Preparation method of nadroparin calcium

Abstract

The invention relates to a preparation method of nadroparin calcium, which comprises the following steps: step 1, degrading heparin sodium, taking the heparin sodium, dissolving the heparin sodium in purified water, adding sodium nitrite after the heparin sodium is dissolved, adding glacial acetic acid in the stirring process, and fully stirring until the heparin sodium and the glacial acetic acid are mixed completely; step 2, reduction of the degradation liquid: sodium borohydride is added into the feed liquid in the step 1 to be dissolved, and reduction liquid is obtained; 3, ultraviolet radiation, wherein sodium borohydride is weighed and added into the reduction liquid for ultraviolet radiation; 4, carrying out fractional precipitation, separation and purification on replacement calcium and ethanol, and collecting secondary lower-layer precipitate; and step 5, filtering and freeze-drying: adding water into the second precipitate for dissolving, and filtering and freeze-drying the dissolved feed liquid. According to the method, the application of an advanced reduction technology is realized, and a reducing agent is added in the ultraviolet radiation process, so that the product stability is improved, the color of feed liquid becomes light, and meanwhile, an independent oxidation decolorization procedure is omitted.

Description

Technical Field

[0001] The invention belongs to the technical field of biopharmaceuticals, and in particular relates to a preparation method of nadroparin calcium. Background Art

[0002] Nadroparin calcium is a type of LMWH. Products prepared by the existing nadroparin calcium preparation process are prone to the disadvantages of poor product stability, dark solution color, and rapid pH drop. In the ultraviolet irradiation process in the existing process, no reducing agent is added for protection. In this way, some unstable structures generated during the ultraviolet irradiation process cannot be reduced and converted online, but remain on the sugar chain for a long time. The presence of these unstable groups will cause the product stability to deteriorate and the solution color to darken, and the stability of the obtained product is poor. Summary of the invention

[0003] (I) Purpose of the invention

[0004] In order to overcome the above disadvantages, the object of the present invention is to provide a method for preparing nadroparin calcium to solve the above technical problems.

[0005] (II) Technical solution

[0006] To achieve the above objectives, the technical solutions provided by this application are as follows:

[0007] A method for preparing nadroparin calcium comprises the following steps:

[0008] Step 1: Degradation of sodium heparin: taking sodium heparin and dissolving it in purified water, adding sodium nitrite after dissolution, adding glacial acetic acid during stirring, and stirring thoroughly until the mixture is completely mixed;

[0009] Step 2: reduction of the degradation solution, adding sodium borohydride to the feed solution of step 1 to dissolve and obtain a reduction solution;

[0010] Step 3: UV irradiation: weigh sodium borohydride at 0.5% W / W of the weight of heparin sodium, add it to the reducing solution for UV irradiation, control the liquid temperature at 10° C. to 30° C., the pH value at 9.0 to 11.0, and the UV irradiation time at 60 min to 80 min;

[0011] Step 4: replacement calcium conversion and ethanol graded precipitation separation and purification, adding calcium chloride to the feed solution of step 3, stirring evenly, the stirring time is not less than 1 hour, then adding 1.0-2.0 ethanol, stirring for not less than 30 minutes, standing for not less than 2 hours, extracting the upper layer of ethanol after standing, collecting the first precipitate of the lower layer, dissolving the first precipitate of the lower layer in water, adding calcium chloride, stirring evenly, the stirring time is not less than 1 hour, then adding ethanol, stirring fully, standing for not less than 2 hours, then extracting the upper layer of ethanol, and collecting the second lower precipitate;

[0012] Step 5: filtration and freeze-drying: the second precipitate is dissolved in water and then filtered, and the filtered liquid is freeze-dried.

[0013] Preferably, the sodium nitrite in step 1 is 1.0% to 5.0% of the weight of the heparin sodium, and the glacial acetic acid is added in an amount of 1% to 5% of the volume of the feed liquid.

[0014] Preferably, the stirring time in step 1 is 60 min to 80 min, and the temperature of the feed liquid is controlled at 20±5° C. during the reaction.

[0015] Preferably, the sodium borohydride in step 2 is 0.5% to 2.5% of the weight of heparin sodium. W / W .

[0016] Preferably, the reaction time in step 2 is 8 to 12 hours, and during the reaction, the temperature is controlled at 10 to 30° C. and the pH value is controlled at 9.0 to 11.0.

