Troxerutin freeze-dried product, preparation method thereof and application of troxerutin freeze-dried product in plasma virus inactivation

Abstract

The invention relates to a troxerutin freeze-dried product, a preparation method thereof and application of the troxerutin freeze-dried product in plasma virus inactivation. The preparation method comprises the following specific steps: providing a split charging liquid obtained by ultrafiltration of a troxerutin raw material medicine for injection, or providing a troxerutin injection; and putting the split charging liquid or the troxerutin injection into a freeze dryer for freeze-drying. Through the freeze-drying preparation process, the prepared troxerutin freeze-dried product is excellent in appearance property and good in redissolving performance before and after sterilization, meets the ethylene oxide sterilization requirement, is high in stability and can be stored for a long time, and the application of the troxerutin freeze-dried product in plasma virus inactivation is effectively guaranteed; and the safety and effectiveness of the troxerutin freeze-dried product in plasma virus inactivation can be improved.

Description

Technical Field

[0001] The present invention relates to the technical field of virus inactivation, in particular to a troxerutin freeze-dried product and a preparation method thereof and application thereof in plasma virus inactivation. Background Art

[0002] Troxerutin, molecular formula C 33 H 42 O 19 Troxerutin, CAS number 7085-55-4. As a drug, it inhibits platelet aggregation and prevents thrombosis. It also counteracts vascular damage caused by serotonin and bradykinin, increases capillary resistance, reduces capillary permeability, and prevents edema caused by increased vascular permeability. It has a significant protective effect against acute ischemic brain injury. As a plasma protective agent, troxerutin effectively quenches reactive oxygen species (ROS) generated during the UVC light inactivation of plasma viruses, thereby preventing damage to the active ingredients in plasma.

[0003] Currently, troxerutin is available in tablets, injections, and lyophilized powder injections. Single-use plasma virus inactivation devices containing troxerutin require ethylene oxide (EO) sterilization during the final production stage. The chemical reaction between ethylene oxide and water requires that products suitable for EO sterilization must contain no or minimal moisture. Troxerutin containing moisture reacts with ethylene oxide, affecting its content and composition and producing other substances, increasing risks to the safety and effectiveness of troxerutin products when used in single-use plasma virus inactivation devices. Therefore, strict requirements are imposed on the moisture content of troxerutin added to single-use plasma virus inactivation devices. Furthermore, troxerutin added to single-use plasma virus inactivation devices must pass sterility testing, bacterial endotoxin testing, and particulate matter testing to meet safety standards for injectables.

[0004] In response to this demand, the present invention provides a troxerutin freeze-dried product, a preparation method thereof, and its application in plasma virus inactivation. Summary of the Invention

[0005] Troxerutin used in plasma virus inactivation equipment must ensure strict moisture control, sterility, bacterial endotoxin, and particulate testing to ensure safety and effectiveness.

[0006] To achieve the above object, in a first aspect, the present invention provides a method for preparing a troxerutin freeze-dried product, which comprises the following specific steps: S1. The subpackage obtained after the preparation and ultrafiltration of the troxerutin injection raw material powder, or the troxerutin injection solution provided for direct subpackage; S2. Place the subpackaged liquid or the subpackaged troxerutin injection in a freeze dryer for freeze drying, wherein the freeze drying step comprises: pre-freezing at a temperature of -35°C to -45°C; performing sublimation drying at a temperature of -10°C after pre-freezing; heating to 0°C for desorption drying and sublimation drying; and heating to 25-35°C for desorption drying.

[0007] In one implementation, the freeze-drying step specifically includes: S21, pre-freeze 01 section: Place the aliquot in the freeze dryer and cool the sample to -35°C~-45°C at a cooling rate of 0.8°C / min; S22, pre-freeze 02 section: maintain the temperature at -35℃~-45℃ for 60~240 minutes; S23, sublimation drying section 03: vacuum setting 5~20Pa, heating the sample from -35℃~-45℃ to -10℃ at a heating rate of 0.8℃ / min; S24, sublimation drying section 04: vacuum setting 5~20Pa, temperature at -10℃ for 60 minutes; S25, sublimation drying section 05: vacuum set to 0Pa, temperature at -10℃ maintained for 600~960 minutes; S26, analytical drying section 06: vacuum setting 5~20Pa, heating the sample from -10℃ to 0℃ at a heating rate of 0.8℃ / min; S27, sublimation drying section 07: vacuum setting 5~20Pa, maintain temperature at 0℃ for 120~240 minutes; S28, analytical drying section 08: vacuum setting 0Pa, the sample temperature was raised from 0℃ to 25~35℃ at a heating rate of 0.8℃ / min; S29, analytical drying section 09: vacuum setting 0-5 Pa, maintaining the temperature at 25-35° C. for 60-180 minutes to obtain the troxerutin freeze-dried product.

[0008] In one implementation, the preparation and ultrafiltration of the troxerutin raw material powder for injection specifically include the following steps: S11, adding the troxerutin raw material powder for injection and the lyophilized excipient to water for injection and stirring until completely dissolved to obtain a troxerutin solution; S12, in an ultrafiltration equipment system, ultrafiltering the troxerutin solution to obtain a filtrate; S13, dispensing the filtrate into the release member to obtain a dispensing liquid.

[0009] In one implementation, the concentration of the packaging liquid obtained after the troxerutin raw material powder for injection is prepared and ultrafiltered is 20-100 mg / mL.

[0010] In one implementation, the lyophilization excipient is any one of hydroxyethyl starch, mannitol and glucose, and the mass fraction concentration of the lyophilization excipient is 1-2%.

