Preparation containing vWF and / or FVIII compound and preparation method thereof

Abstract

The invention relates to the field of biological medicine, and provides a preparation containing a vWF and / or FVIII compound and a preparation method thereof, the preparation containing the vWF and / or FVIII compound comprises a protection solution, the protection solution contains albumin and chlorate, and the mass ratio of the albumin to the chlorate is 1.45-3.40. The active recovery rate of the vWF / FVIII compound in the preparation is high, the appearance of the preparation after freeze-drying and dry-heating is free of collapse, atrophy and visible foreign matters, the redissolution time is short, the active recovery rates of vWF and FVIII are up to 99% and 86% respectively, and the preparation is good in stability at a high temperature, safe, effective and low in cost, can be prepared on a large scale and has a good clinical application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a preparation containing a vWF and / or FVIII complex and a preparation method thereof. Background Art

[0002] Von Willebrand Factor (vWF) is a multimeric plasma glycoprotein with a molecular weight ranging from 250 kDa to 20 million kDa, forming the largest molecular weight soluble protein known in plasma. The small molecular weight vWF in plasma is mainly in the form of dimers with a molecular weight of about 500 kDa. Multimers of varying sizes are of great significance in maintaining the normal biological activity of vWF. Plasma vWF plays an important role in primary hemostasis, being responsible for the adhesion of platelets to the surface of damaged blood vessels, and thus forming platelet plugs, which contribute to the formation of fibrin cross-links. In addition, plasma vWF acts as a transporter and stabilizer for coagulation factor VIII.

[0003] The vWF / FVIII (human coagulation factor VIII) complex is a blood product. It is made of cold precipitate separated from healthy human plasma, purified and virus-inactivated. It is a compound factor preparation used for the prevention and treatment of hemophilia A (HA) and von Willebrand disease (VWD). However, the VWF / FⅧ complex is extremely unstable. Once it leaves the natural environment of the human body, it is easily activated and prone to hydrolysis, reduced activity, and changes in appearance. Therefore, pharmaceutical manufacturers add stabilizers during separation and purification and make freeze-dried preparations. On the one hand, it can make the product able to withstand freeze-drying and dry heat treatment, ensuring that the quality does not change, and does not affect the virus inactivation effect of dry heat treatment; on the other hand, freeze-dried preparations are easy to transport and store. Before clinical use, a certain amount of sterile water for injection is added according to the specifications, and after reconstitution, it can be used for intravenous administration to patients. It is easy to use, has stable quality, and greatly improves safety.

[0004] The dry heat treatment stabilizer for albumin-free human coagulation factor VIII and vWF complex or human coagulation factor VIII preparation disclosed in CN102580062B has poor protection effect on human coagulation factor VIII during 80°C dry heat treatment, and the activity recovery rate of human coagulation factor VIII is less than 35%. It does not mention whether the problem of preparation instability is solved, and is not suitable for the actual production of the product. Patent CN116715751A uses a stabilizer containing sodium citrate, calcium chloride, and arginine hydrochloride for dry heat treatment, but the reconstitution time is long. The freeze-dried preparation is treated at 80°C for 72 hours to inactivate the virus. The reconstitution time of the obtained preparation is less than 1 minute, and the activity yield of vWF / FVIII has not been verified, and the preparation stability time is short.

[0005] Based on the above problems existing in the prior art, there is still a need to develop new human von Willebrand factor complexes. Summary of the invention

[0006] This application is based on the inventor's discovery and understanding of the following problems:

[0007] The VWF / FⅧ complex is extremely unstable. Once it leaves the natural environment of the human body, it is easily activated and prone to hydrolysis, reduced activity, changes in appearance, etc. The inventors of the present application have conducted a large number of actual experiments and surprisingly found that the mass ratio of albumin and chloride salt has an important influence on the stability of the von Willebrand factor / FⅧ complex preparation. The activity recovery rate of human von Willebrand factor / FⅧ in the vWF and / or FVIII complex preparation prepared therefrom is high, and the appearance after freeze-drying and dry-heating has no collapse, no atrophy, no visible foreign matter, and the re-dissolution time is less than 30s. The activity recovery rate of vWF is as high as 99%, and the activity recovery rate of FVIII is as high as 86%. It has good stability at 5°C to 40°C, is safe and effective, has low cost, can be prepared on a large scale, and has good clinical application prospects.

