Recombinant blood coagulation factor VIII preparation and preparation method and application thereof

Abstract

The invention provides a recombinant blood coagulation factor VIII preparation and a preparation method and application thereof, and belongs to the field of biological medicine. The recombinant blood coagulation factor VIII preparation provided by the invention is prepared from the following components: 500 to 1500 IU / mL of recombinant blood coagulation factor VIII, 20 to 100 mM of glycine, 50 to 100 mM of raffinose, 2 to 10 mM of divalent calcium salt, 0.5 to 5 percent of dextran-40 and 0.05 to 0.15 percent of tween-80, the preparation is simple in component, so that the recombinant blood coagulation factor VIII is long in preservation time and stable in activity, and can be used for preparing drugs for treating hemophilia.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to recombinant coagulation factor VIII, and in particular to a recombinant coagulation factor VIII preparation and a preparation method and application thereof. Background Art

[0002] Hemophilia is an inherited bleeding disorder caused by mutations in coagulation factor genes, which can lead to low or insufficient levels of coagulation factors in the body, resulting in bleeding. A deficiency or insufficiency of coagulation factor F VIII causes hemophilia A.

[0003] Coagulation factor VIII (rFVIII) is a cofactor in the cascade of reactions that lead to coagulation during blood coagulation. Coagulation factors can typically be obtained from human plasma. The preparation of coagulation factors using recombinant DNA technology avoids potential contamination associated with blood products and eliminates the need for a single source of coagulation factor VIII, effectively avoiding supply constraints caused by the use of voluntary plasma donations as a raw material. Lyophilized preparations of coagulation factor VIII prepared using recombinant DNA technology can be administered therapeutically for acute bleeding or as regular, intermittent dosing to prevent internally controlled bleeding.

[0004] Coagulation factor VIII produced using recombinant DNA technology is susceptible to degradation, aggregation, and inactivation, making it relatively unstable during storage. To improve its stability, human serum albumin is often added to the formulation as a stabilizer. While one of the advantages of rFVIII products is that they are not derived from human plasma, potentially avoiding contamination from human plasma, the addition of human serum albumin negates the advantage of recombinant production methods that lack any human plasma as a raw material. Due to its complex protein structure and large molecular weight, FVIII is highly unstable and susceptible to inactivation during the collection and extraction processes of the raw plasma. Furthermore, the packaging, storage, and infusion methods of the drug can also lead to a loss of its coagulation activity. Therefore, improving the storage stability of FVIII is particularly important.

[0005] Prior art CN106139127B discloses a lyophilized formulation of recombinant coagulation factor VIII, comprising 50-1500 IU / mL of recombinant coagulation factor VIII, 5-50 mM buffer, 5-100 mM purity stabilizer, 1-5 mM activity stabilizer, 150-350 mM solubilizer, and 0.005-0.05 wt% surfactant. The buffer is preferably a histidine buffer, preferably at a concentration of 10 mM. The purity stabilizer is preferably sucrose or trehalose, preferably at a concentration of 10 mM. The activity stabilizer is preferably a divalent calcium salt, such as calcium chloride, calcium gluconate, or calcium glucarate, preferably at a concentration of 2.5 mM-5 mM. The solubilizer is preferably sodium chloride, preferably at a concentration of 300 mM. The surfactant is preferably polysorbate 80, preferably at a concentration of 0.01 wt%. This recombinant coagulation factor VIII lyophilized preparation does not contain albumin. After being reconstituted in water, the purity of the main peak detected by SEC-HPLC drops below 92.5% within 30 days, and the activity remains above 50% within 30 days, resulting in poor long-term storage effect.

[0006] Prior art CN112138149A discloses a recombinant coagulation factor VIII formulation comprising (a) a recombinant FVIII protein; (b) one or more sugars or sugar alcohols selected from mannitol or sucrose; (c) one or more amino acids selected from glycine or arginine; (d) sodium chloride or potassium chloride; and (e) a divalent cation salt. Preferably, the formulation comprises 20-30 mmol / L histidine, 100-150 mmol / L sodium chloride, 2-8 mmol / L calcium chloride, 3-6% w / v mannitol, 1-1.8% w / v arginine hydrochloride, 0.01%-0.03% polysorbate 80, a pH of 6.5-7.5, and a recombinant FVIII protein concentration of 1000 IU. After storage at 25°C for 5 days, the purity of the main peak decreased to below 90% as determined by SEC-HPLC, and the activity also decreased to below 90%, indicating poor long-term storage performance. Summary of the Invention