[0011] In one implementation, in S12, the ultrafiltration condition is: 0.1~2.5m 2 The filter membrane package is made of PES and 10KD. The inlet pressure in the ultrafiltration system is 1~3bar, the reflux pressure is 0.5~1bar, and the flow rate is 16~64L / m 2 .

[0012] In one implementation, in S1, the concentration of the troxerutin injection is 20-100 mg / mL.

[0013] In one implementation, in S1, the troxerutin injection includes troxerutin and excipients, and the excipients include water for injection, disodium hydrogen phosphate, sodium dihydrogen phosphate, disodium edetate, sodium hydroxide and hydrochloric acid.

[0014] In a second aspect, the present invention further provides a troxerutin freeze-dried product, which is prepared by the above-mentioned method for preparing the troxerutin freeze-dried product.

[0015] In a third aspect, the present invention also provides the use of any of the above-mentioned troxerutin freeze-dried products in plasma virus inactivation.

[0016] In one implementation, the application specifically includes: adding the troxerutin freeze-dried product to plasma as a plasma protective agent, placing the mixed bag containing plasma and troxerutin in an inactivation cabinet, and irradiating with UVC light to obtain virus-inactivated plasma, wherein the dosage of the added troxerutin is 0.2-1.4 g / L, and the radiation dosage of the UVC light is 0.1-1.5 J / cm 2 .

[0017] Beneficial effects: The preparation method of the troxerutin freeze-dried product provided by the present invention can freeze-dry the troxerutin raw material for injection and the troxerutin injection. Through its freeze-drying preparation process, the prepared troxerutin freeze-dried product can be effectively used in plasma virus inactivation equipment. Before and after the plasma virus inactivation equipment is sterilized with ethylene oxide, the troxerutin freeze-dried product has excellent appearance and good resolubility, meeting the requirements of ethylene oxide sterilization of the equipment. It has high stability and can be stored for a long time, effectively ensuring the use of the troxerutin freeze-dried product in plasma virus inactivation. The present invention also provides a method for using the troxerutin freeze-dried product in plasma virus inactivation. 254nm UVC irradiation can effectively inactivate enveloped indicator viruses and non-enveloped indicator viruses. At the same time, the added troxerutin freeze-dried product effectively protects the plasma protein components, making plasma virus inactivation safe and effective. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a flow chart of the steps of the preparation method of the troxerutin freeze-dried product provided by the present invention; Figure 2 A flow chart of the steps for preparing and ultrafiltration of the troxerutin raw material powder for injection provided by the present invention; Figure 3 A flow chart of the freeze-drying steps provided by the present invention; Figure 4 This is the shape of 2.0 mL of the troxerutin bulk drug substance provided in Example 1 after ultrafiltration and before lyophilization; Figure 5 The shape of 2.0 mL of the troxerutin bulk drug substance provided in Example 1 after preparation, ultrafiltration, and freeze-drying; Figure 6 This is the shape of 1.0 mL of the troxerutin bulk drug substance provided in Example 2 after ultrafiltration and before lyophilization; Figure 7 The shape of 1.0 mL of the troxerutin bulk drug substance provided in Example 2 after preparation, ultrafiltration, and freeze-drying; Figure 8 This is the shape of the 2.0 mL troxerutin injection provided in Example 4 before lyophilization; Figure 9 This is the shape of the 2.0 mL lyophilized troxerutin injection provided in Example 4; Figure 10 This is the shape of the 0.5 mL troxerutin injection provided in Example 6 before lyophilization; Figure 11 This is the shape of the 0.5 mL lyophilized troxerutin injection provided in Example 6; Figure 12 This is a morphological diagram of normal growth of BHK-21 cells; Figure 13This is a picture of the pathological and death morphology of BKH-21 cells infected with EMCV virus.

[0019] The purpose, features and advantages of the present invention will be further described with reference to the accompanying drawings and in conjunction with the embodiments. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. In addition, the descriptions of the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" described below mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the technical features involved in the various embodiments of the present invention can be combined with each other as long as they do not conflict with each other.

[0021] See also Figure 1-Figure 3 , Figure 1 This is a flow chart of the steps of the preparation method of the troxerutin freeze-dried product provided by the present invention. Figure 2 A flow chart of the steps for preparing and ultrafiltration of the troxerutin raw material powder for injection provided by the present invention; Figure 3 The present invention provides a process for preparing a troxerutin freeze-dried product, which comprises the following specific steps: S1. Provide the prepared and ultrafiltered troxerutin injection bulk drug powder, or provide the troxerutin injection solution for direct packaging; S2. Place the subpackaged liquid or the subpackaged troxerutin injection in a freeze dryer for freeze drying, wherein the freeze drying step comprises: pre-freezing at a temperature of -35°C to -45°C; performing sublimation drying at a temperature of -10°C after pre-freezing; heating to 0°C for desorption drying and sublimation drying; and heating to 25-35°C for desorption drying.

[0022] Specifically, the freeze-drying step includes: S21, pre-freeze 01 section: Place the aliquot in the freeze dryer and cool the sample to -35°C~-45°C at a cooling rate of 0.8°C / min; S22, pre-freeze 02 section: maintain the temperature at -35℃~-45℃ for 60~240 minutes; S23, sublimation drying section 03: vacuum setting 5~20Pa, heating the sample from -35℃~-45℃ to -10℃ at a heating rate of 0.8℃ / min; S24, sublimation drying section 04: vacuum setting 5~20Pa, temperature at -10℃ for 60 minutes; S25, sublimation drying section 05: vacuum set to 0Pa, temperature at -10℃ maintained for 600~960 minutes; S26, analytical drying section 06: vacuum setting 5~20Pa, heating the sample from -10℃ to 0℃ at a heating rate of 0.8℃ / min; S27, sublimation drying section 07: vacuum setting 5~20Pa, maintain temperature at 0℃ for 120~240 minutes; S28, analytical drying section 08: vacuum setting 0Pa, the sample temperature was raised from 0℃ to 25~35℃ at a heating rate of 0.8℃ / min; S29, analytical drying section 09: vacuum setting 0-5 Pa, maintaining the temperature at 25-35° C. for 60-180 minutes to obtain the troxerutin freeze-dried product.