[0008] Therefore, in the first aspect of the present invention, the present invention provides a preparation containing vWF and / or FVIII complex. According to an embodiment of the present invention, the preparation comprises a protective solution, wherein the protective solution contains albumin and chloride salt, and the mass ratio of the albumin to the chloride salt is 1.45 to 3.40.

[0009] In the present application, the inventors screened and optimized the mass ratio of albumin and chloride salt, as well as the components and contents in the preparation, and found that the mass ratio of albumin and chloride salt has an important influence on the stability of the preparation of vWF and / or FVIII complex. Chloride salt can increase the osmotic pressure of the drug and the degree of hydration of the protein, protect the protein space chain from being directly exposed to the environment, and thus maintain the activity of the protein. At the same time, it can also make the freeze-dried product appear loose mesh-like, significantly shortening the re-dissolution time. Albumin, as a natural protective agent, can reduce the impact of the product on the severe conditions of the freeze-drying and dry heat processes. Thus, the preparation of the above-mentioned vWF and / or FVIII complex obtained can effectively ensure the activity of the human vascular hemophilia factor / human coagulation factor VIII complex, and it is safe and effective, low-cost, can be prepared on a large scale, and has good prospects for clinical application.

[0010] According to an embodiment of the present invention, the above-mentioned preparation containing vWF and / or FVIII complex may also include at least one of the following additional technical features:

[0011] According to an embodiment of the present invention, the mass ratio of albumin to chloride salt is 1.54 to 3.33.

[0012] According to an embodiment of the present invention, the mass ratio of albumin to chloride salt is 1.71 to 3.07.

[0013] According to an embodiment of the present invention, the mass ratio of albumin to chloride salt is 2.04 to 2.73.

[0014] According to an embodiment of the present invention, the mass ratio of albumin to chloride salt is 2.39 to 2.73;

[0015] According to some preferred embodiments of the present invention, the mass ratio of albumin to chloride salt is 2.73.

[0016] According to an embodiment of the present invention, the albumin is selected from any one or more of ovalbumin, bovine albumin, human albumin, and recombinant human albumin, preferably human albumin.

[0017] According to an embodiment of the present invention, the chloride salt comprises sodium chloride.

[0018] According to an embodiment of the present invention, the protective solution further contains amino acids and / or citrate.

[0019] It should be noted that amino acids provide excipients during the freeze-drying process, and the preparation has good clarity after reconstitution. At the same time, they also act as protein protectants. The amino and carboxyl groups they contain can make freeze-dried products and products have strong buffering properties. Citrate can be used as a buffer system, which can combine with calcium ions in the blood, thereby reducing the calcium concentration in the blood, helping to prevent blood coagulation, and has the effects of anticoagulant and stabilizer. In addition, citrate can reduce the formation of blood clots by inhibiting the activity of thrombin.

[0020] According to an embodiment of the present invention, the amino acid is selected from any one or more of glycine, histidine, lysine, arginine and tryptophan.

[0021] According to an embodiment of the present invention, the amino acid is glycine, histidine or lysine.

[0022] According to some specific embodiments of the present invention, the amino acid is glycine.

[0023] According to some preferred embodiments of the present invention, the citrate comprises sodium citrate.

[0024] According to an embodiment of the present invention, the mass percentage of the sodium citrate is 0.1% to 1.5%.

[0025] According to some preferred embodiments of the present invention, the mass percentage of sodium citrate is 0.29% to 0.89%.

[0026] According to an embodiment of the present invention, the mass percentage of the amino acid is 0.05% to 0.4%.

[0027] According to an embodiment of the present invention, the mass percentage of the amino acid is 0.1% to 0.3%.

[0028] According to some preferred embodiments of the present invention, the mass percentage of the amino acid is 0.15% to 0.25%.

[0029] According to an embodiment of the present invention, the albumin content is 0.2% to 2%.

[0030] According to an embodiment of the present invention, the mass percentage of the sodium chloride is 0.1% to 1.0%, preferably 0.29% to 0.65%.

