Efgonyl lyophilized powder

Abstract

The application discloses a lyophilized powder of Evf and a preparation method thereof. The lyophilized powder comprises recombinant human acidic fibroblast growth factor, excipient, protein protective agent, pH buffer and lyophilized preparation maintenance agent. The lyophilized powder prepared by the method has high stability and is not easy to be broken into powder in the shaking process, so that electrostatic is avoided due to the friction between the broken powder and the bottle wall in the storage and transportation process, and the product quality is improved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a shape-preserving lyophilized powder formulation of Evgiev and its preparation method. Background Technology

[0002] Aifjifu typically refers to topical lyophilized recombinant human acidic fibroblast growth factor. Its main functions and effects include promoting wound healing, improving second-degree burns, treating chronic ulcers, relieving pressure ulcers, and treating diabetic ulcers. This medication promotes wound healing and generally does not irritate the skin or mucous membranes. When second-degree burns occur, symptoms such as redness and pain may occur; this medication can be used to treat these symptoms, reduce pain, and gradually heal the burn. It can also be used to treat chronic ulcers, effectively controlling their progression. This medication also has good efficacy in treating pressure ulcers, effectively inhibiting further aggravation of skin ulceration and relieving skin pain and blisters. In patients with a history of diabetes who have not received timely blood sugar control, skin ulcers may develop, mainly on the lower limbs and feet; timely use of this medication can suppress the condition.

[0003] The active ingredient in Efgiev is recombinant human acidic fibroblast growth factor (FGF). Fibroblast growth factors (FGFs) are multifunctional and potent cytokines, a class of polypeptide growth factors with pro-proliferative and differentiation-promoting activity on various cell types derived from the mesoderm and neuroectoderm. These include several members such as recombinant human acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF). Fibroblast growth factors promote the growth and proliferation of skin tissue. By binding to specific receptors on the cell surface, they regulate the division, proliferation, growth, and differentiation of epithelial, endothelial, and stromal cells in the skin, promoting cell metabolism and enhancing oxidation. They can promote the rapid growth and proliferation of cells related to skin damage and regulate the synthesis, secretion, and decomposition of the intercellular matrix. They can promote the regeneration of stratum corneum cells, accelerate the repair of the stratum corneum and stroma, and promote the growth of human skin cells. They can enhance the synthesis of proteins and cell metabolism in skin cells, thus delaying skin cell aging and promoting the repair and growth of epidermal cells.

[0004] Commercially available fibroblast growth factors include recombinant human acidic fibroblast growth factor (rh-aFGF) lyophilized formulations and basic fibroblast growth factor (bFGF, or FGF-2) eye drops and gels. The rh-aFGF lyophilized formulation is a solid, white or off-white, loose solid. To use, pour the matching solvent into the vial containing the rh-aFGF lyophilized formulation, shake well, and spray the solution directly onto the cleaned wound. Alternatively, cover the wound with appropriately sized sterile gauze, apply the solution evenly to the gauze, and bandage appropriately. This allows for rapid and efficient healing of skin wounds and is suitable for the rapid repair of various minimally invasive skin injuries. Compared to solutions or gels, lyophilized formulations have advantages such as faster absorption, higher activity, simpler composition, and greater skin-friendliness. Furthermore, the dosage can be adjusted according to the size of the wound, which helps improve the effective utilization rate of a single dose.

[0005] During the initial formulation screening process, the inventors discovered that conventional recombinant human acidic fibroblast growth factor lyophilized formulations maintain a certain overall state in the lyophilization workshop after lyophilization. However, under vibration conditions or during the process of transporting the lyophilized formulations from the lyophilization workshop to the packaging workshop by machine, the overall state of the lyophilized formulations is difficult to maintain, resulting in broken and powdery forms. During this process, the friction between the powder and the packaging bottle can easily generate static electricity, causing the powder to be further adsorbed and agglomerated inside the packaging bottle, affecting the use of the drug. Summary of the Invention

[0006] This invention addresses the shortcomings of existing technologies by studying the composition and freeze-drying process parameters of lyophilized formulations, and provides a recombinant human acidic fibroblast growth factor lyophilized formulation with suitable shape retention and its preparation method. The shape retention of this invention refers to the stability of the lyophilized formulation during vibration or shaking, preventing it from breaking into powder and avoiding or reducing static electricity generated by friction between the powder and the bottle wall.

[0007] Specifically, the present invention provides a lyophilized formulation of recombinant human acidic fibroblast growth factor, wherein the lyophilized formulation comprises recombinant human acidic fibroblast growth factor, excipients, protein protectants, pH buffers, and lyophilization preservation agents.

[0008] Furthermore, the present invention provides a lyophilized formulation 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.

[0009] Furthermore, the present invention provides a lyophilized formulation of recombinant human acidic fibroblast growth factor, wherein the protein protectant is selected from human serum albumin.

