Low molecular weight heparin injection and method for preparing the same

Abstract

The application discloses a low-molecular-weight heparin injection and a preparation method thereof, and relates to the technical field of pharmaceutical preparations. The low-molecular-weight heparin injection provided by the application comprises low-molecular-weight heparin calcium, an auxiliary material and water for injection, wherein the auxiliary material is composed of glutathione or functionalized glutathione and tea polyphenol. The low-molecular-weight heparin calcium is used as an active ingredient to be mixed with the water for injection to prepare an injection, and the auxiliary material is introduced to increase the stability, so that the prepared low-molecular-weight heparin injection has better anti-Xa activity and long-term stability, is beneficial to improving the pharmaceutical efficacy of the injection and preventing the safety, effectiveness and quality controllability of the medicine from being affected due to the poor stability of the low-molecular-weight heparin injection, and is helpful to the safe use of the low-molecular-weight heparin injection.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical preparation, in particular to a low molecular weight heparin injection and a preparation method thereof. BACKGROUND

[0002] Low molecular weight heparin injection (LMWH) is an anticoagulant drug obtained by chemical or enzymatic cleavage from ordinary heparin (UFH). Compared with unfractionated heparin, the anti-Xa activity is much higher than the anti-IIa activity, and the anti-Xa / IIa ratio is 2-4:1, so it has the characteristics of strong anticoagulation and relatively low risk of bleeding. The half-life of low molecular weight heparin injection is extended to 4-6h, which can be injected subcutaneously once or twice a day, without the need for routine coagulation monitoring, and is widely used for the prevention and treatment of deep vein thrombosis, pulmonary embolism, acute coronary syndrome and thromboembolism during pregnancy. Nadroparin calcium is one of the active ingredients, which is obtained by calcium salt of nitrous acid degradation of porcine intestinal mucosa heparin. Its calcium ion form can reduce the local irritation of subcutaneous injection and reduce the risk of hematoma.

[0003] In the production process, when heparin raw materials are cleaved by nitrous acid, beta-elimination or oxidative depolymerization to generate low molecular weight heparin, improper production process control can cause the loss of sulfate group, isomerization of terminal residue or molecular weight drift, thereby weakening the anti-Xa activity. In addition, during the storage of low molecular weight heparin calcium injection, the sulfate group may also be lost, and factors such as temperature, light, container-drug interaction and microbial contamination during storage can also cause the injection to undergo hydrolysis, oxidation and other conditions, resulting in a significant decrease in anti-Xa activity, which has a great impact on the effectiveness and safety of the drug.

[0004] Therefore, it is of great significance to develop a new preparation method that can improve the stability of low molecular weight heparin injection to improve the safety of low molecular weight heparin injection. SUMMARY

[0005] In view of the above defects of the prior art, the present application provides a low molecular weight heparin injection and a preparation method thereof. The present application uses low molecular weight heparin calcium as the active ingredient and mixes it with water for injection to prepare the injection, and introduces excipients to increase the anti-Xa activity and stability, so that the prepared low molecular weight heparin injection has good efficacy and long-term stability, which is beneficial to prevent the influence of poor stability of low molecular weight heparin injection on the safety, effectiveness and quality controllability of the drug, and helps the safe use of low molecular weight heparin injection.

[0006] To achieve the above-mentioned purpose, the present application provides a preparation method of low molecular weight heparin injection, comprising the following steps:

[0007] (1) Liquid preparation: the prescription amount of low molecular weight heparin calcium, excipient, injection water, stirring to make it completely dissolved, to get mixed liquor; adjust the pH of the mixed liquor, constant volume, filtration; the potency of the low molecular weight heparin injection is 4500-5700 IU / mL; the amount of excipient is 0.5-1 wt%; the excipient is composed of glutathione or functional glutathione and tea polyphenol mixture;

[0008] (2) Cleaning and sterilization of low borosilicate glass ampoule: after ultrasonic cleaning, injection water flushing and compressed air drying, the low borosilicate glass ampoule enters the tunnel type sterilization and drying machine, preheats in the preheating section, sterilizes in the high temperature section, cools in the cooling section, and then enters the filling and sealing room for standby;

[0009] (3) Filling and sealing: the cleaned and sterilized low borosilicate glass ampoule and the filtered drug solution are sent into the filling and sealing room. After adjusting the machine to normal, the filling is carried out according to the filling post operation procedure, and the filling is sealed at the same time;

[0010] (4) Leak detection: remove damaged and unqualified products;

[0011] (5) Lamp inspection and leak detection: the samples that pass the leak detection are transferred into the lamp inspection room, and are subjected to lamp inspection and leak detection respectively, and unqualified products are removed;

[0012] (6) Labeling: the semi-finished products that pass the lamp inspection are sent into the packaging room for labeling;

[0013] (7) Packaging: the semi-finished products with labels are packaged into small boxes and large boxes, and then are stored in the warehouse.