[0031] According to an embodiment of the present invention, the protective solution improves the activity recovery rate of the liquid composition containing vWF or vWF / FVIII complex. In one embodiment of the present invention, the vWF activity is 10-500 IU / mL; optionally, the vWF activity is 20-300 IU / mL; optionally, the vWF activity is 30-100 IU / mL; according to some specific embodiments of the present invention, the vWF activity is 50 IU / mL.

[0032] According to an embodiment of the present invention, the FVIII activity is 10-500 IU / mL; preferably, the FVIII activity is 15-300 IU / mL; more preferably, the FVIII activity is 20-100 IU / mL; according to some specific embodiments of the present invention, the FVIII activity is preferably 25 IU / mL.

[0033] 400, 420, 425, 445, 455, 475, 480, 490, 495, 500 IU / mL, etc.

[0034] The vWF activity and FVIII activity described in the present invention are merely for the purpose of describing specific embodiments and should not be considered as limiting the vWF and FVIII activities described in the present invention.

[0035] In a second aspect of the present invention, the present invention provides a method for preparing a preparation containing a vWF and / or FVIII complex. According to an embodiment of the present invention, the method comprises:

[0036] 1) Dissolving the cryoprecipitate;

[0037] 2) performing virus inactivation, column chromatography purification, and ultrafiltration dialysis on the product obtained in step 1) in sequence to obtain a protein solution containing human von Willebrand factor and / or human coagulation factor VIII complex;

[0038] 3) adding a protective solution to the protein solution for freeze drying and dry heat treatment.

[0039] According to an embodiment of the present invention, the above method may further include at least one of the following additional technical features:

[0040] According to an embodiment of the present invention, before carrying out step 2), the product obtained in step 1) is subjected to impurity removal and filtration treatment in advance.

[0041] According to an embodiment of the present invention, the virus inactivation is performed using the S / D method.

[0042] According to an embodiment of the present invention, the column chromatography is anion exchange chromatography.

[0043] According to an embodiment of the present invention, the buffer used in the column chromatography purification includes at least one of an equilibration buffer, a washing buffer and an elution buffer.

[0044] According to an embodiment of the present invention, the equilibration buffer, the washing buffer and the elution buffer include at least one of chloride salt, citrate and amino acid.

[0045] According to an embodiment of the present invention, the mass ratio of chloride, citrate and amino acid in the equilibration buffer is (5-10): (1-5): (5-15).

[0046] According to an embodiment of the present invention, the mass ratio of chloride, citrate and amino acid in the washing buffer is (15-20): (1-5): (5-15).

[0047] According to an embodiment of the present invention, the mass ratio of chloride, citrate and amino acid in the elution buffer is (17-25): (1-5): (5-15).

[0048] According to an embodiment of the present invention, the pH of the equilibration buffer, the washing buffer and / or the elution buffer is 6.5-7.5.

[0049] According to an embodiment of the present invention, each kilogram of the balancing buffer comprises: 5 to 10 g of chloride salt, 1 to 5 g of citrate, and 5 to 15 g of amino acids.

[0050] According to an embodiment of the present invention, each kilogram of washing buffer solution includes: 15-20 g sodium chloride, 1-5 g citrate, and 5-15 g amino acids.

[0051] According to an embodiment of the present invention, each kilogram of elution buffer comprises: 17-25 g sodium chloride, 1-5 g citrate, and 5-15 g amino acids.

[0052] According to some specific embodiments of the present invention, the chloride salt in the equilibration buffer, the washing buffer and the elution buffer comprises sodium chloride.

[0053] According to an embodiment of the present invention, the amino acids in the equilibration buffer, the washing buffer and the elution buffer are selected from glycine, histidine, lysine, arginine and tryptophan.

[0054] According to some specific embodiments of the present invention, the amino acid in the equilibration buffer, the washing buffer and the elution buffer is glycine, histidine or lysine.

[0055] According to an embodiment of the present invention, the amino acid in the equilibration buffer, the washing buffer and the elution buffer is glycine.

[0056] According to some preferred embodiments of the present invention, the citrate in the equilibration buffer, the washing buffer and the elution buffer comprises sodium citrate.

[0057] According to an embodiment of the present invention, the vWF titer in the protein solution in step 2) is not less than 60 IU / mL.

[0058] According to an embodiment of the present invention, the protective solution comprises albumin and chloride salt, and the mass ratio of the albumin to the chloride salt is 1.45 to 3.40.