[0010] Furthermore, the present invention provides a lyophilized formulation of recombinant human acidic fibroblast growth factor, wherein 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.

[0011] Furthermore, the present invention provides a lyophilized formulation of recombinant human acidic fibroblast growth factor, wherein the lyophilized formulation retainer is selected from polyacrylamide.

[0012] Furthermore, the present invention provides a lyophilized formulation of recombinant human acidic fibroblast growth factor, wherein each 100 μg of recombinant human acidic fibroblast growth factor (rhAFGF) or recombinant human acidic fibroblast growth factor (rhAFGF) stock solution 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 retainer.

[0013] Furthermore, the present invention provides a lyophilized formulation of recombinant human acidic fibroblast growth factor (rhAFGF), wherein each 100 μg of recombinant human acidic fibroblast growth factor (rhAFGF) stock solution 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.

[0014] Furthermore, the present invention provides a lyophilized formulation of recombinant human acidic fibroblast growth factor (rhAFGF), wherein each 100 μg of recombinant human acidic fibroblast growth factor (rhAFGF) stock solution 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 maintainer.

[0015] Furthermore, the present invention provides a lyophilized formulation of recombinant human acidic fibroblast growth factor, wherein each 100 μg of recombinant human acidic fibroblast growth factor (rhAFGF) or recombinant human acidic fibroblast growth factor (rhAFGF) stock solution 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 retainer.

[0016] Furthermore, the present invention provides a method for preparing a lyophilized formulation of recombinant human acidic fibroblast growth factor, the method comprising the following steps:

[0017] (a) Mix the prescribed amounts of excipients, protein protectants, pH buffers and lyophilization preservers thoroughly, and dissolve them in water for injection;

[0018] (b) Add the prescribed amount of recombinant human acidic fibroblast growth factor and bring to a final volume.

[0019] (c) Filter, then transfer the filtrate to a container for filling, and then freeze-dry to obtain a freeze-dried formulation.

[0020] Furthermore, the present invention provides a lyophilized formulation of recombinant human acidic fibroblast growth factor prepared by the above method.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. The product prepared by this invention has high stability and remains stable during vibration or shaking. The freeze-dried formulation has a certain degree of adhesion to the bottle wall and is not easily broken into powder, thus avoiding or reducing static electricity generated by friction between the powder and the bottle wall during storage and transportation.

[0023] 2. The method of the present invention has a high collapse retention rate during the preparation process, resulting in a product with good appearance and high yield. Detailed Implementation

[0024] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below in conjunction with specific embodiments.

[0025] The embodiments are intended to illustrate the present invention. Fine adjustments to the relative proportions of active ingredients and excipients are all within the scope of protection of the present invention. Lyophilized formulations that are scaled up or down proportionally according to the prescription proportions are all within the scope of protection of the present invention.

[0026] The rhAFGF stock solution used in this invention is from Shanghai Tengrui Pharmaceutical Co., Ltd., and the rhAFGF stock solution contains 2.93 mg / ml of rhAFGF protein (specific activity of 2.9 × 10⁻⁶). 5 U / mg).

[0027] Example 1

[0028] Freeze-dried prescription:

[0029]

[0030] Weigh the materials according to the prescribed ratio. Add mannitol, disodium hydrogen phosphate, sodium dihydrogen phosphate, and polyacrylamide to the vials in that order. Add 50% water for injection and shake to dissolve, obtaining diluent 1. Add rhAFGF stock solution and human serum albumin according to the prescribed ratio, and dilute to volume with the remaining water for injection to obtain a semi-finished product. Then, filter through two 0.22µm PVDF sleeve filters, fill the vials, and partially seal with sterilized and dried rubber stoppers. The vials are then fed into a freeze dryer via an automatic feeding and discharging system. The freezing parameters are as follows, yielding the lyophilized formulation:

[0031]

[0032] Example 2:

[0033] Freeze-dried prescription:

[0034]

[0035] The freeze-drying process is as described in Example 1.

[0036] Example 3:

[0037] Freeze-dried prescription:

[0038]

[0039] The freeze-drying process is as described in Example 1.

[0040] Comparative Example 1:

[0041] The freeze-drying formulation and freeze-drying process parameters are the same as in Example 2, except that the freeze-drying formulation does not contain the freeze-drying agent polyacrylamide.

[0042] Comparative Example 2:

[0043] The freeze-drying formulation and freeze-drying process parameters are the same as in Example 2, except that the relative amount of polyacrylamide, the freeze-drying agent, in the freeze-drying formulation is adjusted to 0.5 mg.

[0044] Comparative Example 3:

[0045] The freeze-drying formulation and freeze-drying process parameters are the same as in Example 2, except that the relative amount of polyacrylamide, the freeze-drying agent, in the freeze-drying formulation is 5 mg.

