Evogil freeze-dried powder

Abstract

The invention discloses EvoGiv freeze-dried powder and a preparation method thereof. The freeze-dried powder comprises a recombinant human acidic fibroblast growth factor, an excipient, a protein protective agent, a pH buffer agent and a freeze-dried preparation retaining agent, the freeze-dried powder preparation product prepared by the method is high in stability, is not easy to break into powder in the vibration process, avoids static electricity generated by friction between the broken medicine powder and a bottle wall in the storage and transportation process, and is beneficial to improving the product quality.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to a shape-keeping Evogeneik freeze-dried powder preparation and a preparation method thereof. BACKGROUND

[0002] Evogeneik generally refers to a freeze-dried recombinant human acidic fibroblast growth factor for external use. The main functions and effects of Evogeneik are to promote wound healing, improve second-degree burns, treat chronic ulcer wounds, relieve bedsores and treat diabetic ulcers. Evogeneik can promote wound healing and generally does not irritate the skin and mucous membrane tissue. When the skin is second-degree burned, it may cause redness and pain, and Evogeneik can be used to treat the symptoms and reduce pain, so that the burn gradually recovers. If there is a chronic ulcer wound, Evogeneik can also be used to treat the condition and effectively control the development of the disease. Evogeneik also has good effects on treating bedsores and can effectively inhibit the further aggravation of skin ulceration and relieve skin pain, blistering and other phenomena. If a person has a history of diabetes and does not receive timely sugar control treatment, skin ulcers may occur, mainly in the lower limbs and feet. Timely use of Evogeneik can inhibit the disease; The active ingredient of Evogeneik is recombinant human acidic fibroblast growth factor. Fibroblast growth factor (FGFs) is a multifunctional powerful cytokine, which is a kind of polypeptide growth factor that has mitogenic differentiation activity on multiple types of cells derived from mesoderm and neural ectoderm, including recombinant human acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF) and multiple members. Fibroblast growth factor can promote the growth and proliferation of skin tissue. It regulates the division, proliferation and growth and differentiation of skin epithelial, endothelial and stromal cells by combining with specific receptors on the cell surface, promotes cell metabolism and enhances oxidation. It can promote the rapid growth and proliferation of cells related to skin damage, regulate the synthesis, secretion and decomposition of intercellular matrix, promote the regeneration of keratinocytes, accelerate the repair of the keratin layer and the stromal layer of the skin, promote the growth of human skin cells, enhance the synthesis of protein and cell metabolism of skin cells, delay skin cell aging, and promote the repair and growth of epidermal cells.

[0003] The commercially available fibroblast growth factor includes a lyophilized preparation of recombinant human acidic fibroblast growth factor (rh-aFGF) and an eye drop, a gel and the like of basic fibroblast growth factor (bFGF or FGF-2), wherein the lyophilized preparation of rh-aFGF is a solid white or white-like loose solid, a solvent matched with the lyophilized preparation of rh-aFGF is poured into a vial containing the lyophilized preparation of rh-aFGF, the medicine liquid is directly sprayed on the wound after debridement after shaking, or a sterile gauze of an appropriate size is covered on the wound, the medicine liquid is uniformly added to the gauze, and the wound is appropriately bandaged, so that the skin wound can be quickly and efficiently healed, and the lyophilized preparation is suitable for the quick repair of various skin microtraumas.

[0004] In the previous preparation screening process, the inventor found that the conventional lyophilized preparation of recombinant human acidic fibroblast growth factor can maintain a certain overall state after the completion of lyophilization in a freeze-drying workshop, but the overall state of the lyophilized preparation is difficult to maintain under vibration conditions or during the machine transportation from the freeze-drying workshop to the packaging workshop, and the lyophilized preparation is broken and in a powder state, and the medicine powder is easily rubbed with the packaging bottle to generate static electricity, so that the powder is further adsorbed and agglomerated in the packaging bottle, and the use of the medicine is affected. SUMMARY

[0005] The present application is directed to the deficiencies of the prior art, and the components and lyophilization process parameters of the lyophilized preparation are researched, and a lyophilized preparation of recombinant human acidic fibroblast growth factor with suitable shape retention and a preparation method thereof are provided, and the shape retention of the present application refers to that the lyophilized preparation can be kept stable and is not easy to be broken and formed into powder during vibration or oscillation, so that the static electricity generated by the friction between the powder and the bottle wall is avoided or reduced.