[0059] Since the human von Willebrand factor / human coagulation factor VIII complex is unstable, a stabilizer needs to be added during separation and purification so that the product can withstand freeze-drying and dry heat treatment without affecting the virus inactivation effect of dry heat treatment. At present, there are no vWF and FVIII preparations in China, and the easy appearance of visible foreign matter in the preparation is also a technical problem that is difficult to solve in this field. In the present application, the inventor screened and optimized the mass ratio of albumin and chloride salt and the various components and contents in the preparation, and found that the mass ratio of albumin and chloride salt has an important influence on the stability of the preparation of vWF and / or coagulation factor VIII complex, which can solve the technical problem of unstable preparation that has not been solved in this field. Thus, the above-mentioned preparation of vWF and / or coagulation factor VIII complex obtained can effectively ensure the activity of human von Willebrand factor / human coagulation factor VIII complex, and it is safe and effective, low cost, can be prepared on a large scale, and has good clinical application prospects.

[0060] According to an embodiment of the present invention, the mass ratio of albumin to chloride salt is 1.54 to 3.33.

[0061] According to an embodiment of the present invention, the mass ratio of albumin to chloride salt is 1.71 to 3.07.

[0062] According to an embodiment of the present invention, the mass ratio of albumin to chloride salt is 2.04 to 2.73.

[0063] According to an embodiment of the present invention, the mass ratio of albumin to chloride salt is 2.39 to 2.73;

[0064] According to an embodiment of the present invention, the mass ratio of albumin to chloride salt is 2.73.

[0065] According to an embodiment of the present invention, the albumin is selected from any one or more of ovalbumin, bovine albumin, human albumin, and recombinant human albumin, preferably human albumin.

[0066] According to an embodiment of the present invention, the chloride salt comprises sodium chloride.

[0067] According to an embodiment of the present invention, the active protection solution further contains amino acids and / or citrate.

[0068] It should be noted that amino acids provide excipients during the freeze-drying process, and the preparation has good clarity after reconstitution. At the same time, they also act as protein protectants. The amino and carboxyl groups they contain can make freeze-dried products and products have strong buffering properties. Citrate can be used as a buffer system, which can combine with calcium ions in the blood, thereby reducing the calcium concentration in the blood, helping to prevent blood coagulation, and has the effects of anticoagulant and stabilizer. In addition, citrate can reduce the formation of blood clots by inhibiting the activity of thrombin.

[0069] According to an embodiment of the present invention, the amino acid is selected from glycine, histidine, lysine, arginine, and tryptophan.

[0070] According to an embodiment of the present invention, the amino acid is glycine, histidine or lysine.

[0071] According to an embodiment of the present invention, the amino acid is glycine.

[0072] According to some preferred embodiments of the present invention, the citrate comprises sodium citrate.

[0073] According to an embodiment of the present invention, the albumin content is 0.2% to 2%.

[0074] According to an embodiment of the present invention, the mass percentage of the sodium chloride is 0.1% to 1.0%, preferably 0.29% to 0.65%.

[0075] According to an embodiment of the present invention, the mass percentage of the sodium citrate is 0.1% to 1.5%.

[0076] According to an embodiment of the present invention, the mass percentage of the sodium citrate is 0.29% to 0.89%.

[0077] According to an embodiment of the present invention, the mass percentage of the amino acid is 0.05% to 0.4%.

[0078] According to an embodiment of the present invention, the mass percentage of the amino acid is 0.1% to 0.3%.

[0079] According to an embodiment of the present invention, the mass percentage of the amino acid is 0.15% to 0.25%.

[0080] According to an embodiment of the present invention, the vWF:Rco titer in the protein solution added to the protective solution in step 3) is 50 to 70 IU / mL.