[0046] Comparative Example 4:

[0047] The freeze-drying formulation and freeze-drying process parameters are the same as in Example 2, except that after filtration, the filtrate is directly cooled to -35°C, the vacuum freeze-drying device is turned on, and the freeze-dried preparation is obtained; then the temperature is raised to 25°C and vacuum dried to constant weight.

[0048] Test Example 1: Retention Test of Lyophilized Formulations

[0049] The test laboratory floor consisted of a 2 mm thick PVC floor covering laid on a regular cement floor using an adhesive method. Ten portions of each freeze-dried formulation prepared in each example and comparative example were placed on a 100 cm high stainless steel table and dropped ten times from the table to the ground. The shape of the freeze-dried formulation was observed, as described in Table 1 below.

[0050] Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Properties One sample had a minor crack, the others were intact. 10 samples intact 10 samples intact 10 samples were made into powder. Six samples turned into powder, and four samples were severely broken. 10 samples intact 10 samples intact

[0051] Test Example 2: Relative Activity Test

[0052] The lyophilized formulations prepared in Examples 1-3 and Comparative Examples 1-4 were stored in a sealed container at 40°C for 6 months. Water for injection was added to bring the volume to 2 ml, and the mixture was thoroughly shaken until dissolved. Recombinant human acidic fibroblast growth factor (AIF) previously filled into vials was also taken. The activities of the lyophilized formulations prepared in Examples 1-3 and Comparative Examples 1-4, and the recombinant human AIF before lyophilization, were tested respectively. Taking the activity of recombinant human AIF before lyophilization as 100%, the relative activity of the lyophilized formulations prepared in Examples 1-3 and Comparative Examples 1-4 after being stored in a sealed container at 40°C for 6 months was tested according to the following method.

[0053] The bioactivity of recombinant human acidic fibroblast growth factor (AGF) was determined using the MTT assay. Balb / c 3T3 cells were used, with Promega's rhaFGF as the standard. Balb / c 3T3 cells in logarithmic growth phase were adjusted to a concentration of 7000 cells / 100 μl in 96-well plates. The cells were cultured at 37°C and 5% CO2 for 24 h, then the medium was changed to 1640 medium containing 0.4% fetal bovine serum, followed by 24 h of starvation culture. The above solution was then diluted 4-fold and added to each well in parallel groups of 3 wells. After 48–72 h of culture, 20 ml of MTT (5 mg / ml) was added, and the cells were cultured for 4 h. The culture medium was then discarded, and 150 μl of DMSO was added to each well. After incubation at room temperature for 40 min, the a value (A value) was measured at 570 nm. A curve of aFGF concentration based on the A value was plotted, and the relative activity was calculated, as shown in Table 2 below.

[0054] 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%

[0055] The above data show that the freeze-dried product prepared by this invention still maintains high biological activity after being stored in a sealed container at 40°C for 6 months.

[0056] Test Example 3: Reconstitution Time Test

[0057] Take the lyophilized formulations prepared in Examples 1-3 and Comparative Examples 1-4, add 10 ml of 10 mmol / L phosphate buffer (pH 7.0), shake manually, and use a stopwatch to time the time until the solution becomes clear and transparent, as shown in Table 3:

[0058] Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Dissolution time 19 seconds 20 seconds 23 seconds 16 seconds 17 seconds 45 seconds 21 seconds

[0059] The above data shows that the proportion of lyophilization stabilizer and lyophilization parameters can affect the reconstitution time of lyophilized formulations.

[0060] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention 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; The excipients are selected from one or more of histidine, mannitol, trehalose, sucrose, sorbitol, maltose, and lactose. The protein protectant mentioned is selected from human serum albumin; The freeze-dried formulation retainer is selected from polyacrylamide; The aforementioned p H buffer is selected from a combination of dihydrogen phosphate and dihydrogen phosphate; The lyophilized powder formulation contains, for every 100 μg of recombinant human acidic fibroblast growth factor (rhAFGF) stock solution, 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 maintainer.

2. The lyophilized powder formulation of Evgiev according to claim 1, characterized in that... The pH buffer is selected from a combination of dihydrogen phosphate and dihydrogen phosphate, wherein 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.

3. The lyophilized powder formulation of Evgeny according to claim 1, characterized in that... The lyophilized powder formulation contains, for every 100 μg of recombinant human acidic fibroblast growth factor (rhAFGF) stock solution, 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 maintainer.

4. The lyophilized powder formulation of Evgeny according to claim 1, characterized in that... The lyophilized powder 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.

5. The method for preparing the Evgiev lyophilized powder formulation according to any one of claims 1-4, the method comprising the following steps: (a) The prescribed amount of excipients, p The H buffer and lyophilization retainer are mixed thoroughly and dissolved in water for injection. (b) Add the prescribed amount of recombinant human acidic fibroblast growth factor and protein protectant, 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 powder formulation.

6. The Evgiev lyophilized powder formulation prepared according to claim 5.

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