[0017] Beneficial effects:

[0018] The invention realizes the application of advanced reduction technology. During the ultraviolet irradiation process, a reducing agent is added to improve the product stability, lighten the color of the feed liquid, and omit the separate oxidation decolorization process. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a schematic diagram of the process structure of the present invention. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical solutions and advantages of the present invention more clear, the following is a detailed description of the present invention in conjunction with the specific implementation methods and with reference to the attached drawings. Figure 1 , the present invention is further described in detail. It should be understood that these descriptions are exemplary only and are not intended to limit the scope of the present invention. In addition, in the following description, descriptions of well-known structures and technologies are omitted to avoid unnecessary confusion of the concept of the present invention.

[0021] Example 1

[0022] The present invention provides a method for preparing nadroparin calcium, comprising the following steps:

[0023] Step 1: Degradation of sodium heparin: 1 kg of sodium heparin is taken and dissolved in 10 L of purified water. After dissolution, 10 g of sodium nitrite is added. During stirring, 500 ml of glacial acetic acid is added. The mixture is stirred for 60 min until the mixture is completely mixed. During the reaction, the temperature of the liquid is controlled at 20° C.

[0024] Step 2: Reduction of the degradation solution: 5 g of sodium borohydride was added to the solution of step 1 to dissolve, and the reaction was carried out for 10 h. During the reaction, the temperature was controlled at 20° C. and the pH was controlled at 11.0 to obtain a reduction solution;

[0025] Step 3: UV irradiation: weigh 5 g of sodium borohydride, add it to the reducing solution to dissolve, and then irradiate it with UV light. During the process, the temperature of the feed solution was controlled at 20° C., the pH value was 9.0, and the UV irradiation time was 70 min.

[0026] Step 4: replacement calcium conversion and ethanol graded precipitation separation and purification, add 500g of calcium chloride to the feed solution of step 3, stir evenly, the stirring time is not less than 1 hour, then add 10L of ethanol, stir for not less than 30 minutes, let stand for not less than 2 hours, extract the upper layer of ethanol after standing, collect the first precipitate of the lower layer, dissolve the first precipitate of the lower layer in water, add calcium chloride, stir evenly, the stirring time is not less than 1 hour, then add ethanol, stir well, let stand for not less than 2 hours, then extract the upper layer of ethanol, and collect the second lower precipitate;

[0027] Step 5: Filtration and freeze-drying: the second precipitate is dissolved in water and then filtered, and the filtered liquid is freeze-dried to obtain 720 g of nadroparin calcium finished product.

[0028] Example 2

[0029] The present invention provides a method for preparing nadroparin calcium, comprising the following steps:

[0030] Step 1 Degradation of sodium heparin: 1 kg of sodium heparin is taken and dissolved in 12 L of purified water. After dissolution, 30 g of sodium nitrite is added. During stirring, 300 ml of glacial acetic acid is added. The mixture is stirred for 70 min until the mixture is completely mixed. During the reaction, the temperature of the liquid is controlled at 22° C.

[0031] Step 2: Reduction of the degradation solution: 15 g of sodium borohydride was added to the solution of step 1 to dissolve, and the reaction was carried out for 8 hours. During the reaction, the temperature was controlled at 25° C. and the pH was controlled at 9.0 to obtain a reduction solution;

[0032] Step 3: UV irradiation: weigh 5 g of sodium borohydride, add it to the reducing solution to dissolve, and then irradiate it with UV light. During the process, the temperature of the feed solution was controlled at 30° C., the pH value was 11.0, and the UV irradiation time was 60 min.

[0033] Step 4: replacement calcium conversion and ethanol graded precipitation separation and purification, add 500g of calcium chloride to the feed solution of step 3, stir evenly, the stirring time is not less than 1 hour, then add 18L of ethanol, stir for not less than 30 minutes, let stand for not less than 2 hours, extract the upper layer of ethanol after standing, collect the first precipitate of the lower layer, dissolve the first precipitate of the lower layer in water, add calcium chloride, stir evenly, the stirring time is not less than 1 hour, then add ethanol, stir well, let stand for not less than 2 hours, then extract the upper layer of ethanol, and collect the second lower precipitate;

[0034] Step 5: Filtration and freeze-drying: the second precipitate is dissolved in water and then filtered, and the filtered liquid is freeze-dried to obtain 710 g of nadroparin calcium finished product.

[0035] Example 3

[0036] The present invention provides a method for preparing nadroparin calcium, comprising the following steps:

[0037] Step 1: Degradation of sodium heparin: 1 kg of sodium heparin is taken and dissolved in 14 L of purified water. After dissolution, 50 g of sodium nitrite is added. During stirring, 140 ml of glacial acetic acid is added. The mixture is stirred for 80 min until the mixture is completely mixed. During the reaction, the temperature of the feed liquid is controlled at 24° C.