[0081] According to an embodiment of the present invention, the freeze-drying process comprises placing the protein solution added with the protective solution in a freeze-drying device, and the freeze-drying conditions include:

[0082] (1) Lower the temperature to -2°C to -10°C and keep it warm for no less than 80 minutes;

[0083] (2) Lower the temperature to -20°C to -40°C within 40 to 60 minutes, and keep warm for no less than 10 minutes;

[0084] (3) Lower the temperature to -40°C to -60°C within 20 to 40 minutes, and keep warm for no less than 280 minutes;

[0085] (4) Evacuate the air and keep the vacuum degree below 50Pa;

[0086] (5) Raise the temperature to -20°C to -40°C within 350 to 550 minutes, and keep it at this temperature for no less than 580 minutes;

[0087] (6) Raise the temperature to -4°C to 4°C within 900 to 1060 minutes, and keep warm for no less than 750 minutes;

[0088] (7) Raise the temperature of the board layer to 7-15°C within 150-200 minutes, and keep it warm for no less than 100 minutes;

[0089] (8) Raise the temperature of the board layer to 20-40°C within 200-280 minutes, and keep it warm for no less than 450 minutes;

[0090] (9) Pump down to the extreme vacuum and prepare to go out.

[0091] The beneficial effects of the present invention include at least:

[0092] The invention adds a certain proportion of albumin and sodium chloride to prepare a preparation containing a vWF and / or FVIII complex. The preparation process is simple, the product purity is effectively improved, and the high activity of vWF and / or human coagulation factor VIII is retained. The activity recovery rate of vWF is as high as 99%, the activity recovery rate of FVIII is as high as 86%, and the reconstitution time is less than 30 seconds. The appearance after freeze-drying and dry-heating has no collapse, no atrophy, and no visible foreign matter. The invention is safe and effective with good stability. Under the conditions of 5±3°C and 25±2°C, the complex preparation of the invention can be stored for 12 months, and under the conditions of 40±2°C, the complex preparation of the invention can be stored for 9 months. The preparation cost of the invention is low, and large-scale preparation can be carried out, so the invention has good clinical application prospects.

[0093] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0094] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0095] Figure 1 The freeze-dried appearance of test group 6 and comparative examples 1 to 3, from left to right are test group 6, comparative example 1, comparative example 2, comparative example 3, comparative example 4;

[0096] Figure 2 This is the appearance picture of the experimental group 6 and comparative examples 1 to 3 after reconstitution, from top to bottom and from left to right, they are the experimental group 6, comparative example 1, comparative example 2, comparative example 3, and comparative example 4. DETAILED DESCRIPTION

[0097] Embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present invention, but should not be understood as limiting the present invention.

[0098] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In the description of the present invention, the meaning of "plurality" and "multiple" is at least two or two, such as two, two, three, three, etc., unless otherwise clearly and specifically defined.

[0099] The term "lyophilization" or "freeze drying" encompasses the process of removing liquid from a dissolved or at least partially dissolved composition under conditions involving at least one step of cooling the dissolved / partially dissolved solution to ice followed by vacuum drying.

[0100] In this document, the terms “include”, “have” or “comprises” are open expressions, that is, they include the contents specified in the present invention but do not exclude other contents.

[0101] As used herein, the term "optionally" generally means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0102] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0103] The data measured in the specific embodiments of the present invention are all average values ​​obtained from three groups of samples.

[0104] Unless otherwise specified, all reagents used in the examples can be purchased from commercial sources.

[0105] The present invention is described below with reference to specific embodiments. It should be noted that these embodiments are merely illustrative and do not limit the present invention in any way.

[0106] Example 1 Preparation of lyophilized / dry heat formulation of vWF / FVIII complex

[0107] This embodiment provides a preparation of a vWF / FVIII complex, which specifically includes the following steps:

[0108] 1. Preparation of stock solution

[0109] (1) Dissolution of cryoprecipitate: fresh frozen plasma from healthy subjects was taken out, thawed and centrifuged to obtain cryoprecipitate, and then dissolved by adding dissolution buffer (13.3 g 75% ethanol and 850 IU heparin sodium / kg dissolution buffer) in an amount 3 to 6 times the weight of the cryoprecipitate.

[0110] (2) Adsorption: Take the dissolved solution and add 100 to 800 g of 2% aluminum hydroxide gel per kg of cold precipitate for adsorption treatment.

[0111] (3) Virus inactivation

[0112] The adsorbed product obtained in step 2) is centrifuged to collect the centrifugal supernatant. After filtering the centrifugal supernatant, 11% S / D solution is added to the supernatant at a mass ratio of 1:10. After mixing, the supernatant has a pH value of 6.5-7.5, a protein content of 1.0 g / L-10.0 g / L, a TNBP concentration of 0.2%-0.4%, and a polysorbate 80 concentration of 0.7%-1.3%.