[0023] The preparation method of the troxerutin freeze-dried product provided by the present invention can obtain a high-quality troxerutin preparation, ensure the stability and effectiveness of troxerutin, extend the shelf life of the drug, and can be effectively applied to plasma virus inactivation equipment. Among them, in the freeze-drying step, by controlling the heating and cooling rates, specifically cooling and heating at a rate of 0.8°C / min, it helps to avoid crystallization or thermal stress of the sample due to excessively rapid temperature changes, thereby ensuring the drying quality and stability of the product. At the same time, specific holding times are set in the pre-freezing and sublimation drying stages to ensure that the sample is fully frozen and fully sublimated and dried, thereby ensuring the quality and stability of the product. In addition, different vacuum degrees are set at different stages, which helps to control the evaporation rate of water in the sample and avoid the generation of gas bubbles, thereby ensuring the uniformity and integrity of the product. The freeze-drying method provided by the present invention also sets specific temperature ranges and holding times at different stages, which helps to control the drying speed and degree of drying of the product, thereby ensuring the quality and stability of the product.

[0024] Specifically, the preparation and ultrafiltration of the troxerutin raw material powder for injection specifically include the following steps: S11, adding the troxerutin raw material powder for injection and the lyophilized excipient to water for injection and stirring until completely dissolved to obtain a troxerutin solution; S12, in an ultrafiltration equipment system, ultrafiltering the troxerutin solution to obtain a filtrate; S13, dispensing the filtrate into the freeze-drying container of the release component to obtain a dispensing solution.

[0025] Ultrafiltration of troxerutin API can remove particulates and bacterial endotoxins, ensuring the safety of the resulting lyophilized product. Ultrafiltration is a membrane filtration method for molecules with molecular weights ranging from 1 kD to 1000 kD. It typically operates using a tangential flow mechanism, where the material flows tangentially to the membrane. This tangential flow mode is less prone to clogging than traditional dead-end filtration (using filter cartridges, carbon rods, etc.), resulting in higher efficiency. Using a 10 kD membrane, the material flows tangentially to the membrane, achieving filtration efficiency. Due to the low molecular weight of troxerutin and excipients, they pass smoothly through the membrane and are collected at the permeate end. Endotoxins and particulates, however, are trapped on the membrane surface due to their larger molecular weight and return to the reflux end. Traditional activated carbon endotoxin removal methods suffer from poor selectivity and tend to adsorb active ingredients. Furthermore, residual activated carbon in the purified solution is difficult to remove, impacting product quality, yield, and cost.

[0026] In S11, the concentration of the packaging liquid obtained after the preparation and ultrafiltration of the troxerutin injection raw material powder is 20-100 mg / mL, the lyophilization excipient is any one of hydroxyethyl starch, mannitol and glucose, and the mass fraction concentration of the lyophilization excipient is 1-2%.

[0027] In S12, the ultrafiltration conditions are: 0.1~2.5m 2 The filter membrane package is made of PES and 10KD. The inlet pressure in the ultrafiltration system is 1~3bar, the reflux pressure is 0.5~1bar, and the flow rate is 16~64L / m 2 By setting the ultrafiltration conditions, the ultrafiltration process can be ensured to be efficient and stable, thereby ensuring the stable quality of the prepared freeze-dried products.

[0028] Furthermore, in S1, the troxerutin injection is directly dispensed into a lyophilization container of the release member to obtain a dispensed solution. The concentration of the troxerutin injection is 20 to 100 mg / mL. The troxerutin injection comprises troxerutin and excipients, wherein the excipients comprise water for injection, disodium hydrogen phosphate, sodium dihydrogen phosphate, disodium edetate, sodium hydroxide, and hydrochloric acid.

[0029] In a second aspect, the present invention further provides a troxerutin freeze-dried product, which is prepared by the above-mentioned method for preparing the troxerutin freeze-dried product.

[0030] In a third aspect, the present invention further provides an application of any of the above-mentioned troxerutin freeze-dried products in plasma virus inactivation. The application specifically comprises: adding the troxerutin freeze-dried product to plasma as a plasma protective agent, placing a mixed bag containing plasma and troxerutin in an inactivation cabinet, and irradiating with UVC light to obtain virus-inactivated plasma, wherein the dosage of the added troxerutin is 0.2 to 1.4 g / L, and the radiation dosage of the UVC light is 0.1 to 1.5 J / cm 2, the wavelength of the UVC light is 254nm. Application of the troxerutin freeze-dried product provided by the present invention in plasma virus inactivation can enhance the protective effect of plasma and improve the effectiveness and safety of blood products. By setting a specific application scheme for plasma virus inactivation, the effectiveness and safety of the troxerutin freeze-dried product can be ensured, thereby guaranteeing the quality and safety of plasma products.

[0031] The present invention provides multiple examples to illustrate the technical solution in detail. In the present invention, not all combinations of orthogonal experiments are shown, and only some representative results are shown to illustrate the basic trends. The sources of raw materials and equipment in the following examples are: Troxerutin injection API comes from Sichuan Xieli Pharmaceutical Company; Troxerutin injection was obtained from Wuhan Fuxing Biopharmaceutical Company; The ultrafiltration equipment was from Dongfulong Technology Group Co., Ltd., model: HIT00300M; The freeze-drying equipment was from Ningbo Xinzhi Freeze-Drying Equipment Co., Ltd., model: Scientz-50F.