[0014] Preferably, the excipient is composed of glutathione and tea polyphenol in a mass ratio of 4-6:1-4; or, composed of functional glutathione and tea polyphenol in a mass ratio of 4-6:1-4.

[0015] Preferably, the preparation method of the functional glutathione comprises the following steps:

[0016] S1, 0.18-0.22 g of polyethylene glycol active ester is added to 8-12 mL of N,N-dimethylformamide, ultrasonic oscillation is carried out, and a polyethylene glycol active ester solution is obtained; 0.03-0.031 g of glutathione is added to 4-6 mL of PBS buffer solution with pH of 8.3-8.7, and stirring and dissolving are carried out at 22-27°C for 8-15 min, and a glutathione solution is obtained;

[0017] S2, 1-2 mL of polyethylene glycol active ester solution is added dropwise into 5-8 mL of glutathione solution, and the rotation speed is kept at 200-500 rpm; after the dropwise addition is completed, 45-55 μL of triethylamine is added, and the reaction is continuously stirred at 22-27℃ for 1.5-3 h; then the reaction solution is dialyzed, and then the dialyzed solution is concentrated under reduced pressure to obtain a concentrated solution; the concentrated solution is freeze-dried to obtain functionalized glutathione.

[0018] Preferably, the polyethylene glycol active ester is selected from one of NHS-PEG-NHS, Biotin-PEG-NHS.

[0019] Preferably, the ultrasonic oscillation in step S1 is ultrasonic oscillation at an ultrasonic power of 200-500 W for 10-15 min.

[0020] Preferably, in step (1), the pH of the mixed liquid is adjusted to 6.0-8.0 by using 0.1-0.5 mol / L dilute hydrochloric acid or sodium hydroxide aqueous solution.

[0021] Preferably, in step (1), the filtration is first pre-filtered through a 0.45 μm filter, and then sterilized by passing through two 0.22 μm sterilization filters.

[0022] Preferably, in step (2), the sterilization temperature of the high-temperature section is 290-310℃, and the sterilization time is 30-35 min.

[0023] The application also provides a low molecular weight heparin injection prepared by the above method.

[0024] The application has the following beneficial effects:

[0025] Compared with the prior art, the application prepares a low molecular weight heparin injection and provides a preparation method thereof. The low molecular weight heparin injection provided by the application comprises low molecular weight heparin calcium, excipients, and water for injection. In the preparation process of the low molecular weight heparin injection, the low molecular weight heparin calcium is used as an active ingredient, mixed with water for injection to prepare an injection solution, and glutathione or functionalized glutathione is introduced and used in cooperation with tea polyphenol as an excipient, which significantly improves the anti-Xa activity and stability of the low molecular weight heparin injection. This is not only beneficial to improve the efficacy of the low molecular weight heparin injection, but also beneficial to prevent the influence of poor stability of the low molecular weight heparin injection on the safety, effectiveness, and quality controllability of the drug, and conducive to the safe use of the low molecular weight heparin injection. DETAILED DESCRIPTION

[0026] Parameters for using specific chemicals, sources.

[0027] Low molecular weight heparin calcium, 75 IU / mg, the component with a weight average molecular weight less than 8000 Da is not less than 70% of the total amount;

[0028] Glutathione, G-SH (reduced form), purity ≥98%, commercially available;

[0029] Tea polyphenol, purity ≥99%, commercially available.