[0006] Specifically, the present application provides a lyophilized preparation of recombinant human acidic fibroblast growth factor, wherein the lyophilized preparation comprises recombinant human acidic fibroblast growth factor, an excipient, a protein protective agent, a pH buffer and a lyophilized preparation retention agent.

[0007] Further, the present application provides a lyophilized preparation of recombinant human acidic fibroblast growth factor, wherein the excipient is selected from one or more of histidine, mannitol, trehalose, sucrose, sorbitol, maltose and lactose.

[0008] Further, the present application provides a lyophilized preparation of recombinant human acidic fibroblast growth factor, wherein the protein protective agent is selected from human blood albumin.

[0009] Further, the present application provides a lyophilized preparation of recombinant human acidic fibroblast growth factor, wherein the pH buffer is selected from the group consisting of a combination of dihydrogen phosphate and hydrogen phosphate; preferably, the dihydrogen phosphate is selected from sodium dihydrogen phosphate or potassium dihydrogen phosphate, and the hydrogen phosphate is selected from sodium hydrogen phosphate or potassium hydrogen phosphate.

[0010] Further, the present application provides a lyophilized preparation of recombinant human acidic fibroblast growth factor, wherein the lyophilized preparation maintaining agent is polyacrylamide.

[0011] Further, the present application provides a lyophilized preparation of recombinant human acidic fibroblast growth factor, wherein the lyophilized preparation contains 50-120 mg of excipient, 10-30 mg of protein protecting agent, 1.50-2.50 mg of pH buffer, and 1-4 mg of lyophilized preparation maintaining agent per 100 ug of recombinant human acidic fibroblast growth factor (rhAFGF) or rhAFGF stock solution.

[0012] Further, the present application provides a lyophilized preparation of recombinant human acidic fibroblast growth factor, wherein the lyophilized preparation contains 50-120 mg of excipient, 10-30 mg of protein protecting agent, 0.60-1.40 mg of dihydrogen phosphate, 1.50-2.50 mg of hydrogen phosphate, and 1-4 mg of lyophilized preparation maintaining agent per 100 ug of recombinant human acidic fibroblast growth factor (rhAFGF) or rhAFGF stock solution.

[0013] Further, the present application provides a lyophilized preparation of recombinant human acidic fibroblast growth factor, wherein the lyophilized preparation contains 70-90 mg of excipient, 15-25 mg of protein protecting agent, 0.80-1.20 mg of dihydrogen phosphate, 1.50-2.00 mg of hydrogen phosphate, and 2-3 mg of lyophilized preparation maintaining agent per 100 ug of recombinant human acidic fibroblast growth factor (rhAFGF) or rhAFGF stock solution.

[0014] Further, the present application provides a lyophilized preparation of recombinant human acidic fibroblast growth factor, wherein the lyophilized preparation contains 80 mg of excipient, 20 mg of protein protecting agent, 1.08 mg of dihydrogen phosphate, 1.73 mg of hydrogen phosphate, and 3.00 mg of lyophilized preparation maintaining agent per 100 ug of recombinant human acidic fibroblast growth factor (rhAFGF) or rhAFGF stock solution.

[0015] Further, the present application provides a preparation method of the lyophilized preparation of the recombinant human acidic fibroblast growth factor, which comprises the following steps: (a) uniformly mixing the prescription amount of excipient, protein protective agent, pH buffer and freeze-drying maintaining agent, and dissolving in water for injection; (b) adding the prescription amount of the recombinant human acidic fibroblast growth factor, and constant volume; (c) filtering, then transferring the filtrate to a container for filling, and then performing freeze-drying to obtain the lyophilized preparation.

[0016] Further, the present application provides the lyophilized preparation of the recombinant human acidic fibroblast growth factor prepared by the above method.

[0017] Compared with the prior art, the present application has the following beneficial effects: 1. The product prepared by the present application has high stability, and remains stable during vibration or oscillation. The lyophilized preparation has certain adhesion to the bottle wall, and is not easy to break to form powder, thereby avoiding or reducing the static electricity generated by the friction between the powder and the bottle wall during storage and transportation. 2. The method of the present application has high collapse retention ratio during preparation, and the prepared product has good appearance and high yield. DETAILED DESCRIPTION

[0018] In order to make the technical means, creative features, purposes and effects of the present application easy to understand, the present application is further described below in combination with specific embodiments.

[0019] It is to be understood that the relative ratio of the active ingredient and the excipient can be adjusted, and the lyophilized preparation prepared by equal proportion enlargement or reduction according to the prescription ratio is also within the protection scope of the present application.