[0032] Example 1 This embodiment provides a process for producing a troxerutin freeze-dried product, which is prepared by preparing and ultrafiltration of the troxerutin injection raw material and then freeze-drying.

[0033] The specific steps of preparing and ultrafiltration of the troxerutin API to obtain the packaging solution include: S11. Add approximately 1500 mL of water for injection to the dispensing tank, add 100 g of troxerutin injection bulk drug powder and 30 g of hydroxyethyl starch, and stir until completely dissolved. Add more water for injection to a total volume of 2000 mL. Continue stirring for 10 minutes to obtain the initial troxerutin solution.

[0034] S12. Turn on the ultrafiltration system and set the inlet pressure to 1 bar, the outlet pressure to 0.5 bar, and the flow rate to 16 L / m 2 , open the inlet, outlet and reflux valves, start the equipment, and circulate the filtration until there is no liquid in the liquid preparation tank to obtain the ultrafiltered troxerutin solution.

[0035] S13. Dispense the ultrafiltered troxerutin solution into the release member at a rate of 2.0 mL / vial to obtain 2.0 mL / vial of troxerutin solution.

[0036] The release member containing the dispensing liquid is placed in a freeze dryer for freeze drying, and the freeze drying step includes: S21, pre-freeze 01 section: place the aliquot in the freeze dryer and cool the sample to -35°C at a cooling rate of 0.8°C / min; S22, pre-freeze 02 section: maintain the temperature at -35°C for 60 minutes; S23, sublimation drying section 03: vacuum setting 5Pa, the sample temperature was raised from -35°C to -10°C at a heating rate of 0.8°C / min; S24, sublimation drying section 04: vacuum setting 5Pa, temperature maintained at -10°C for 60 minutes; S25, sublimation drying section 05: vacuum setting 0Pa, temperature -10℃ maintained for 600 minutes; S26, analytical drying section 06: vacuum setting 5Pa, the sample temperature was raised from -10°C to 0°C at a heating rate of 0.8°C / min; S27, sublimation drying section 07: vacuum setting 5Pa, temperature maintained at 0℃ for 120 minutes; S28, analytical drying section 08: vacuum setting 0Pa, the sample temperature was raised from 0°C to 25°C at a heating rate of 0.8°C / min; S29, analytical drying section 09: vacuum setting 0 Pa, maintaining the temperature at 25° C. for 60 minutes to obtain the troxerutin freeze-dried product.

[0037] See the final product Figure 4 and Figure 5 , Figure 4 This is the shape of the 2.0 mL / tube troxerutin before freeze-drying provided in Example 1. Figure 5 This is the shape of the 2.0 mL / vial troxerutin provided in Example 1 after freeze-drying. The troxerutin contained in the release element of Example 1 is 100 mg / vial.

[0038] Example 2 This embodiment provides a process for producing a troxerutin freeze-dried product, which is prepared by preparing and ultrafiltration of the troxerutin injection raw material and then freeze-drying.

[0039] S11, the preparation steps are the same as those in Example 1.

[0040] S12, the ultrafiltration step is the same as in Example 1.

[0041] S13. Dispense the ultrafiltered troxerutin solution into the release member at a rate of 1.0 mL / vial to obtain 1.0 mL / vial of troxerutin dispensing solution.

[0042] S2. Place the release member containing the sub-packaging liquid in a freeze dryer for freeze drying. The freeze drying steps are the same as those in Example 1 and will not be repeated here.

[0043] See the final product Figure 6 and Figure 7 , Figure 6This is the shape of 1.0 mL / tube of troxerutin before freeze-drying provided in Example 2. Figure 7 This is the shape of the 1.0 mL / vial troxerutin after freeze-drying provided in Example 2. The release element of Example 2 contains 50 mg / vial troxerutin.

[0044] Example 3 This embodiment provides a process for producing a troxerutin freeze-dried product, which is prepared by preparing and ultrafiltration of the troxerutin injection raw material and then freeze-drying.

[0045] S11, the preparation steps are the same as those in Example 1.

[0046] S12. Turn on the ultrafiltration system and set the inlet pressure to 3 bar, the outlet pressure to 1 bar, and the flow rate to 64 L / m 2 , open the inlet, outlet and reflux valves, start the equipment, and circulate the filtration until there is no liquid in the liquid preparation tank to obtain the ultrafiltered troxerutin solution.

[0047] S13. Dispense the ultrafiltered troxerutin solution into the release member at a rate of 0.5 mL / vial to obtain 0.5 mL / vial of troxerutin solution.

[0048] S2. Place the release member containing the sub-packaging liquid in a freeze dryer for freeze drying. The freeze drying steps are the same as those in Example 1 and will not be repeated here.

[0049] The amount of troxerutin contained in the release element of Example 3 is 25 mg / vial.

[0050] Example 4 This embodiment provides a process for preparing a troxerutin freeze-dried product, wherein the troxerutin injection solution is directly dispensed into a release member for freeze-drying. The process specifically includes the following steps: S1. Take a commercially available troxerutin injection with a specification of 2 ml:100 mg, and dispense it into the release member at a volume of 2.0 ml per vial to obtain a troxerutin injection aliquot of 2.0 mL / vial.