[0030] Example 1, a preparation method of a low molecular weight heparin injection, comprising the following steps:

[0031] (1) Liquid preparation: 74.7 g of low molecular weight heparin calcium (calculated based on the potency of low molecular weight heparin injection of 5600 IU / mL) is weighed and added to 950 g of water for injection, and stirred to completely dissolve to obtain a mixed drug solution; the pH of the mixed drug solution is adjusted to 7 with 0.5 mol / L sodium hydroxide aqueous solution, and then the mixed drug solution is diluted to 1 L with water for injection; and then it is first pre-filtered through a 0.45 μm filter, and then sterilized and filtered through two 0.22 μm sterilization filters;

[0032] (2) Cleaning and sterilization of low borosilicate glass ampoules: after being cleaned by ultrasonic wave, rinsed with water for injection, and dried by compressed air, the low borosilicate glass ampoules enter a tunnel type sterilization and drying machine, are preheated in a preheating section, sterilized at 300°C for 30 min in a high temperature section, and then sent to a filling and sealing room after cooling in a cooling section for standby;

[0033] (3) Filling and sealing: the cleaned and sterilized low borosilicate glass ampoules and the drug solution after sterilization and filtration are sent to the filling and sealing room, and after the machine is adjusted to be normal, the filling is performed according to the filling post operation procedures, and the filling is simultaneously sealed by fusion;

[0034] (4) Leak detection: damaged and unqualified products are removed through leak detection;

[0035] (5) Lamp inspection and leak detection: the samples after leak detection are transferred to a lamp inspection room, and are subjected to lamp inspection and leak detection, respectively, and unqualified products are removed;

[0036] (6) Labeling: the semi-finished products after lamp inspection are sent to a packaging room for labeling;

[0037] (7) Packaging: the semi-finished products after labeling are packaged into small boxes and large cases, and then are stored in a warehouse.

[0038] Example 2, a preparation method of a low molecular weight heparin injection, comprising the following steps:

[0039] (1) Liquid preparation: 74.7 g of low molecular weight calcium heparin (calculated based on the potency of low molecular weight heparin injection of 5600 IU / mL) and 8 g of auxiliary materials are weighed, 950 g of water for injection is added, and stirring is performed to completely dissolve them, to obtain a mixed drug solution; the pH of the mixed drug solution is adjusted to 7 with 0.5 mol / L sodium hydroxide aqueous solution, and then the mixed drug solution is diluted to 1 L with water for injection; and then the mixed drug solution is first pre-filtered through a 0.45 μm filter, and then sterilized and filtered through two 0.22 μm sterilization filters;

[0040] (2) Cleaning and sterilization of low borosilicate glass ampoules: After the low borosilicate glass ampoules are ultrasonically cleaned, rinsed with water for injection, and dried with compressed air, they are introduced into a tunnel-type sterilization and drying machine, preheated in a preheating section, sterilized at 300°C for 30 min in a high-temperature section, and then cooled in a cooling section before being sent to a filling and sealing room for standby;

[0041] (3) Filling and sealing: The cleaned and sterilized low borosilicate glass ampoules and the drug solution after sterilization and filtration are sent to the filling and sealing room, and after the machine is adjusted to normal, filling is performed according to the filling post operating procedures, and the filling is simultaneously sealed by fusion;

[0042] (4) Leak detection: damaged and unqualified products are removed through leak detection;

[0043] (5) Lamp inspection and leak detection: the samples after leak detection are introduced into a lamp inspection room, and lamp inspection and leak detection are performed on them, respectively, and unqualified products are removed;

[0044] (6) Labeling: the semi-finished products after lamp inspection are sent to a packaging room for labeling;

[0045] (7) Packaging: the semi-finished products after labeling are packaged into small boxes and large cases, and then stored in a warehouse.

[0046] The auxiliary materials are composed of glutathione and tea polyphenol at a mass ratio of 5:3.

[0047] Example 3, a preparation method of low molecular weight heparin injection, differs from example 2 only in that the auxiliary materials are composed of functionalized glutathione and tea polyphenol at a mass ratio of 5:3.