[0038] Step 2: Reduction of the degradation solution: 25 g of sodium borohydride was added to the solution of step 1 to dissolve, and the reaction was carried out for 12 h. During the reaction, the temperature was controlled at 19° C. and the pH was controlled at 10.0 to obtain a reduction solution;

[0039] Step 3: UV irradiation: weigh 5 g of sodium borohydride, add it to the reducing solution to dissolve, and then irradiate it with UV light. During the process, the temperature of the feed solution was controlled at 10° C., the pH value was 10.0, and the UV irradiation time was 80 min.

[0040] Step 4: replacement calcium conversion and ethanol graded precipitation separation and purification, add 500g of calcium chloride to the feed solution of step 3, stir evenly, the stirring time is not less than 1 hour, then add 28L of ethanol, stir for not less than 30 minutes, let stand for not less than 2 hours, extract the upper layer of ethanol after standing, collect the first precipitate of the lower layer, dissolve the first precipitate of the lower layer in water, add calcium chloride, stir evenly, the stirring time is not less than 1 hour, then add ethanol, stir well, let stand for not less than 2 hours, then extract the upper layer of ethanol, and collect the second lower precipitate;

[0041] Step 5: Filtration and freeze-drying: the second precipitate is dissolved in water and then filtered, and the filtered liquid is freeze-dried to obtain 732 g of nadroparin calcium finished product.

[0042] Example 4

[0043] Step 1 Degradation: Take 1kg of sodium heparin, add 10L of purified water to dissolve, weigh 30g of sodium nitrite, dissolve and add. Add 100ml of glacial acetic acid to the sodium heparin solution under stirring, stir and react for 70min, and control the liquid temperature at 20℃ during the reaction.

[0044] Step 2: Reduction: weigh 10 g of sodium borohydride, add it to the feed solution to dissolve, and react for 10 hours. During the reaction, control the temperature at 20° C. and the pH at 10.0 to obtain a reduced solution.

[0045] Step 3: UV irradiation: UV irradiation is performed, during which the feed liquid temperature is controlled at 20° C. and the pH is 10.0 for 70 minutes.

[0046] Step 4: Replace calcium and separate and purify by ethanol graded precipitation, add 500g calcium chloride and dissolve it. Stir the reaction for ≥1 hour. Add 15L ethanol to the liquid, stir for 30 minutes, let stand for ≥2 hours, control the temperature at 25°C during the standing process, extract the upper layer of ethanol, collect the lower layer of precipitate, dissolve the first lower layer of precipitate in water, add calcium chloride, stir evenly, stir for not less than 1 hour, then add ethanol, stir well, let stand for not less than 2 hours, then extract the upper layer of ethanol, and collect the second lower layer of precipitate;

[0047] Step 5: Filter, dissolve the second precipitate in water and filter, freeze-dry the filtered liquid, and obtain 702 g of nadroparin calcium finished product after freeze-drying.

[0048] According to Examples 1-4, four batches of nadroparin calcium finished products were prepared, including three test batches and one control batch. The control process was the same as that of Examples 1-3 except that no reducing agent was added during the ultraviolet irradiation.

[0049]

[0050]

[0051]

[0052] Table 1 Test results of finished products of Examples 1 to 4

[0053] As can be seen from the data in Table 1, after quality testing, all indicators of the nadroparin calcium finished products prepared in Examples 1 to 3 meet the quality standards, which are China's proposed registration standards, proving that the method can produce products of high quality; the nadroparin calcium prepared in Example 4 does not meet the standards because the solution color is darker than the yellow No. 1 standard colorimetric solution, and a nadroparin calcium finished product of qualified quality cannot be obtained. Through the control experiment of Example 4, it can be concluded that after adding sodium borohydride during ultraviolet irradiation, the solution color can be improved and a finished product that meets the quality standards can be prepared.

[0054] In order to further study the stability of the product, we conducted stability tests of Examples 1 to 4. The specific test items include influencing factor test, accelerated test and long-term stability test. The specific test conditions and test results are shown in Tables 2 and 3. The influencing factor high temperature stability test of Example 1 was placed at 60°C to simulate more severe test conditions, and the influencing factor high temperature stability test of Example 4 was placed at 50°C.