[0051] The release member containing the troxerutin injection subpackage is placed in a freeze dryer for freeze drying, and step S2 specifically includes: S21, pre-freeze 01 section: place the aliquot in the freeze dryer and cool the sample to -45°C at a cooling rate of 0.8°C / min; S22, pre-freezing section 02: maintain the temperature at -45°C for 240 minutes; S23, sublimation drying section 03: vacuum setting 20 Pa, the sample temperature was raised from -45 °C to -10 °C at a heating rate of 0.8 °C / min; S24, sublimation drying section 04: vacuum setting 20Pa, temperature -10℃ maintained for 60 minutes; S25, sublimation drying section 05: vacuum setting 0Pa, temperature -10℃ maintained for 960 minutes; S26, analytical drying section 06: vacuum setting 20Pa, the sample temperature was raised from -10°C to 0°C at a heating rate of 0.8°C / min; S27, sublimation drying section 07: vacuum setting 20Pa, temperature maintained at 0℃ for 240 minutes; S28, analytical drying section 08: vacuum setting 0Pa, the sample temperature was raised from 0°C to 35°C at a heating rate of 0.8°C / min; S29, analytical drying section 09: vacuum setting 5 Pa, maintaining the temperature at 35° C. for 180 minutes to obtain the troxerutin freeze-dried product.

[0052] See the final product Figure 8 and Figure 9 , Figure 8 This is the shape of the 2.0 mL / tube troxerutin before freeze-drying provided in Example 4. Figure 9 This is the shape of the 2.0 mL / vial troxerutin after freeze-drying provided in Example 4. The troxerutin contained in the release element of Example 4 is 100 mg / vial.

[0053] Example 5 This embodiment provides a process for preparing a troxerutin freeze-dried product, wherein the troxerutin injection solution is directly dispensed into a release member for freeze-drying. The process specifically includes the following steps: S1. Take a commercially available troxerutin injection with a specification of 2 ml:100 mg, and dispense it into the release member at a volume of 1.0 ml per vial to obtain a troxerutin injection aliquot of 1.0 mL / vial.

[0054] S2. Place the release member containing the troxerutin injection subpackage in a freeze dryer for freeze drying. The freeze drying step is the same as that in Example 4 and will not be repeated here.

[0055] The amount of troxerutin contained in the release element of Example 5 is 50 mg / piece.

[0056] Example 6 This embodiment provides a process for preparing a troxerutin freeze-dried product, wherein the troxerutin injection solution is directly dispensed into a release member for freeze-drying. The process specifically includes the following steps: S1. Take a commercially available troxerutin injection with a specification of 2 ml:100 mg, and dispense 0.5 ml per vial into the release member to obtain 0.5 mL / vial of troxerutin injection aliquot.

[0057] S2. Place the release member containing the troxerutin injection subpackage in a freeze dryer for freeze drying. The freeze drying step is the same as that in Example 4 and will not be repeated here.

[0058] See the final product Figure 10 and Figure 11 , Figure 10 This is the shape of the 0.5 mL / tube troxerutin before freeze-drying provided in Example 6. Figure 11 This is the shape of the 0.5 mL / tube troxerutin after freeze-drying provided in Example 6. The troxerutin contained in the release element of Example 6 is 25 mg / tube.

[0059] The troxerutin freeze-dried products provided in Examples 1 to 6 were tested according to Part II of the Pharmacopoeia of the People's Republic of China (2020 Edition) before and after ethylene oxide sterilization. The test results are shown in Table 1, Table 2-1, and Table 2-2. Table 1 Test results of troxerutin freeze-dried products prepared by different process methods

[0060]

[0061] The bacterial endotoxin levels in each example before and after sterilization were all lower than 0.5 EU / ml.

[0062] As can be seen from Table 1, the troxerutin freeze-dried product prepared by the preparation method provided by the present invention has excellent appearance and good resolubility before and after sterilization, and meets all standards and the requirements for ethylene oxide sterilization.

[0063] Table 2-1 Stability test results of troxerutin freeze-dried products prepared by different process methods (Examples 1-3)

[0064] Table 2-2 Stability test results of troxerutin freeze-dried products prepared by different process methods (Examples 4-6)

[0065] The bacterial endotoxin levels in each example before and after sterilization were all lower than 0.5 EU / ml.

[0066] It can be seen from Table 2-1 and Table 2-2 that the troxerutin product prepared by the preparation method provided by the present invention meets the requirements for various detection indicators under accelerated test conditions and has good stability.

[0067] The lyophilized products of troxerutin of Examples 1, 2, and 3 are provided for use in plasma virus inactivation, which are used to inactivate enveloped indicator viruses and non-enveloped indicator viruses. In this solution, the plasma virus inactivation method provided by the present invention and the application of troxerutin in plasma virus inactivation are experimented.

[0068] Provide main reagents, raw materials and instruments: The plasma used in the experiment comes from the blood collection and supply institutions of the cooperative scientific research; Sindbis virus (SINV), encephalomyocarditis virus (EMCV, moderately resistant), and porcine parvovirus (PPV, highly resistant) were purchased from the China Veterinary Microbiology Culture Collection Center; Vero cells, BHK-21 cells, and ST cells (ATCC CRL-1746) were purchased from the China Veterinary Microbial Culture Collection. The blood component virus inactivation cabinet was developed by Lanbo Medical Technology Development (Suzhou) Co., Ltd. The disposable plasma virus inactivation device was developed by Lanbo Medical Technology Development (Suzhou) Co., Ltd., wherein the release element contains 100 mg / vial of the troxerutin freeze-dried product of Example 1; the release element contains 50 mg / vial of the troxerutin freeze-dried product of Example 2; and the release element contains 25 mg / vial of the troxerutin freeze-dried product of Example 3. The fully automatic coagulation analyzer is a Sysmex brand, and the coagulation reagent is a Siemens brand; The fully automatic biochemical analyzer and reagents are Shandong Waters brand; The remaining reagents and instruments were all conventional domestic models.