[0048] The preparation method of the functionalized glutathione comprises the following steps:

[0049] S1, 0.2 g of Biotin-PEG-NHS (molecular weight: 2000, catalog number: 80030411-2000, Guangzhou Carbohydrate Technology Co., Ltd.) is added to 10 mL of N,N-dimethylformamide, and ultrasonic oscillation is performed at an ultrasonic power of 200 W for 10 min to obtain a polyethylene glycol active ester solution; 0.0307 g of glutathione is weighed and added to 5 mL of PBS buffer solution (0.01 mol / L) with a pH of 8.5, and stirring and dissolution are performed at 25°C for 10 min to obtain a glutathione solution;

[0050] S2, 1 mL of polyethylene glycol active ester solution was added dropwise to 5 mL of glutathione solution, and the rotation speed was kept at 300 rpm; after the addition was completed, 50 μL of triethylamine (0.1 mol / L) was added, and the reaction was continued at 25°C for 2 h; then the reaction solution was transferred to a 10 kDa dialysis bag, dialyzed against water at 4°C for 24 h, and then the dialysate was concentrated to 1 / 5 of the original volume under reduced pressure at 40°C to obtain a concentrated solution; the concentrated solution was freeze-dried to obtain functionalized glutathione.

[0051] Example 4, a preparation method of a low molecular weight heparin injection, which is only different from example 2 in that the adjuvant is composed of functionalized glutathione and tea polyphenol in a mass ratio of 5:3.

[0052] The preparation method of the functionalized glutathione comprises the following steps:

[0053] S1, 0.2 g of Biotin-PEG-NHS (molecular weight: 5000, catalog number: 80030411-5000, Guangzhou Carbohydrate Technology Co., Ltd.) was added to 10 mL of N,N-methyl formamide, and ultrasonic oscillation was carried out at an ultrasonic power of 200 W for 10 min to obtain a polyethylene glycol active ester solution; 0.0307 g of glutathione was weighed and added to 5 mL of PBS buffer solution (0.01 mol / L) with a pH of 8.5, and stirred and dissolved at 25°C for 10 min to obtain a glutathione solution;

[0054] S2, 1 mL of polyethylene glycol active ester solution was added dropwise to 5 mL of glutathione solution, and the rotation speed was kept at 300 rpm; after the addition was completed, 50 μL of triethylamine (0.1 mol / L) was added, and the reaction was continued at 25°C for 2 h; then the reaction solution was transferred to a 10 kDa dialysis bag, dialyzed against water at 4°C for 24 h, and then the dialysate was concentrated to 1 / 5 of the original volume under reduced pressure at 40°C to obtain a concentrated solution; the concentrated solution was freeze-dried to obtain functionalized glutathione.

[0055] Example 5, a preparation method of a low molecular weight heparin injection, which is only different from example 2 in that the adjuvant is composed of functionalized glutathione and tea polyphenol in a mass ratio of 5:3.

[0056] The preparation method of the functionalized glutathione comprises the following steps:

[0057] S1, 0.2 g of NHS-PEG-NHS (molecular weight: 2000, catalog number: 80020105-2000, Guangzhou City Carbohydrate Technology Co., Ltd.) was added to 10 mL of N,N-dimethylformamide, and ultrasonic oscillation was performed at 200 W for 10 min to obtain a polyethylene glycol active ester solution; 0.0307 g of glutathione was weighed and added to 5 mL of PBS buffer solution (0.01 mol / L) with a pH of 8.5, and was stirred and dissolved at 25°C for 10 min to obtain a glutathione solution;

[0058] S2, 1 mL of the polyethylene glycol active ester solution was added dropwise to 5 mL of the glutathione solution, and the stirring speed was maintained at 300 rpm; after the dropwise addition was completed, 50 μL of triethylamine (0.1 mol / L) was added, and the reaction was continued at 25°C for 2 h; then the reaction solution was transferred to a 10 kDa dialysis bag, and dialysis was performed at 4°C for 24 h using water, and then the dialysate was concentrated to 1 / 5 of the original volume under reduced pressure at 40°C to obtain a concentrated solution; the concentrated solution was freeze-dried to obtain functionalized glutathione.