[0007] To address the poor long-term storage performance of existing technologies, the present invention provides a recombinant factor VIII preparation, its preparation method, and its application. The present invention provides a recombinant factor VIII preparation comprising 500-1500 IU / mL of recombinant factor VIII, 20-100 mM glycine, 50-100 mM raffinose, 2-10 mM divalent calcium salt, 0.5%-5% dextran-40, and 0.05%-0.15% Tween-80. This preparation has simple ingredients, ensures long shelf life, and maintains stable activity of the recombinant factor VIII, making it suitable for use in the preparation of a drug for the treatment of hemophilia.

[0008] To achieve the above object, the technical solution adopted by the present invention is as follows: In one aspect, the present invention provides a recombinant coagulation factor VIII preparation comprising 500-1500 IU / mL recombinant coagulation factor VIII, 20-100 mM glycine, 50-100 mM raffinose, 2-10 mM divalent calcium salt, 0.5%-5% dextran-40, and 0.05%-0.15% Tween-80.

[0009] Preferably, the composition includes 800-1200 IU / mL recombinant coagulation factor VIII, 40-80 mM glycine, 60-80 mM raffinose, 4-8 mM divalent calcium salt, 2.0%-4.0% dextran-40 and 0.10%-0.12% Tween-80.

[0010] Preferably, the composition includes 1000 IU / mL recombinant coagulation factor VIII, 50 mM glycine, 80 mM raffinose, 6 mM divalent calcium salt, 3.0% dextran-40 and 0.10% Tween-80.

[0011] Preferably, the divalent calcium salt is selected from any one or more of calcium chloride, calcium sulfate, calcium gluconate and calcium glucarate.

[0012] Preferably, the divalent calcium salt is calcium chloride.

[0013] In another aspect, the present invention provides a method for preparing the above-mentioned recombinant coagulation factor VIII preparation, comprising the following steps: S1. preparing recombinant coagulation factor VIII and obtaining recombinant coagulation factor VIII stock solution after purification; S2. Add glycine, raffinose, divalent calcium salt, dextran-40, and Tween-80 to the recombinant coagulation factor VIII stock solution, and adjust the pH to 7.0 with a pH regulator to obtain a semi-finished product; S3. Freeze-dry the semi-finished product to obtain a recombinant coagulation factor VIII preparation.

[0014] Preferably, the purification in step S1 comprises purification by anion exchange chromatography.

[0015] Preferably, the pH adjuster in step S2 is hydrochloric acid, and the pH is adjusted to 7.0.

[0016] Preferably, the freeze-drying in step S3 comprises the following steps: (1) Lower the freeze-drying chamber temperature to at least -45°C for 2-4 hours; (2) Raise the temperature of the freeze-drying chamber to a range of -15°C to -35°C and maintain for 1-3 hours; (3) Lower the freeze-drying chamber temperature to at least -45°C for 2-4 hours; (4) After the freeze dryer pressure is reduced to 0.1 mBar, the temperature is raised to the range of -32°C to -35°C for drying; (5) After drying, the temperature is raised to 25°C and maintained for 15 hours. The temperature increase rate is preferably 0.2°C / min.

[0017] On the other hand, the present invention provides the use of the above-mentioned recombinant coagulation factor VIII preparation or the above-mentioned preparation method, wherein the use includes the preparation of a drug for treating hemophilia.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The recombinant coagulation factor VIII preparation provided by the present invention has simple ingredients, ensures that the recombinant coagulation factor VIII has a long shelf life, and its activity is stable for not less than 2 months, and can be used to prepare medicines for treating hemophilia. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The preparation prepared in Example 1 (contained in a vial). DETAILED DESCRIPTION

[0020] To make the objects, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the examples. Where specific conditions are not specified in the examples, the methods are carried out according to conventional conditions or the conditions recommended by the manufacturer. All reagents or instruments not specified by the manufacturer are commercially available pharmaceutical grade products. To better illustrate the present invention, numerous specific details are provided in the following specific embodiments. The specific examples described herein are intended only to explain the present invention and are not intended to constitute any limitation thereto.