[0113] (4) Anion exchange chromatography

[0114] After the virus is extinguished by the S / D method, the solution is clarified and filtered, and then the Fractogel EMD TMAE gel is used as a filler for column chromatography. After the injection is completed, the buffer solution prepared with different formulas is used for washing and elution, and the eluate is collected. The buffer solution formula is as follows:

[0115] Balance solution (taking the preparation of 1 kg solution as an example): 7.0 g sodium chloride, 2.94 g sodium citrate, 9 g glycine, 0.147 g calcium chloride dihydrate; and adjust the pH to 7.0 ± 0.05 with 1 mol / L hydrochloric acid / sodium hydroxide;

[0116] Washing liquid (taking the preparation of 1 kg solution as an example): 17.25 g sodium chloride, 2.94 g sodium citrate, 9 g glycine, 0.147 g calcium chloride dihydrate; and adjust the pH to 7.0 ± 0.05 with 1 mol / L hydrochloric acid / sodium hydroxide;

[0117] Eluent (taking the preparation of 1 kg solution as an example): 21.65 g sodium chloride, 2.94 g sodium citrate, 9 g glycine, 0.147 g calcium chloride dihydrate; and adjust the pH to 7.0 ± 0.05 with 1 mol / L hydrochloric acid / sodium hydroxide.

[0118] (5) Ultrafiltration dialysis

[0119] The protein solution obtained after elution with the above elution buffer was subjected to ultrafiltration and dialysis, and concentrated to a vWF titer of ≥65 IU / mL.

[0120] (6) Preparation of semi-finished products

[0121] The protein solution containing human von Willebrand factor and / or human coagulation factor VIII complex after ultrafiltration and dialysis is prepared into a semi-finished product according to a vWF:Rco titer of 60±5 IU / mL, and human albumin is added according to a final concentration of 0.5% to 0.8% of human albumin per kg of protein solution. Stir evenly and adjust the pH to 7.0±0.1.

[0122] (7) Freeze-drying, dry heat inactivation

[0123] The product of step (6) is sterilized, packaged, freeze-dried, and then dry-heat-inactivated at 80° C. for 72 hours to obtain the finished product, wherein the freeze-drying process is as follows:

[0124] ① Drugs should be placed in the cabinet at room temperature (10-30℃);

[0125] ② The temperature of the board layer drops to -6℃, and the insulation time is not less than 90 minutes;

[0126] ③ Reduce the plate temperature to -32°C within 45 minutes, and keep warm for no less than 15 minutes;

[0127] ④ Reduce the plate temperature to -50℃ within 30 minutes, and keep warm for no less than 300 minutes;

[0128] ⑤ Start to evacuate the vacuum to 10Pa and keep the vacuum below 50Pa;

[0129] ⑥Raise the board temperature to -30℃ within 450min, and keep it warm for no less than 600min;

[0130] ⑦ Raise the temperature of the board layer to 0℃ within 1020min, and the insulation time shall not be less than 780min;

[0131] ⑧Raise the temperature of the board layer to 10℃ within 180min, and keep it warm for no less than 120min;

[0132] ⑨Raise the temperature of the board to 30℃ within 240min, and keep it warm for no less than 480min;

[0133] ⑩ Draw the ultimate vacuum, press the plug, take out the cabinet, and roll the cap.

[0134] (8) Redissolution

[0135] Reconstitute with sterile water for injection, the reconstitution volume is 10mL / bottle, each bottle contains 500IU vWF / 250IU FVIII.

[0136] Example 2 Optimization design of the composite preparation of the present invention

[0137] This example optimizes the formulation design based on Example 1, and refers to the preparation method of Example 1, except that in the preparation process of the semi-finished product in step (6), the protein solution containing vWF and / or FⅧ complex after ultrafiltration and dialysis is prepared into a semi-finished product according to the vWF:Rco titer of 60±5IU / mL, and then the components are added to each kg of the protein solution according to the components and addition amounts shown in Table 2. Stir evenly, adjust the pH to 7.0±0.1, and then freeze-dry and dry heat inactivate after the preparation is completed.

