High-activity hirudin freeze-drying preparation as well as preparation method and application thereof

Abstract

The invention provides a high-activity hirudin freeze-drying preparation as well as a preparation method and application thereof, and the preparation method comprises the following steps: (1) introducing a hirudin gene to a chassis cell to construct a recombinant engineering strain; (2) inoculating the engineering strain into a prepared culture medium, carrying out fermentation culture at a set temperature, after the thallus density reaches a target density, switching to methanol induced expression, and after continuous induction is carried out for a preset time, extracting a fermentation liquid; (3) performing ultrafiltration concentration and chromatographic purification on the obtained fermentation liquor to obtain a purified hirudin solution; and (4) adding a stabilizer into the obtained hirudin solution, pre-freezing to completely cure the hirudin solution, and drying to obtain the target hirudin freeze-dried preparation. According to the invention, the high-activity, high-purity, high-stability and low-cost industrial production of the hirudin freeze-drying preparation is realized through collaborative optimization of genetic engineering strain construction, high-density fermentation, multi-step purification and freeze-drying processes.

Description

Technical Field

[0001] This invention relates to the field of hirudin preparation technology, and in particular to a highly active hirudin freeze-dried formulation, its preparation method and application. Background Technology

[0002] Hirudin, the most potent thrombin-specific inhibitor discovered to date, exhibits antithrombotic activity 12-15 times greater than heparin (Markwardt et al., 1989). According to a 2024 WHO report, there are over 520 million cardiovascular disease patients worldwide, requiring an annual dose of 380 tons of anticoagulants. Natural hirudin, due to the absence of thrombocytopenia risk, is considered a preferred alternative to heparin. However, traditional leech extraction methods yield only 1 mg of pure product from 1000 medical leeches (Chinese Pharmaceutical Journal, 2023), resulting in low yields that cannot meet clinical needs. Summary of the Invention

[0003] The purpose of this invention is to provide a highly active hirudin freeze-dried formulation, its preparation method and application, which can achieve high activity, high purity, high stability and low cost industrial production of hirudin freeze-dried formulation.

[0004] To achieve the above objectives, the present invention provides the following technical solution:

[0005] In a first aspect, the present invention relates to a method for preparing a highly active hirudin lyophilized formulation, comprising the following steps:

[0006] (1) The hirudin gene was introduced into the chassis cells to construct a recombinant engineered strain;

[0007] (2) The engineered strain is inoculated into the prepared culture medium and fermented at a set temperature. After the cell density reaches the target density, the expression is switched to methanol-induced expression and the fermentation broth is extracted after a preset induction time.

[0008] (3) The obtained fermentation broth was concentrated by ultrafiltration and purified by chromatography to obtain a purified hirudin solution;

[0009] (4) Add a stabilizer to the obtained hirudin solution, pre-freeze to completely solidify the hirudin solution, and obtain the target hirudin freeze-dried formulation after drying.

[0010] Further settings: Step (1) includes:

[0011] Obtain the hirudin gene and select a compatible vector based on the chassis cells to carry the hirudin gene;

[0012] The vector carrying the hirudin gene was used to form a recombinant plasmid, which was then introduced into the chassis cells to screen and verify recombinant engineered strains that expressed hirudin protein.

[0013] Further steps: In the recombinant plasmid formed by the vector carrying the hirudin gene, the hirudin gene is inserted into the vector by restriction endonuclease or Gibson assembly. The vector is then transformed into competent cells for plasmid amplification, and sequencing is used to verify the correctness of the inserted hirudin gene sequence.

[0014] Further steps: The recombinant plasmid is introduced into the chassis cells using electroporation or thermal shock, wherein the chassis cells include at least one of yeast, Escherichia coli, Bacillus subtilis, and cyanobacteria.

[0015] Further setup: In step (2), the engineered strain is placed in a sterilized fermenter for fermentation. After sterilization, the fermenter is cooled to 25-28°C before the engineered strain is introduced. The engineered strain is introduced at 5%-10% of the fermenter volume. The fermentation continues until the cell density reaches OD200. 600 When the concentration is 20-30, switch to methanol-induced expression. During the methanol-induced phase, the residual methanol concentration is controlled to be 0.5-1% by feeding, and the dissolved oxygen is maintained at 20-30%.