[0059] Comparative Example 1, a preparation method of a low molecular weight heparin injection, comprising the following steps:

[0060] (1) Liquid preparation: 74.7 g of low molecular weight heparin calcium (calculated based on a low molecular weight heparin injection potency of 5600 IU / mL) and 8 g of functionalized glutathione were weighed and added to 950 g of water for injection, and stirred to completely dissolve to obtain a mixed drug solution; the pH of the mixed drug solution was adjusted to 7 using a 0.5 mol / L sodium hydroxide aqueous solution, and then water for injection was added to bring the mixed drug solution to a volume of 1 L; then the mixed drug solution was first pre-filtered through a 0.45 μm filter, and then sterilized by filtering through two 0.22 μm sterilization filters;

[0061] (2) Cleaning and sterilization of low borosilicate glass ampoules: after ultrasonic cleaning, water for injection rinsing, and compressed air drying, the low borosilicate glass ampoules were preheated in the preheating section, sterilized at 300°C for 30 min in the high temperature section, and then sent to the filling and sealing room after cooling in the cooling section for standby;

[0062] (3) Filling and sealing: the cleaned and sterilized low borosilicate glass ampoules and the sterilized drug solution were sent to the filling and sealing room, the machine was adjusted to normal operation, and then filling was performed according to the filling post operating procedures, and the filling was simultaneously sealed by melting;

[0063] (4) Leak detection: damaged and unqualified products were removed;

[0064] (5) Lamp inspection and leak detection: the samples that passed the leak detection were transferred to the lamp inspection room, and were subjected to lamp inspection and leak detection, respectively, and unqualified products were removed;

[0065] (6), labeling: the semi-finished product passed the lamp inspection is sent to the packaging room for labeling;

[0066] (7), packaging: the semi-finished product with labels is packed into small boxes and large boxes, and then stored in the warehouse.

[0067] The preparation method of the functionalized glutathione is consistent with Example 3.

[0068] Comparative Example 2, a preparation method of a low molecular weight heparin injection, comprising the following steps:

[0069] (1), liquid preparation: 74.7 g of low molecular weight heparin calcium (calculated based on the potency of low molecular weight heparin injection of 5600 IU / mL) and 8 g of tea polyphenol are weighed, 950 g of water for injection is added, and stirring is performed to completely dissolve them, to obtain a mixed drug solution; the pH of the mixed drug solution is adjusted to 7 with 0.5 mol / L sodium hydroxide aqueous solution, and then the mixed drug solution is diluted to 1 L with water for injection; then, it is first pre-filtered through a 0.45 μm filter, and then sterilized and filtered through two 0.22 μm sterilization filters;

[0070] (2), cleaning and sterilization of low borosilicate glass ampoules: after the low borosilicate glass ampoules are ultrasonically cleaned, rinsed with water for injection, and dried with compressed air, they are sent into a tunnel type sterilization and drying machine, preheated in a preheating section, sterilized at 300°C for 30 min in a high temperature section, and then sent into a filling and sealing room after cooling in a cooling section for standby;

[0071] (3), filling and sealing: the cleaned and sterilized low borosilicate glass ampoules and the sterilized and filtered drug solution are sent into the filling and sealing room, the machine is adjusted to be normal, and then filling is performed according to the filling and sealing post operation procedures, and the filling is simultaneously sealed by fusion;

[0072] (4), leak detection: damaged and unqualified products are removed through leak detection;

[0073] (5), lamp inspection for leak detection: the samples that pass the leak detection are sent into a lamp inspection room, and lamp inspection and leak detection are performed on them respectively, and unqualified products are removed;

[0074] (6), labeling: the semi-finished product passed the lamp inspection is sent to the packaging room for labeling;

[0075] (7), packaging: the semi-finished product with labels is packed into small boxes and large boxes, and then stored in the warehouse.

[0076] Comparative Example 3, a preparation method of a low molecular weight heparin injection, comprising the following steps:

[0077] (1) Liquid preparation: 74.7 g of low molecular weight calcium heparin (calculated based on the potency of low molecular weight heparin injection of 5600 IU / mL) and 8 g of glutathione are weighed, 950 g of water for injection is added, and stirring is performed to completely dissolve them, to obtain a mixed drug solution; the pH of the mixed drug solution is adjusted to 7 using 0.5 mol / L sodium hydroxide aqueous solution, and then the mixed drug solution is diluted to 1 L with water for injection; and then the solution is first pre-filtered through a 0.45 μm filter, and then sterilized through two 0.22 μm sterilization filters;

[0078] (2) Cleaning and sterilization of low borosilicate glass ampoules: after the low borosilicate glass ampoules are subjected to ultrasonic cleaning, water for injection rinsing, and compressed air drying, they are introduced into a tunnel-type sterilization and drying machine, preheated in a preheating section, sterilized at 300°C for 30 min in a high-temperature section, and then cooled in a cooling section before being sent to a filling and sealing room for standby;