[0021] The present invention provides a recombinant coagulation factor VIII preparation, the components of which are 500-1500 IU / mL recombinant coagulation factor VIII, 20-100mM glycine, 50-100mM raffinose, 2-10mM divalent calcium salt, 0.5%-5% dextran-40 (purchased from Yisheng Biotechnology Co., Ltd., product number 61209ES60), and 0.05%-0.15% Tween-80; wherein the divalent calcium salt is preferably calcium chloride.

[0022] Experimental methods: (1) Preparation method of recombinant coagulation factor VIII liquid preparation: Purification is performed using anion exchange chromatography, based on the competitive interaction between the recombinant coagulation factor and the charged ions in the buffer medium used, to obtain a stock solution of recombinant coagulation factor VIII. The concentration of recombinant coagulation factor VIII in the stock solution should be above 1000 IU / mL. The required amounts of various excipients are calculated based on the volume of the stock solution (see the table below) and added to the stock solution. The concentrations of all components in the solution are adjusted, and the pH is adjusted to 7.0 with hydrochloric acid to obtain a semi-finished product. The semi-finished product is aseptically divided into brown penicillin bottles, partially stoppered, and freeze-dried to obtain the finished lyophilized preparation of recombinant coagulation factor VIII.

[0023] (2) Freeze-drying method: A recombinant coagulation factor VIII liquid preparation is freeze-dried using a freeze dryer. The freeze-drying steps include: (1) lowering the temperature of the freeze drying chamber of the freeze dryer to at least -45°C and maintaining it for 2 hours; (2) raising the temperature of the freeze drying chamber to -15°C to -35°C (preferably -25°C) and maintaining it for 1 to 3 hours; (3) lowering the temperature of the freeze drying chamber to at least -45°C and maintaining it for 2 hours. During this freezing process, the temperature rise and fall rate is preferably 0.5°C to 1.0°C per minute. (4) After the freeze dryer pressure is reduced to about 0.1 mBar, the temperature is raised to between -32°C and -35°C for a primary drying step. This step takes up to about 50 hours; (5) After the primary drying step, the temperature is raised to 25°C and maintained for 15 hours. The temperature rise rate is preferably 0.2°C / minute.

[0024] Data analysis was performed using professional data processing software, and significance analysis was performed using one-way ANOVA test, with P < 0.05 indicating a significant difference.

[0025] Preparation of the preparations in the examples and comparative examples The components of the liquid preparations of recombinant coagulation factor VIII in this example and comparative example are shown in Table 1. Except for the different components, the preparation methods of Examples 2-3 and Comparative Examples 1-9 are the same as those of Example 1 to prepare recombinant coagulation factor VIII (lyophilized) preparations. Figure 1 This is a bottle of the preparation prepared in Example 1. The only difference between Example 8 and Example 1 is that raffinose is replaced with an equal molar amount of sucrose, and the only difference between Comparative Example 9 and Example 1 is that Tween-80 is replaced with an equal mass of polysorbate 80.

[0026] Table 1 Recombinant coagulation factor VIII liquid formulations of Examples and Comparative Examples

[0027] Note: NA means this ingredient is not added.

[0028] Test example: Verification of the efficacy of different groups of recombinant coagulation factor VIII preparations The recombinant coagulation factor VIII preparations in different groups were stored at 4°C in the dark for a certain period of time. After being taken out, they were reconstituted with purified water for effect verification. The purity of the recombinant coagulation factor VIII was detected by SEC-HPLC. The activity of the recombinant coagulation factor VIII was detected using a kit (purchased from Rossix, catalog number REF 800070). Three parallel experiments were set up for each group. The results are presented as the mean ± standard deviation. See Tables 2 and 3.

[0029] Table 2 SEC-HPLC purity (%)

[0030] Note: Data in the same column marked with the same letters indicate that there is no significant difference between the data (P>0.05), and data marked with different letters indicate that there is a significant difference between the data (P<0.05).

[0031] The results of the SEC-HPLC method for detecting the purity of recombinant coagulation factor VIII preparations in different groups are shown in Table 2. It can be seen that the purity of Examples 1-3 is significantly improved compared with that of Comparative Examples 1-9, and the purity can be maintained at above 96% after storage at 4°C for 60 days.

[0032] Table 3 Biological activity stability (IU / mL)

[0033] Note: Data in the same column marked with the same letters indicate that there is no significant difference between the data (P>0.05), and data marked with different letters indicate that there is a significant difference between the data (P<0.05).