[0069] Part I: UVC photoinactivation of plasma and virus detection: Preparation of infected plasma: Take a blood bag containing 180ml of plasma and add 20ml of indicator virus solution to the blood bag at a ratio of 1:9 to plasma. Mix thoroughly. For enveloped indicator viruses, choose Sindbis virus (SINV); for non-enveloped indicator viruses, choose encephalomyocarditis virus (EMCV) or porcine parvovirus (PPV).

[0070] According to the instructions of the "Disposable Plasma Virus Inactivation Device", the contaminated plasma bag is connected to the device, and the plasma is allowed to flow through the release piece containing the troxerutin freeze-dried product into the mixing bag to obtain the contaminated plasma to be treated containing troxerutin.

[0071] 1. Sample Treatment: In this invention, not all combinations of orthogonal concentrations are presented; only some representative results are shown to illustrate the basic trends. Specifically, plasma was treated with toxins according to the proportions. Samples of troxerutin freeze-dried products from different embodiments were provided to verify their inactivation effects against Sindbis virus (SINV), encephalomyocarditis virus (EMCV), and porcine parvovirus (PPV). The inactivation treatment methods were as follows: Sample 1: No troxerutin was added to the contaminated plasma (i.e., concentration was 0.0 g / L), inactivated, and irradiated at a dose of 0.5 J / cm 2 .

[0072] Sample 2: The lyophilized product of Troxerutin of Example 1 was added to the infected plasma to a concentration of 0.5 g / L of Troxerutin. The plasma was inactivated and irradiated at a dose of 0.5 J / cm 2 .

[0073] Sample 3: The lyophilized product of Troxerutin of Example 2 was added to the contaminated plasma to a concentration of 0.25 g / L of Troxerutin. The plasma was inactivated and irradiated at a dose of 0.5 J / cm 2 .

[0074] Sample 4: The lyophilized product of Troxerutin of Example 3 was added to the infected plasma to a concentration of 0.125 g / L of Troxerutin. The plasma was inactivated and irradiated at a dose of 0.5 J / cm 2 .

[0075] 2. Inactivation process: S1. The inactivation cabinet is powered on and self-checked to be ready for use; S2. Hang the mixed bag containing the contaminated plasma on the hook above the inactivation cabinet, insert the UVC light component into the illumination position of the irradiation unit, install the upper end of the light component into the flow control caliper, place the collection bag in the collection position below the irradiation unit, close the door of the illumination cabinet, and start the inactivation program; S3. Plasma flows through the irradiation unit at a set fixed flow rate, receives UVC light treatment, and is then collected in a collection bag, thereby obtaining virus-inactivated plasma. Specifically, the UVC light treatment time is 120-1200 seconds, and the wavelength of the UVC light is 254 nm. By controlling the time the plasma flows through the irradiation unit, the radiation dose received by the plasma is calculated under a fixed illumination intensity. S4. Heat-seal the collection bag catheter and use a sterile syringe to draw 10 ml of sample from the collection bag for testing; S5. After the treatment is completed, all items that have come into contact with the virus liquid must be soaked in acid for disinfection to prevent the virus from leaking out.

[0076] 3. Collect the inactivated plasma, which is the virus-inactivated plasma, and take samples for testing.

[0077] 4. Virus detection and results Vero cells, BHK-21 cells, and ST cells (ATCC CRL-1746) were used to culture Sindbis virus (SINV), encephalomyocarditis virus (EMCV), and porcine parvovirus (PPV), respectively.

[0078] (1) Cells for detection were cultured at a rate of 0.5 to 1.5 × 10 5 / mL density was added to a 96-well (8×12) cell culture plate, 100 μL per well; the culture plate was placed in a 37°C, 5% CO2 incubator for 12 to 24 hours. Figure 12 This is a morphological diagram of normal growth of BHK-21 cells.

[0079] (2) The infected plasma samples, the virus-inactivated plasma samples and the virus positive control samples were serially diluted in a gradient manner, with the dilution multiple being 10 times, i.e.: 10 -1 ~10 -10 .

[0080] (3) Add 10 -1 ~10 -10 Serial serial dilutions of virus, 100 μL per well; columns 1 and 12 are normal cell controls.

[0081] (4) The treated culture plates were cultured in a 37°C, 5% CO2 incubator, and the cell pathology was observed daily. The number of pathological cell wells was recorded until the cells in the control wells could no longer maintain normal morphology.

[0082] (5) Calculate the titer of residual virus in each sample (TCID) based on the number of lesion wells with a cell lesion rate of more than 50%. 50 ), Figure 13 This is a picture of the pathological and death morphology of BKH-21 cells infected with EMCV virus.

[0083] (6) According to the formula: virus titer reduction value = zero-point control virus titer - virus titer after UVC light treatment, calculate the virus titer reduction value of the infected plasma sample before and after virus inactivation.

[0084] The test results are shown in Table 3 Table 3. Residual virus titer values ​​(TCID) of samples 1-4 50 )Test results table

[0085] From the data in Table 3, we can see that the UVC irradiation dose at 254nm is 0.5J / cm 2Under the condition that no troxerutin was added to the infected plasma, and in the experimental groups with different concentrations of troxerutin, the virus titer reduction value after inactivation treatment was ≥4 logs, showing a good inactivation effect; Part II: UVC light treatment of plasma and detection of plasma-related in vitro functional indicators: 1. Treatment of plasma samples: In this invention, not all combinations of orthogonal experiments of ratio concentrations are shown. Only some representative results are shown to illustrate the basic trend. Specifically, four 200ml portions of fresh frozen plasma were provided by a blood collection and supply institution for cooperative scientific research. Each portion of plasma was divided into two 100ml portions, one of which was added with the lyophilized product of Example 2, and the other was not added with troxerutin. Among them: Sample 1-1: Plasma without troxerutin, inactivated, irradiated at a dose of 0.32 J / cm 2 .