[0138] Table 1

[0139]

[0140]

[0141] Example 3

[0142] The quality of the products obtained by freeze-drying and dry-heat inactivation of the semi-finished products prepared with different formulations in Example 2 was judged by the following indicators: 1. appearance; 2. reconstitution time; 3. potency recovery rate, and the recovery rate detection was carried out by conventional methods in the art.

[0143] The corresponding test results of each group are shown in Table 2.

[0144] Table 2

[0145]

[0146]

[0147] As shown in Table 2, when the dosage of glycine and sodium citrate was changed in test groups 1 to 3 and the mass ratio of human albumin: sodium chloride remained unchanged, the product appearance and reconstitution time were qualified, the vWF activity recovery rate was stable at more than 95%, and the FVIII activity recovery rate was stable at more than 77%.

[0148] When the mass ratio of human albumin: sodium chloride in test groups 4 to 9 was in the range of 1.53 to 3.33, the activity recovery rates of vWF and FVIII in the products were relatively high, among which the activity recovery rate of vWF was higher than 92% and reached a maximum of 99%, and the activity recovery rate of FVIII was as low as 77% and as high as 86%.

[0149] In test groups 10 and 11, after glycine was replaced by histidine and lysine, respectively, the obtained vWF activity recovery rate was stabilized at more than 91%, and the FVIII activity recovery rate was stabilized at more than 77%.

[0150] When the ratio of human albumin:sodium chloride in Comparative Examples 1 and 3 is lower than 1.45 and the ratio in Comparative Example 2 is higher than 3.33, the obtained vWF recovery rate is lower than 92%, the FVIII activity recovery rate is lower than 70%, and the appearance shows problems of collapse and atrophy, which cannot meet the production requirements.

[0151] Example 4 Detection of the stability of the vWF / FVIII complex preparation of the present invention

[0152] The vWF / coagulation factor VIII complex preparations prepared by adding the freeze-dried / dry-heat inactivated protection solutions prepared according to the formulas shown in Table 1 were placed at 5±3°C, 25±2°C, and 40±2°C for stability testing. The preparations in the test groups all had good stability, and the stability test results of the products obtained after freeze-drying of the preparations in the test group 6 (the titer recovery rate at 0 months was the titer obtained after freeze-drying and dry-heating) are shown in Table 3.

[0153] Table 3: Stability test of products prepared with protective solution in test group 6

[0154]

[0155]

[0156] As can be seen from Table 3, under the condition of 5±3°C, the complex preparation is stored for one year, and its vWF titer recovery rate can still be maintained at more than 97%, and the FVIII titer recovery rate is stable at more than 84%. Under the conditions of 25±2°C and 40±2°C accelerated stability test, the vWF titer recovery rate and the FVIII titer recovery rate can still be maintained at a relatively high level; This indicates that at a relatively high temperature, the preparation of the present invention can still maintain the activity of the vWF / FVIII factors.

[0157] In summary, the preparation of the present invention can effectively maintain the activity of vWF / FVIII factors, the appearance and visible foreign matter of the obtained preparation meet the requirements, the preparation has good stability under conventional storage conditions and high temperature conditions, the activity recovery rate is high, and the preparation has good industrial application prospects.

[0158] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", "some implementation schemes" or "some examples" etc. means 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 specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, unless they are contradictory.

[0159] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A preparation containing vWF and / or FVIII complex, characterized in that The invention comprises a protective solution, wherein the protective solution contains albumin and chloride salt, and the mass ratio of the albumin to the chloride salt is 1.45-3.

40.

2. The preparation according to claim 1, characterized in that The mass ratio of albumin to chloride salt is 1.54 to 3.33; Optionally, the mass ratio of albumin to chloride salt is 1.71 to 3.07; Optionally, the mass ratio of albumin to chloride salt is 2.04 to 2.73; Preferably, the mass ratio of albumin to chloride salt is 2.39 to 2.73; Preferably, the mass ratio of albumin to chloride salt is 2.73; Optionally, the albumin is selected from any one or more of ovalbumin, bovine albumin, human albumin, and recombinant human albumin, preferably human albumin; Optionally, the chloride salt comprises sodium chloride.