[0034] The results of the activity stability test of recombinant coagulation factor VIII preparations in different groups are shown in Table 3. It can be seen that, compared with Example 1, Comparative Example 4 does not add raffinose, dextran 40, and Tween 80; Comparative Examples 1-3 respectively add only raffinose, dextran 40, and Tween 80, and the activity on day 60 is improved compared with Comparative Example 4; Comparative Examples 5-7 respectively add raffinose + dextran 40, raffinose + Tween 80, and dextran 40 + Tween 80, and their activities on day 60 are improved on the basis of Comparative Examples 1-3. , but within the expected improvement range; compared with Comparative Examples 5-7, the activity of Example 1 on the 60th day was significantly improved, and the improvement range was higher than the cumulative improvement range of raffinose, dextran 40 and Tween 80 alone, indicating that raffinose, dextran 40 and Tween 80 have a synergistic effect; in Comparative Example 8, after raffinose was replaced with an equal molar amount of sucrose, the synergistic effect of Example 1 was not achieved; in Comparative Example 9, after Tween 80 was replaced with an equal mass of polysorbate 80, the synergistic effect of Example 1 was also not achieved.

[0035] In summary, the preparation provided by the present invention has simple ingredients, and the recombinant coagulation factor VIII can be stably stored for at least 2 months, and can be used to prepare medicines for treating hemophilia.

[0036] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A recombinant coagulation factor VIII preparation, characterized in that: The components include 500-1500 IU / mL recombinant coagulation factor VIII, 20-100mM glycine, 50-100mM raffinose, 2-10mM divalent calcium salt, 0.5%-5% dextran-40 and 0.05%-0.15% Tween-80.

2. The recombinant coagulation factor VIII preparation according to claim 1, characterized in that The components include 800-1200 IU / mL recombinant coagulation factor VIII, 40-80mM glycine, 60-80mM raffinose, 4-8mM divalent calcium salt, 2.0%-4.0% dextran-40 and 0.10%-0.12% Tween-80.

3. The recombinant coagulation factor VIII preparation according to claim 2, characterized in that Components include 1000 IU / mL recombinant coagulation factor VIII, 50 mM glycine, 80 mM raffinose, 6 mM divalent calcium salt, 3.0% dextran-40, and 0.10% Tween-80.

4. The recombinant coagulation factor VIII preparation according to claim 3, characterized in that The divalent calcium salt is selected from any one or more of calcium chloride, calcium sulfate, calcium gluconate and calcium glucarate.

5. The recombinant coagulation factor VIII preparation according to claim 4, characterized in that The divalent calcium salt is calcium chloride.

6. The method for preparing the recombinant coagulation factor VIII preparation according to any one of claims 1 to 5, characterized in that: The following steps are involved: S1. preparing recombinant coagulation factor VIII and obtaining recombinant coagulation factor VIII stock solution after purification; S2. Add glycine, raffinose, divalent calcium salt, dextran-40, and Tween-80 to the recombinant coagulation factor VIII stock solution, and adjust the pH to 7.0-8.0 with a pH regulator to obtain a semi-finished product; S3. Freeze-dry the semi-finished product to obtain a recombinant coagulation factor VIII preparation.

7. The preparation method according to claim 6, characterized in that The purification in step S1 includes purification by anion exchange chromatography.

8. The preparation method according to claim 6, characterized in that In step S2, the pH adjuster is hydrochloric acid, and the pH is adjusted to 7.

0.

9. The preparation method according to claim 6, characterized in that The freeze drying in step S3 comprises the following steps: (1) Lower the freeze-drying chamber temperature to at least -45°C for 2-4 hours; (2) Raise the temperature of the freeze-drying chamber to a range of -15°C to -35°C and maintain for 1-3 hours; (3) Lower the freeze-drying chamber temperature to at least -45°C for 2-4 hours; (4) After the freeze dryer pressure is reduced to 0.1 mBar, the temperature is raised to the range of -32°C to -35°C for drying; (5) After drying, the temperature is raised to 25°C and maintained for 15 hours. The temperature increase rate is preferably 0.2°C / min.

10. Use of the recombinant coagulation factor VIII preparation according to any one of claims 1 to 5 or the preparation method according to any one of claims 6 to 9, characterized in that: The application includes preparing medicine for treating hemophilia.

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