[0016] Further steps: In step (3), the extracted fermentation broth is first centrifuged and filtered at 0.45 μm, then concentrated using an ultrafiltration membrane with a molecular weight cutoff of 3-10 kDa, and then purified in two steps by cation exchange chromatography and heparin affinity chromatography to obtain a hirudin solution with a purity greater than or equal to the target purity.

[0017] Further configuration: The stabilizer comprises mannitol at a volume content of 5-10% and sucrose at a volume content of 1-5%.

[0018] Further steps: In step (4), the hirudin solution with added stabilizer is pre-frozen to -40°C, and then dried once at -20°C and twice at 25°C to obtain the freeze-dried formulation.

[0019] As a second aspect, the present invention relates to a highly active hirudin freeze-dried formulation, which is prepared by the above-mentioned method for preparing highly active hirudin freeze-dried formulation.

[0020] As a third aspect, the application of a highly active hirudin freeze-dried formulation as described above in medical aesthetic preparations.

[0021] Compared with the prior art, the solution of the present invention has the following advantages:

[0022] 1. In the preparation method of the highly active hirudin freeze-dried formulation involved in this invention, the high activity, high purity, high stability and low cost industrial production of the hirudin freeze-dried formulation are achieved through the synergistic optimization of genetically engineered strain construction, high-density fermentation, multi-step purification and freeze-drying process.

[0023] 2. In the preparation method of the highly active hirudin freeze-dried formulation involved in this invention, the activity and purity of the obtained hirudin are significantly improved by efficient expression through genetic engineering and two-step chromatographic purification, and the batch stability is high with an activity difference of <±5%, which is far lower than the instability of natural extraction methods.

[0024] 3. In the preparation method of the highly active hirudin freeze-dried preparation involved in this invention, the production cycle can be greatly shortened by fermentation technology, the production capacity is controllable, the raw materials used are low cost and renewable, and are not limited by leech resources, thus having good advantages for large-scale production.

[0025] 4. In the preparation method of the highly active hirudin freeze-dried formulation involved in this invention, the chassis cell and carrier system are mature and can be quickly adapted to the production of other recombinant proteins.

[0026] 5. In the preparation method of the highly active hirudin freeze-dried formulation involved in this invention, the use of freeze-drying technology to preserve hirudin can efficiently retain active ingredients, improve product quality, extend shelf life and improve production efficiency.

[0027] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and will become apparent from the description or may be learned by practice of the invention. Attached Figure Description

[0028] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments taken in conjunction with the accompanying drawings, wherein:

[0029] Figure 1 This is a process flow diagram of a method for preparing a highly active hirudin freeze-dried formulation according to the present invention. Detailed Implementation

[0030] Embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0031] Those skilled in the art will understand that, unless specifically stated otherwise, the terms "described" and "the" as used herein may also include plural forms. It should be further understood that the term "comprising" as used in this specification means the presence of the described features, parts, and / or components, but does not exclude implementation as other features, parts, components, and / or combinations thereof supported by this art. The term "and / or" as used herein refers to at least one of the items defined by the term; for example, "A and / or B" can be implemented as "A," or as "B," or as "A and B."

[0032] In the description of this invention, the terms "center," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," and "outer," etc., indicate directions or positional relationships based on exemplary directions or positional relationships shown in the accompanying drawings. They are used to facilitate the description or simplification of embodiments of the invention and are not intended to indicate or imply that the device or component referred to must have a specific orientation or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the invention.

[0033] The terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0034] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0035] Example 1

[0036] This embodiment relates to a method for preparing a highly active hirudin freeze-dried formulation. Please refer to... Figure 1 It includes the following steps:

[0037] (1) The hirudin gene was introduced into the chassis cells to construct a recombinant engineered strain.

[0038] First, the hirudin gene can be obtained. Specifically, mRNA can be extracted from leech salivary gland cells using the natural gene extraction method, and cDNA can be synthesized through reverse transcription to obtain the hirudin gene sequence. Alternatively, the hirudin gene can be amplified using chemical synthesis methods and PCR technology.