[0020] The rhAFGF stock solution used in the present application is from Shanghai Tengrui Pharmaceutical Co., Ltd., and the protein content of the rhAFGF stock solution is 2.93 mg / ml (specific activity is 2.9×10 5 U / mg).

[0021] Example 1 Lyophilization prescription:

[0022] The materials were weighed proportionally, and mannitol, disodium hydrogen phosphate, sodium dihydrogen phosphate, and polyacrylamide were added into a vial according to the prescription proportion, 50% water for injection was added, and the mixture was shaken to dissolve, to obtain diluent 1. rhAFGF stock solution and human serum albumin were added according to the prescription proportion, and the mixture was diluted to the volume with the remaining water for injection to obtain a semi-finished product. The semi-finished product was filtered through two 0.22 µm PVDF sleeve filters, filled into a vial, semi-capped with a sterilized rubber stopper, and then introduced into a freeze-drying machine through an automatic feeding and discharging system. The freezing parameters were as follows, to obtain a freeze-dried preparation:

[0023] Example 2: Freeze-drying prescription:

[0024] The freeze-drying process was the same as in Example 1.

[0025] Example 3: Freeze-drying prescription:

[0026] The freeze-drying process was the same as in Example 1.

[0027] Comparative Example 1: The freeze-drying prescription and freeze-drying process parameters were the same as in Example 2, except that the freeze-dried preparation stabilizer polyacrylamide was not included in the freeze-drying prescription.

[0028] Comparative Example 2: The freeze-drying prescription and freeze-drying process parameters were the same as in Example 2, except that the relative amount of the freeze-dried preparation stabilizer polyacrylamide in the freeze-drying prescription was adjusted to 0.5 mg.

[0029] Comparative Example 3: The freeze-drying prescription and freeze-drying process parameters were the same as in Example 2, except that the relative amount of the freeze-dried preparation stabilizer polyacrylamide in the freeze-drying prescription was 5 mg.

[0030] Comparative Example 4: The freeze-drying prescription and freeze-drying process parameters were the same as in Example 2, except that the filtrate was directly cooled to -35°C after filtration, the vacuum freeze-drying device was started, and the freeze-dried preparation was obtained. Then the temperature was increased to 25°C, and the vacuum dried to a constant weight.

[0031] Test Example 1: Freeze-dried preparation stability test The test laboratory ground was a 2 mm thick PVC floor leather laid on a common cement floor by bonding. Ten freeze-dried preparations prepared in each example and comparative example were placed on a 100 cm high stainless steel table, and the freeze-dried preparations were dropped to the ground ten times, and the shape of the freeze-dried preparations was observed. The results are shown in Table 1. Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Properties 1 sample had slight cracking, others were intact 10 samples were intact 10 samples were intact 10 samples were powdery 6 samples were powdery, 4 samples were severely cracked 10 samples were intact 10 samples were intact Test Example 2: Relative activity test The lyophilized preparations prepared in Examples 1-3 and Comparative Examples 1-4 were stored at 40°C in a closed condition for 6 months, and water for injection was added to make up to 2 ml, and shaken well to dissolve. The recombinant human acidic fibroblast growth factor filled in a vial before lyophilization was taken, and the activities of the lyophilized preparations prepared in Examples 1-3 and Comparative Examples 1-4 and the recombinant human acidic fibroblast growth factor before lyophilization were tested, respectively. The activity of the human recombinant human acidic fibroblast growth factor before lyophilization was taken as 100%, and the relative activity of the lyophilized preparations prepared in Examples 1-3 and Comparative Examples 1-4 stored at 40°C in a closed condition for 6 months was tested according to the following method.

[0032] The bioactivity of the recombinant human acidic fibroblast growth factor was determined by MTT cell culture method. That is, Balb / c 3T3 cells were used, and the rhaFGF of Promega Company was used as a standard when determination. The logarithmic proliferation period of Balb / c 3T3 cells was adjusted to a cell concentration, and was added to a 96-well plate (7000 cells / 100 μl per well). After being cultured at 37°C in a 5% CO2 condition for 24 h, the cells were starved and cultured for 24 h after being replaced with 1640 medium containing 0.4% fetal bovine serum. The above solution was diluted by 4 times according to a certain concentration, and was added to each parallel group of 3 wells. After being cultured for 48-72 h, 20 ml of MTT (5 mg / ml) was added, and the culture solution was discarded after being cultured for 4 h. 150 μl of DMSO was added to each well, and was placed at room temperature for 40 min. The A value was measured at 570 nm wavelength, and the corresponding A value-aFGF concentration curve was drawn, and the relative activity was calculated, as shown in Table 2 below: Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Relative Activity 99.1% 98.2% 97.4% 99.0% 98.9% 81.1% 90.3% The above data show that the lyophilized product prepared in the present application still maintains a high bioactivity after being stored at 40°C in a closed condition for 6 months.