[0079] (3) Filling and sealing: the cleaned and sterilized low borosilicate glass ampoules and the drug solution after sterilization filtration are sent to the filling and sealing room, and after the machine is adjusted to be normal, filling is performed according to the filling post operating procedures, and the filling is simultaneously sealed by fusion;

[0080] (4) Leak detection: damaged and unqualified products are removed through leak detection;

[0081] (5) Lamp inspection and leak detection: the samples after leak detection are introduced into a lamp inspection room, and lamp inspection and leak detection are performed, and unqualified products are removed;

[0082] (6) Labeling: the semi-finished products after lamp inspection are sent to a packaging room for labeling;

[0083] (7) Packaging: the labeled semi-finished products are packaged into small boxes and large cases, and then stored in a warehouse.

[0084] Test Example 1, Anti-Xa activity test

[0085] 1. Solution preparation:

[0086] Tris-Hydroxymethylaminomethane-sodium chloride buffer (pH 7.4): 6.08 g of tris-hydroxymethylaminomethane and 8.77 g of sodium chloride are taken, 500 mL of water is added to dissolve them, 10 g of bovine serum albumin is added, the pH is adjusted to 7.4 with hydrochloric acid, and then diluted to 1000 mL with water;

[0087] Tris-Hydroxymethylaminomethane-ethylenediaminetetraacetic acid disodium buffer (pH 8.4): 5.12 g of sodium chloride, 3.03 g of tris-hydroxymethylaminomethane, and 1.4 g of ethylenediaminetetraacetic acid disodium are taken, 250 mL of water is added to dissolve them, the pH is adjusted to 8.4 with hydrochloric acid, and then diluted to 500 mL with water;

[0088] Antithrombin (ATIII) solution preparation: Take antithrombin (ATIII) (brand: Asnail-Biotechnology, specification 10 IU / bottle) and mix with 10 mL Tris-HCl buffer solution (pH 7.4) to dissolve and prepare 1 IU / mL antithrombin (ATIII) solution;

[0089] Chromogenic substrate S-2765 solution preparation: Mix chromogenic substrate S-2765 (brand: Asnail-Biotechnology, specification 3 mmol / L) with 11.66 mL water to dissolve and prepare 3 mmol / L chromogenic substrate S-2765 solution, which is stored in the dark; before use, dilute it with Tris-HCl-EDTA buffer solution (pH 8.4) to 0.5 mmol / L chromogenic substrate S-2765 solution;

[0090] Factor Xa (FXa) solution: Mix factor Xa (brand: Chromogenix, specification 71 nkat / bottle) with 11 mL Tris-HCl buffer solution (pH 7.4) to dissolve and obtain factor Xa (FXa) solution for standby use.

[0091] 2. Detection method

[0092] Low molecular weight heparin standard solution preparation: Take low molecular weight heparin standard (from China Food and Drug Inspection Research Institute, specification 1497 IU / branch) and place it in a 10 mL volumetric flask, dilute to the mark with Tris-HCl buffer solution (pH 7.4), shake well, and prepare a solution containing 0.10 IU anti-Xa factor per 1 mL as a standard solution;

[0093] Test solution preparation: Take about 0.1 g of the product (low molecular weight heparin injection prepared in Examples 1-5 and Comparative Examples 1-3 of the present application), place it in a 50 mL volumetric flask, dilute to the mark with water, shake well, then accurately take 1 mL, dilute with Tris-HCl buffer solution (pH 7.4), and prepare a solution containing 0.10 IU anti-Xa factor per 1 mL;

[0094] Take 8 centrifuge tubes and label them Sa, Sb, Sc, Sd, Ta, Tb, Tc, and Td, respectively. Dilute the standard (S) and test (T) solutions into four different concentrations with a ratio of 1:0.7;

[0095] Take 18 centrifugal tubes, respectively marked as B1, S1, S2, S3, S4, T1, T2, T3, T4, T1', T2', T3', T4', S1', S2', S3', S4', B2, wherein B1, B2 represent blank. And placed in the centrifugal tube rack in the above order, 4 in each row, add test sample solution or standard solution;

