When the sodium content is not greater than 6.7 mg / mL, the quality control is qualified. When the sodium content is greater than 6.7 mg / mL, the quality control is unqualified and the calcium conversion process needs to be continued until the test is qualified.

2. The quality control method for the production process of nadroparin calcium according to claim 1, characterized in that, The nadroparin calcium ultrafiltration concentrate mentioned in step (1) is the concentrate that is concentrated to 5 times the amount of heparin sodium added during production.

3. The quality control method for the production process of nadroparin calcium according to claim 1, characterized in that, In step (2), the flow rate of the inductively coupled plasma atomic emission spectrometer is controlled to be 0.60-0.90 L / min, the flow rate of the plasma gas is 10.0-15.0 L / min, and the flow rate of the auxiliary gas is 0.80-1.30 L / min.

4. The quality control method for the production process of nadroparin calcium according to claim 1, characterized in that, During the inductively coupled plasma atomic emission spectrometer test described in step (2), the pump speed is controlled at 10-13 rpm.

5. The quality control method for the production process of nadroparin calcium according to claim 1, characterized in that, In step (2), the RF power of the inductively coupled plasma atomic emission spectrometer is controlled to be 1.10-1.40kW.

6. The quality control method for the production process of nadroparin calcium according to claim 1, characterized in that, In step (2), the plasma preheating time is controlled to be 15-25 min and the reading time is 3-6 s during the inductively coupled plasma atomic emission spectrometer test.

7. The quality control method for the production process of nadroparin calcium according to claim 1, characterized in that, In step (2), the inductively coupled plasma atomic emission spectrometer is used to control the detection wavelength at 589.592 nm and the observation method is axial.

8. The quality control method for the production process of nadroparin calcium according to claim 1, characterized in that, The sodium standard solution used for sodium content detection in step (2) has the following concentrations: 0, 0.1 ppm, 0.2 ppm, 0.4 ppm, 0.6 ppm, 0.8 ppm, and 1.2 ppm.

9. The quality control method for the production process of nadroparin calcium according to claim 1, characterized in that, When testing the sample described in step (2), the sample volume is 0.05 mL, and then diluted with ultrapure water to 500 mL.

10. The quality control method for the production process of nadroparin calcium according to claim 1, characterized in that, The method for continuing calcium conversion in step (3) is as follows: first, continuously add an equal volume of 5% calcium chloride solution for ultrafiltration for 1-2 hours, and then continuously add an equal volume of purified water for ultrafiltration for 0.5-1 hours.

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