[0086] Sample 1-2: Follow the instructions for the "Disposable Plasma Virus Inactivation Device" and connect the plasma bag to the device. Allow the plasma to flow through the release element containing the lyophilized troxerutin product of Example 2 and into the mixing bag to obtain a plasma containing 0.5 g / L troxerutin. The inactivation procedure was performed with an irradiation dose of 0.32 J / cm 2 .

[0087] Sample 2-1: Plasma without troxerutin, inactivated, irradiated at a dose of 0.50 J / cm 2 .

[0088] Sample 2-2: Plasma was treated according to the method of Sample 1-2 to obtain a plasma containing 0.5 g / L of troxerutin. The plasma was inactivated and irradiated at a dose of 0.50 J / cm 2 .

[0089] Sample 3-1: Plasma without troxerutin, treated with inactivation procedure, irradiated with a dose of 1.01 J / cm 2 .

[0090] Sample 3-2: Plasma was treated according to the method of Sample 1-2 to obtain a plasma containing 0.5 g / L of troxerutin. The plasma was inactivated and irradiated at a dose of 1.01 J / cm 2 .

[0091] Sample 4-1: Plasma without troxerutin, treated with inactivation procedure, irradiated with a dose of 1.51 J / cm 2 .

[0092] Sample 4-2: Plasma was treated according to the method of Sample 1-2 to obtain a plasma containing 0.5 g / L of troxerutin. The plasma was inactivated and irradiated at a dose of 1.51 J / cm2 .

[0093] 2. Inactivation process: S1. The inactivation cabinet is powered on and self-checked to be ready for use; S2. Hang the mixed bag containing plasma on the hook above the inactivation cabinet, insert the UVC irradiation component into the irradiation position of the irradiation unit, install the upper end of the irradiation component on the flow control caliper, place the collection bag in the collection position below the irradiation unit, close the irradiation cabinet door, and start the inactivation program; S3. Plasma flows through the irradiation unit at a set fixed flow rate, receives UVC light treatment, and is then collected into a collection bag, thereby obtaining plasma that has undergone a virus inactivation treatment procedure. Specifically, the UVC light irradiation treatment time is 120-1200 seconds, and the wavelength of the UVC light radiation is 254 nm. By controlling the time the plasma flows through the irradiation unit, the radiation dose received by the plasma is calculated under a fixed light intensity. S4. Heat-seal the collection bag catheter to obtain the plasma in the collection bag that has been treated with the light virus inactivation procedure, and use a sterile syringe to draw samples from the collection bag for testing.

[0094] 3. Detection of plasma-related in vitro functional indicators: All samples were tested for coagulation factor VIII (F VIII) and fibrinogen (Fbg) content before and after UVC light treatment. The recovery rates of F VIII and Fbg before and after light treatment were calculated as follows: recovery = post-treatment content / pre-treatment content * 100%. This combined test was used to compare the protective effects of troxerutin on plasma coagulation factors.

[0095] The test results are shown in Table 4

[0096] Specifically, coagulation factor VIII is an important indicator for evaluating plasma quality. According to the "Fresh Frozen Plasma" item in the "Quality Requirements for Whole Blood and Blood Components GB18469-2012", the factor VIII content is ≥0.7IU / ml. The factor VIII content of the experimental plasma in this embodiment before UVC light treatment meets the requirements, and according to the "Virus Inactivated Fresh Frozen Plasma" item in the "Quality Requirements for Whole Blood and Blood Components GB 18469-2012", the factor VIII content is ≥0.5IU / ml. The results in Table 4 show that under different UVC irradiation doses, the recovery rate of factor VIII with / without troxerutin showed significant differences. At 0.5J / cm 2 The recovery rate of factor VIII in the troxerutin group was 92%, even at a radiation dose of 1.01 J / cm 2At a radiation dose of 0.5 J / cm2, the recovery rate of factor VIII was still 88% and the content was ≥0.5 IU / ml; at .... 2 At a radiation dose of 1.01 J / cm, the recovery rate of factor VIII in the group without troxerutin was 85%, which barely met the quality requirements. 2 At a radiation dose of 1.5, the factor VIII recovery rate was only 70%, and the content was close to the limit of 0.5 IU / ml. This indicates that the addition of troxerutin as a protective agent can effectively protect the activity of factor VIII in plasma. Although various indicators decreased after irradiation, they were able to remain within normal levels.

[0097] Specifically, fibrinogen (Fbg) is an important indicator for evaluating plasma quality. The results in Table 4 show that the recovery rate of fibrinogen (Fbg) under different UVC irradiation doses with and without troxerutin showed significant differences: at 0.5 J / cm 2 The fibrinogen recovery rate in the troxerutin group was 82% even at a radiation dose of 1.01 J / cm 2 At a radiation dose of 0.5 J / cm 2 At a radiation dose of 1.01 J / cm 2 The fibrinogen recovery rate was only 62% at a radiation dose of 1.5 wt%. This indicates that the addition of troxerutin can effectively protect the activity of fibrinogen in plasma. Although various indicators decreased after irradiation, they were basically maintained within the normal range.