[0055]

[0056]

[0057] Table 2 Results of product stability test in Example 1 From the data in Table 2, it can be seen that in the stability test in Example 1, all test items of the stability test including influencing factors, acceleration, and long-term items are in compliance with the provisions of China's proposed registration standards. The data characterizing the activity of the samples, such as the anti-Xa factor potency and the anti-IIa factor potency, have not changed significantly, indicating that the key quality attributes of the samples are very stable; "the clarity and color of the solution" is one of the most intuitive and important manifestations of sample stability. The clarity and color of the sample solution in Example 1 have remained stable under various placement conditions. The pH of the sample has a slight downward trend under the influencing factor 60°C and the accelerated test, but the decline is not large and is within a controllable range. There is no obvious trend of change in other items. In summary, the product prepared by the method of Example 1 has very good stability, controllable quality, and stable key quality parameters.

[0058]

[0059]

[0060]

[0061]

[0062] Table 3 Example 4 (control) product stability test results

[0063] As can be seen from the data in Table 3, the key test items such as the clarity and color of the solution, pH, and free sulfate in the test items of the stability test of Example 4 all have test values ​​that do not meet the Chinese proposed registration standards. The clarity and color of the solution are one of the most intuitive and important manifestations of sample stability; if the pH drops quickly and exceeds the standard range, it will cause changes in the acid-base environment of the sample, directly affecting the quality of the sample; free sulfate is an impurity, and if the content exceeds the standard, it will affect the quality of the sample. In the stability test of Example 4, the clarity and color of the solutions of all samples were unqualified; the pH of high temperature (50°C), long-term, and accelerated tests dropped quickly, and finally all exceeded the standard range; the free sulfate content of high temperature (50°C) and accelerated tests finally exceeded the standard range.

[0064] The above test results prove that if a reducing agent is not added for reduction during ultraviolet irradiation, the obtained sample has poor stability and does not meet the quality requirements of China's proposed registration standards. In summary, the product prepared by our invention of a method in which sodium borohydride is added for reduction during ultraviolet irradiation has high product quality, and all test results meet China's proposed registration standards. This method solves the problems of poor product stability and dark color of the feed liquid. For this reason, this method does not require an additional decolorization step for decolorization, which undoubtedly shortens the process flow and reduces production costs. If it is promoted and used, it can generate greater economic value and social benefits.

[0065] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the existence of other identical elements in the process, method, article or device including the elements.

[0066] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing nadroparin calcium, characterized in that: The following steps are involved: Step 1: Degradation of sodium heparin: taking sodium heparin and dissolving it in purified water, adding sodium nitrite after dissolution, adding glacial acetic acid during stirring, and stirring thoroughly until the mixture is completely mixed; Step 2: reduction of the degradation solution, adding sodium borohydride to the feed solution of step 1 to dissolve and obtain a reduction solution; Step 3: UV irradiation: weigh sodium borohydride at 0.5% W / W of the weight of heparin sodium, add it to the reducing solution for UV irradiation, control the liquid temperature at 10° C. to 30° C., the pH value at 9.0 to 11.0, and the UV irradiation time at 60 min to 80 min; Step 4: replacement calcium conversion and ethanol graded precipitation separation and purification, adding calcium chloride to the feed solution of step 3, stirring evenly, the stirring time is not less than 1 hour, then adding 1.0-2.0 ethanol, stirring for not less than 30 minutes, standing for not less than 2 hours, extracting the upper layer of ethanol after standing, collecting the first precipitate of the lower layer, dissolving the first precipitate of the lower layer with water, adding calcium chloride, stirring evenly, the stirring time is not less than 1 hour, then adding ethanol, stirring fully, standing for not less than 2 hours, then extracting the upper layer of ethanol, and collecting the second lower precipitate; Step 5: filtration and freeze-drying: the second precipitate is dissolved in water and then filtered, and the filtered liquid is freeze-dried.

2. The method for preparing nadroparin calcium according to claim 1, characterized in that: The sodium nitrite in step 1 is 1.0%-5.0% of the weight of the heparin sodium, and the glacial acetic acid is added in an amount of 1%-5% of the volume of the feed liquid.

3. The method for preparing nadroparin calcium according to claim 1, characterized in that: The stirring time in step 1 is 60 min to 80 min, and the temperature of the feed liquid is controlled at 20±5° C. during the reaction.

4. The method for preparing nadroparin calcium according to claim 1, characterized in that: The sodium borohydride in step 2 is 0.5% to 2.5% of the weight of heparin sodium W / W .

5. The method for preparing nadroparin calcium according to claim 1, characterized in that: The reaction time in step 2 is 8 to 12 hours, and the temperature is controlled at 10 to 30° C. and the pH value is controlled at 9.0 to 11.0 during the reaction.

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