3. The preparation according to claim 1, characterized in that The protective solution also contains amino acids and / or citrate; Optionally, the amino acid is selected from any one or more of glycine, histidine, lysine, arginine, and tryptophan; Preferably, the amino acid is glycine, histidine or lysine; More preferably, the amino acid is glycine; Optionally, the citrate comprises sodium citrate.

4. The preparation according to claim 3, characterized in that The mass percentage of the sodium citrate is 0.1% to 1.5%; Preferably, the mass percentage of the sodium citrate is 0.29% to 0.89%; Optionally, the mass percentage of the amino acid is 0.05% to 0.4%; Optionally, the mass percentage of the amino acid is 0.1% to 0.3%; Optionally, the mass percentage of the amino acid is 0.15% to 0.25%.

5. The preparation according to claim 1, characterized in that The albumin mass percentage is 0.2% to 2%; The mass percentage of the sodium chloride is 0.1% to 1.0%, preferably 0.29% to 0.65%.

6. A method for preparing a preparation containing a vWF and / or FVIII complex according to any one of claims 1 to 5, characterized in that: The method comprises: 1) Dissolving the cryoprecipitate; 2) performing virus inactivation, column chromatography purification, and ultrafiltration dialysis on the product obtained in step 1) in sequence to obtain a protein solution containing human von Willebrand factor and / or human coagulation factor VIII complex; 3) adding a protective solution to the protein solution for freeze drying and dry heat treatment.

7. The method according to claim 6, characterized in that The column chromatography is anion exchange chromatography; Optionally, the buffer used in the column chromatography purification includes at least one of an equilibration buffer, a washing buffer and an elution buffer; Optionally, the equilibration buffer, the washing buffer and the elution buffer include at least one of chloride, citrate and amino acid; Optionally, the mass ratio of chloride, citrate and amino acid in the equilibration buffer is (5-10):(1-5):(5-15); Optionally, the mass ratio of chloride, citrate and amino acid in the washing buffer is (15-20): (1-5): (5-15); Optionally, the mass ratio of chloride, citrate and amino acid in the elution buffer is (17-25): (1-5): (5-15); Optionally, the pH of the equilibration buffer, the washing buffer and / or the elution buffer is 6.5 to 7.

5.

8. The method according to claim 7, characterized in that Each kilogram of equilibrium buffer contains: 5-10 g of chloride, 1-5 g of citrate, and 5-15 g of amino acids; Optionally, the washing buffer comprises per kilogram: 15-20 g sodium chloride, 1-5 g citrate, 5-15 g amino acids; Optionally, the elution buffer comprises, per kilogram, 17-25 g sodium chloride, 1-5 g citrate, and 5-15 g amino acids.

9. The method according to claim 8, characterized in that The vWF titer in the protein solution in step 2) is not less than 60 IU / mL; Optionally, the protective solution includes albumin and chloride salt, and the mass ratio of the albumin to the chloride salt is 1.45 to 3.40; Optionally, the protective solution further contains amino acids and / or citrate; Optionally, the amino acid is selected from glycine, histidine, lysine, arginine, tryptophan; Optionally, the citrate comprises sodium citrate; Optionally, the mass percentage of the sodium citrate is 0.1% to 1.5%; Optionally, the mass percentage of the amino acid is 0.05% to 0.4%.

10. The method according to claim 8, characterized in that The freeze-drying process includes placing the protective solution in a freeze-drying device, and the freeze-drying conditions include: (1) Lower the temperature to -2°C to -10°C and keep it warm for no less than 80 minutes; (2) Lower the temperature to -20°C to -40°C within 40 to 60 minutes, and keep warm for no less than 10 minutes; (3) Lower the temperature to -40°C to -60°C within 20 to 40 minutes, and keep warm for no less than 280 minutes; (4) Evacuate the air and keep the vacuum degree below 50Pa; (5) Raise the temperature to -20°C to -40°C within 350 to 550 minutes, and keep it at this temperature for no less than 580 minutes; (6) Raise the temperature to -4°C to 4°C within 900 to 1060 minutes, and keep it warm for no less than 750 minutes; (7) Raise the temperature of the board layer to 7-15°C within 150-200 minutes, and keep it warm for no less than 100 minutes; (8) Raise the temperature of the board layer to 20-40°C within 200-280 minutes, and keep it warm for no less than 450 minutes; (9) Pump down to the extreme vacuum and prepare to go out.

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