[0098] As can be seen from Tables 3 and 4, the dosage of troxerutin in the application of plasma virus inactivation is preferably 0.5 g / L, and the irradiation dose of 254 nm UVC is preferably 0.5 J / cm 2 .

[0099] In summary, the preparation method of the troxerutin freeze-dried product provided by the present invention can perform freeze-drying treatment on the troxerutin raw material for injection and the troxerutin injection. Through its freeze-drying preparation process, the obtained troxerutin freeze-dried product can be effectively used in plasma virus inactivation equipment. The troxerutin freeze-dried product has excellent appearance and good resolubility before and after the plasma virus inactivation equipment is sterilized with ethylene oxide, meets the requirements of ethylene oxide sterilization of the equipment, has high stability, can be stored for a long time, and effectively ensures the use of the troxerutin freeze-dried product in plasma virus inactivation. The preparation method of the troxerutin product provided by the present invention is simple and reasonable and can meet the needs of large-scale production. The present invention also provides an application method of the troxerutin freeze-dried product in plasma virus inactivation. 254nm UVC irradiation can effectively inactivate enveloped viruses and non-enveloped viruses; at the same time, the added troxerutin freeze-dried product effectively protects the plasma protein components, making plasma virus inactivation safer and more effective.

[0100] The above description is only a preferred embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made by using the contents of the present invention description and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for preparing a troxerutin freeze-dried product, characterized in that: The specific steps include: S1. Provide the packaging liquid obtained after the preparation and ultrafiltration of the troxerutin injection raw material powder, or provide the troxerutin injection liquid for direct packaging; S2, placing the subpackaged liquid or the subpackaged troxerutin injection in a freeze dryer for freeze drying, wherein the freeze drying step comprises: pre-freezing at a temperature of -35°C to -45°C; performing sublimation drying at a temperature of -10°C after pre-freezing; heating to 0°C for analytical drying and sublimation drying; heating to 25 to 35°C for analytical drying; Wherein, in S1, specifically includes: S11, adding the troxerutin raw material powder for injection and the lyophilized excipient into water for injection and stirring evenly until completely dissolved to obtain a troxerutin solution; The freeze-drying excipient is any one of hydroxyethyl starch, mannitol and glucose, and the mass fraction concentration of the freeze-drying excipient is 1-2%.

2. The method for preparing the lyophilized troxerutin product according to claim 1, characterized in that: The freeze-drying step specifically comprises: S21, pre-freezing section 01: place the aliquot in a freeze dryer and cool the sample to -35°C~-45°C at a cooling rate of 0.8°C / min; S22, pre-freezing section 02: keep the temperature at -35℃~-45℃ for 60~240 minutes; S23, sublimation drying section 03: vacuum setting 5~20Pa, heating the sample from -35℃~-45℃ to -10℃ at a heating rate of 0.8℃ / min; S24, sublimation drying section 04: vacuum setting 5~20Pa, maintained at -10℃ for 60 minutes; S25, sublimation drying section 05: vacuum setting 0Pa, maintained at -10℃ for 600~960 minutes; S26, analytical drying section 06: vacuum setting 5~20Pa, heating the sample from -10℃ to 0℃ at a heating rate of 0.8℃ / min; S27, sublimation drying section 07: vacuum setting 5~20Pa, maintain at 0℃ for 120~240 minutes; S28, analytical drying section 08: vacuum setting 0Pa, heating the sample from 0℃ to 25~35℃ at a heating rate of 0.8℃ / min; S29, analytical drying section 09: the vacuum is set to 0-5 Pa, and the temperature is maintained at 25-35° C. for 60-180 minutes to obtain the troxerutin freeze-dried product.

3. The method for preparing the lyophilized troxerutin product according to claim 1, characterized in that: The preparation and ultrafiltration of the troxerutin raw material powder for injection specifically further include the following steps: S12, in an ultrafiltration equipment system, ultrafiltering the troxerutin solution to obtain a filtrate; S13, dispensing the filtrate into the freeze-drying container of the release component to obtain dispensing liquid.

4. The method for preparing the lyophilized troxerutin product according to claim 3, characterized in that: In S12, the ultrafiltration conditions are: 0.1~2.5m 2 The filter membrane package is made of PES and 10KD. The inlet pressure in the ultrafiltration system is 1~3bar, the reflux pressure is 0.5~1bar, and the flow rate is 16~64L / m 2 .

5. The method for preparing the lyophilized troxerutin product according to claim 1, characterized in that: In S1, the concentration of the packaging liquid obtained after the troxerutin injection raw material powder is prepared and ultrafiltered, and the concentration of the troxerutin injection is 20~100 mg / mL.

6. The method for preparing the lyophilized troxerutin product according to claim 1, characterized in that: In S1, the troxerutin injection comprises troxerutin and excipients, wherein the excipients comprise water for injection, disodium hydrogen phosphate, sodium dihydrogen phosphate, disodium edetate, sodium hydroxide and hydrochloric acid.

7. A troxerutin freeze-dried product, characterized in that: The product is prepared by the method for preparing the lyophilized troxerutin product according to any one of claims 1 to 6.

8. Use of the lyophilized troxerutin product according to claim 7 in inactivating plasma viruses.

9. The use of the lyophilized troxerutin product according to claim 8 in inactivating plasma viruses, characterized in that: The application specifically includes: adding the lyophilized troxerutin product to plasma as a plasma protective agent, placing the mixed bag containing plasma and troxerutin in an inactivation cabinet, and irradiating with UVC light to obtain virus-inactivated plasma, wherein the dosage of the troxerutin added is 0.2-1.4 g / L, and the radiation dosage of the UVC light is 0.1-1.5 J / cm 2 , the wavelength of the UVC light is 254nm.

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