[0096] After adding the test sample or standard solution, place the tubes in order on the constant temperature mixer, add 50 μL of 1 IU / mL antithrombin solution to each tube, mix (no bubbles are allowed), and equilibrate at 37°C for 1 minute; add 100 μL of factor Xa (FXa) solution to each tube, mix, and equilibrate at 37°C for 1 minute; add 250 μL of chromogenic substrate (S-2765) to each tube, mix, and incubate at 37°C for 4 minutes, then add 375 μL of 30% acetic acid solution to each tube to terminate the reaction. Use a 1 cm light path semi-micro cuvette, with Tris-HCl buffer (pH 7.4) as blank, measure the absorbance of each tube at 405 nm wavelength. The absorbance of the two blank tubes should not have significant difference;

[0097] Take the absorbance as the vertical coordinate and the logarithmic value of the concentration of the standard solution or test sample solution as the horizontal coordinate, respectively make linear regression, according to the 4×4 experimental design of the parallel line principle in the biological assay statistics, use the biological statistics calculation software BS2000 of Chinese Pharmacopoeia to calculate the anti-Xa factor titer and experimental error.

[0098] The test results are shown in Table 1 below.

[0099] Table 1

[0100]

[0101] From Table 1, it can be seen that, compared with Example 1, the anti-Xa activity of Examples 2-5 and Comparative Examples 1-3 is increased, indicating that the introduction of adjuvants is beneficial to enhancing the anti-Xa activity of low molecular weight injection. By comparing Example 2 and Comparative Examples 2-3, it can be seen that the anti-Xa activity of Example 2 is higher than that of Comparative Examples 2-3, indicating that compared with the introduction of glutathione and tea polyphenol as adjuvants alone, the use of glutathione and tea polyphenol as adjuvants has a better effect on improving the anti-Xa activity of the injection, and the reason may be that glutathione targets the protection of sulfate groups through sulfydryl to inhibit the oxidation and shedding of key sites, while tea polyphenol widely eliminates initial free radicals to block the oxidative chain reaction. The synergistic effect of the two on different oxidation stages can effectively stabilize the structure of heparin and help maintain and improve the anti-Xa activity. By comparing Examples 2-5 and Comparative Example 1, it can be seen that the anti-Xa activity of Examples 3-5 is higher than that of Example 2 and Comparative Example 1, and the anti-Xa activity of Example 3 is the highest, indicating that compared with the introduction of functionalized glutathione, glutathione, tea polyphenol as adjuvants alone, or the introduction of glutathione and tea polyphenol as adjuvants, the use of functionalized glutathione and tea polyphenol as adjuvants has a better effect on improving the anti-Xa activity of the injection, which is beneficial to improving the efficacy of low molecular weight heparin injection.

[0102] In Test Example 2, the low molecular weight heparin injection prepared from Examples 1-5 and Comparative Examples 1-3 was used as a sample for long-term stability test. The sample was placed in a long-term condition (25±2℃, 60±5%RH) for 9 months, and samples were taken at 0 month, 3 months, 6 months and 9 months for pH value determination and titer determination, and the color and state of the low molecular weight heparin injection were observed, and the results are shown in Table 2 below:

[0103] Table 2

[0104]

[0105] From Table 2, after 9 months of long-term stability test, it was found that the pH value and potency of Examples 1 and Comparative Examples 2-3 were greater than those of Examples 2-5 and Comparative Example 1, and the color of the low molecular weight heparin injection of Comparative Examples 2-3 appeared yellowish at the 9th month, indicating that the stability of Examples 1 and Comparative Examples 2-3 was poorer than that of Examples 2-5 and Comparative Example 1. It was found that the pH value and potency of Example 2 and Comparative Example 1 were greater than those of Examples 3-5, and the color of the low molecular weight heparin injection of Example 2 appeared yellowish at the 9th month, indicating that compared with the introduction of glutathione and tea polyphenol as a synergistic auxiliary material, the use of functionalized glutathione and tea polyphenol as an auxiliary material had a better effect on the stability improvement of the injection, which was beneficial to the long-term storage of the low molecular weight heparin injection. After 9 months of long-term stability test, the injection of Examples 3-5 remained colorless and clear and transparent, and compared with Example 3, the pH value and potency of Examples 4-5 decreased more greatly, and Example 3 had better long-term stability, indicating that the synergistic effect of the functionalized glutathione prepared by using Biotin-PEG-NHS modified glutathione with a molecular weight of 2000 and tea polyphenol was better, which could better prevent the influence of the poor stability of the low molecular weight heparin injection on the safety, effectiveness and quality controllability of the drug, and was beneficial to the safe use of the low molecular weight heparin injection.