[0033] Test Example 3: Reconstitution time test The lyophilized preparations prepared in Examples 1-3 and Comparative Examples 1-4 were taken, 10 ml of 10 mmol / L phosphate buffer (pH 7.0) was added, and the time to transparent and clear was calculated by a stopwatch, as shown in Table 3: Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Clear Time 19 seconds 20 seconds 23 seconds 16 seconds 17 seconds 45 seconds 21 seconds The above data show that the ratio of the lyophilization stabilizer, the lyophilization parameters, etc. can affect the reconstitution time of the lyophilized preparation.

[0034] The above shows and describes the basic principles and main features of the present application and the advantages of the present application. Those skilled in the art should understand that the present application is not limited to the above embodiments, and the above embodiments and descriptions in the specification are only to illustrate the principles of the present application. Without departing from the spirit and scope of the present application, various changes and improvements can be made to the present application, and these changes and improvements all fall within the scope of the claimed present application. The scope of protection of the present application is defined by the appended claims and their equivalents.

Claims

1. An Avjiv lyophilized powder formulation, wherein the lyophilized powder formulation comprises recombinant human acidic fibroblast growth factor, excipients, protein protectants, pH buffers, and lyophilization preservation agents.

2. The lyophilized powder formulation of Evgiev according to claim 1, characterized in that... The excipients are selected from one or more of histidine, mannitol, trehalose, sucrose, sorbitol, maltose, and lactose.

3. The lyophilized powder formulation of Evgeny according to claim 1, characterized in that... The protein protectant is selected from human serum albumin.

4. The lyophilized powder formulation of Evgeny according to claim 1, characterized in that... The pH buffer is selected from a combination of dihydrogen phosphate and dihydrogen phosphate; preferably, the dihydrogen phosphate is selected from sodium dihydrogen phosphate or potassium dihydrogen phosphate, and the dihydrogen phosphate is selected from disodium hydrogen phosphate or dipotassium hydrogen phosphate.

5. The lyophilized powder formulation of Evgeny according to claim 1, characterized in that... The freeze-dried formulation retainer is selected from polyacrylamide.

6. The lyophilized powder formulation of Evgeny according to claim 1, characterized in that... The lyophilized formulation contains 50-120 mg of excipients, 10-30 mg of protein protectant, 1.50-2.50 mg of pH buffer, and 1-4 mg of lyophilized formulation maintainer per 100 μg of recombinant human acidic fibroblast growth factor (rhAFGF) stock solution.

7. The lyophilized powder formulation of Evgeny according to claim 1, characterized in that... The lyophilized formulation contains 50-120 mg of excipients, 10-30 mg of protein protectant, 0.60-1.40 mg of dihydrogen phosphate, 1.50-2.50 mg of dihydrogen phosphate, and 1-4 mg of lyophilized formulation retainer per 100 μg of recombinant human acidic fibroblast growth factor (rhAFGF) stock solution.

8. The lyophilized powder formulation of Evgeny according to claim 1, characterized in that... The lyophilized formulation contains 70-90 mg of excipients, 15-25 mg of protein protectant, 0.80-1.20 mg of dihydrogen phosphate, 1.50-2.00 mg of dihydrogen phosphate, and 2-3 mg of lyophilized formulation retainer per 100 μg of recombinant human acidic fibroblast growth factor (rhAFGF) stock solution.

9. The lyophilized powder formulation of Evgeny according to claim 1, characterized in that... The lyophilized formulation contains 80 mg of excipients, 20 mg of protein protectant, 1.08 mg of dihydrogen phosphate, 1.73 mg of dihydrogen phosphate, and 3.00 mg of lyophilization preserver per 100 μg of recombinant human acidic fibroblast growth factor (rhAFGF) stock solution.

10. The method for preparing the Evgiev lyophilized powder formulation according to any one of claims 1-9, wherein the method comprises the following steps: (a) Mix the prescribed amounts of excipients, protein protectants, pH buffers and lyophilization preservers thoroughly, and dissolve them in water for injection; (b) Add the prescribed amount of recombinant human acidic fibroblast growth factor and bring to a final volume. (c) Filter, then transfer the filtrate to a container for filling, and then freeze-dry to obtain a freeze-dried formulation.

11. The lyophilized powder formulation of Evgif prepared according to the method of claim 10.

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