Claims

1. A method for preparing a low molecular weight heparin injection, characterized in that: The steps include: (1) Liquid preparation: Weigh the prescribed amount of low molecular weight heparin calcium and excipients, add the prescribed amount of water for injection, stir to completely dissolve, and obtain a mixed liquid; adjust the pH of the mixed liquid, constant volume, and filter; the potency of the low molecular weight heparin injection is 4500-5700 IU / mL; the amount of the excipients is 0.5-1 wt%; the excipients are composed of glutathione or a mixture of functionalized glutathione and tea polyphenols; (2) Cleaning and sterilization of low-borosilicate glass ampoules: After ultrasonic cleaning, injection water flushing, and compressed air drying, the low-borosilicate glass ampoules enter the tunnel-type sterilization dryer, are preheated in the preheating section, sterilized in the high-temperature section, cooled in the cooling section, and then sent to the filling room for standby use; (3) Filling: Send the cleaned and sterilized low-boron silicon glass ampoules and the sterilized and filtered liquid medicine into the filling room. After the machine is debugged and normal, fill it according to the filling position operating procedures, and melt and seal it at the same time; (4) Leak detection: leak detection to remove damaged and unqualified products; (5) Light inspection and leak detection: The samples that have been leak-checked are transferred to the light inspection room, where they are inspected by light and leak-checked, and unqualified products are eliminated; (6) Labeling: Send the semi-finished products that have passed the light inspection to the packaging room for labeling; (7) Packaging: The semi-finished products with labels are packed into small boxes or large cartons and then put into storage; The preparation method of the functionalized glutathione comprises the following steps: S1. Take 0.18-0.22 g of polyethylene glycol active ester and add it to 8-12 mL of N,N-dimethylformamide, and ultrasonically vibrate to obtain a polyethylene glycol active ester solution; weigh 0.03-0.031 g of glutathione and add it to 4-6 mL of PBS buffer solution with a pH of 8.3-8.7, and stir and dissolve it at 22-27°C for 8-15 minutes to obtain a glutathione solution; S2. Add 1-2 mL of polyethylene glycol active ester solution dropwise to 5-8 mL of glutathione solution, maintaining a rotation speed of 200-500 rpm; after the addition is complete, add 45-55 μL of triethylamine, and continue to stir and react at 22-27°C for 1.5-3 hours; then dialyze the reaction solution, and then concentrate the dialyzate under reduced pressure to obtain a concentrated solution; the concentrated solution is freeze-dried to obtain functional glutathione.

2. The method for preparing the low molecular weight heparin injection according to claim 1, wherein: The auxiliary material is composed of glutathione and tea polyphenols mixed in a mass ratio of 4-6:1-4; or, is composed of functionalized glutathione and tea polyphenols mixed in a mass ratio of 4-6:1-4.

3. The method for preparing low molecular weight heparin injection according to claim 1, characterized in that: The polyethylene glycol active ester is selected from one of NHS-PEG-NHS and Biotin-PEG-NHS.

4. The method for preparing low molecular weight heparin injection according to claim 1, characterized in that: The ultrasonic oscillation in step S1 is performed at an ultrasonic power of 200-500 W for 10-15 minutes.

5. The method for preparing low molecular weight heparin injection according to claim 1, characterized in that: In the step (1), the pH of the mixed liquid is adjusted to 6.0-8.0 using 0.1-0.5 mol / L dilute hydrochloric acid or sodium hydroxide aqueous solution.

6. The method for preparing low molecular weight heparin injection according to claim 1, characterized in that: The filtration in step (1) is first pre-filtered through a 0.45 μm filter and then sterilized and filtered through two 0.22 μm sterilizing grade filters.

7. The method for preparing low molecular weight heparin injection according to claim 1, characterized in that: The sterilization temperature of the high temperature section sterilization in step (2) is 290-310°C, and the sterilization time is 30-35 minutes.

8. A low molecular weight heparin injection, characterized in that: The method is prepared by any one of claims 1 to 7.

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