[0039] Simultaneously, a compatible vector is selected based on the chassis cells to carry the hirudin gene. The chassis cells in this invention can be at least one of yeast, Escherichia coli, Bacillus subtilis, and cyanobacteria. When selecting a vector, promoter type, selection markers, protein secretion signals, or vector copy number need to be considered. The vector carrying the hirudin gene is used to form a recombinant plasmid. The hirudin gene is inserted into the vector using restriction endonuclease or Gibson assembly. The plasmid is then amplified by transforming competent cells. Specifically, competent cells can be transformed using chemical transformation or electrotransformation. Single clones are then selected and inoculated into antibiotic culture medium, cultured at 37°C with shaking at 200 rpm for 12-16 hours. The recombinant plasmid is extracted using a plasmid extraction kit to complete plasmid amplification. Sequencing is then used to verify the correctness of the inserted hirudin gene sequence, ensuring the accuracy of the vector construction.

[0040] Recombinant plasmids can be introduced into chassis cells, and hirudin-engineered strains can be constructed using chemical transformation and electrotransformation methods. Then, recombinant engineered strains expressing hirudin protein can be screened and verified. When screening recombinant engineered strains, methods such as antibiotic screening, blue-white screening, colony PCR, and enzyme digestion identification can be used. Hirudin protein expression can then be verified by SDS-PAGE detection and Western blot verification.

[0041] The constructed recombinant engineered strain was stored at -80°C.

[0042] (2) The engineered strain is inoculated into the prepared culture medium and fermented at a set temperature. After the cell density reaches the target density, the expression is switched to methanol and the fermentation broth is extracted after a preset induction time.

[0043] The culture medium raw materials in this embodiment include C 5 / 6 Glucose, C3 glycerol, C1 methane and their derivatives, etc., were used. The culture medium was then sterilized at 121℃. The engineered strain was removed from -80℃, placed at -20℃ for 30 minutes, and then slowly thawed on ice. The thawed engineered strain was then inoculated into the culture medium for initial propagation at an inoculation rate of 1%-5%. The engineered strain was then placed in a sterilized fermenter for fermentation. After sterilization, the fermenter was cooled to 25-28℃ before inoculating with the engineered strain at a rate of 5%-10% of the fermenter volume. Fermentation continued until the cell density reached the OD value. 600 When the concentration is 20-30, switch to methanol-induced expression. During the methanol-induced phase, the residual methanol concentration is controlled to be 0.5-1% by feeding, and the dissolved oxygen is maintained at 20-30%.

[0044] (3) The fermentation broth obtained in step (2) is concentrated by ultrafiltration and purified by chromatography to obtain a purified hirudin solution.

[0045] The extracted fermentation broth is first pretreated by centrifuging at 8000-12000 rpm for 15-20 minutes to remove solid impurities such as bacterial cells and cell debris. Then, a 0.45μm microporous membrane or depth filter is used to further remove suspended particles, preventing clogging of the ultrafiltration membrane in subsequent applications. Additionally, polyacrylamide (PAM) or chitosan can be added to aggregate tiny particles through charge neutralization or bridging, thereby improving solid-liquid separation efficiency.

[0046] When concentrating the fermentation broth by ultrafiltration, an ultrafiltration membrane with a molecular weight cutoff of 3-10 kDa is selected. Under a pressure of 0.5-1.5 bar, the transmembrane pressure is controlled at 0.2-0.5 bar to allow the fermentation broth to pass through the ultrafiltration membrane until the volume is reduced by 5-10 times. Then, the membrane surface is rinsed with pure water to remove residual protein. The broth is then disinfected by circulating a 0.1 mol / L sodium hydroxide solution for 30 minutes. Finally, the pH of the fermentation broth is balanced with a starting buffer.

[0047] For chromatographic purification, a two-step process of cation exchange chromatography and heparin affinity chromatography was employed. Cation exchange chromatography served as the first step, using SP Sepharose Fast Flow or CM Sepharose Fast Flow as the chromatographic medium. The buffer solution was a 20 mmol / L phosphate solution with a pH of 6.0. During sample loading, the ultrafiltration concentrate was diluted to a conductivity ≤5 mS / cm, and the flow rate was 1-2 mL / min. Subsequently, heparin affinity chromatography was performed using Heparin Sepharose 6Fast Flow as the chromatographic medium. The high affinity of heparin for hirudin allows for specific binding, thereby further purifying the fermentation broth to obtain hirudin with a purity greater than or equal to the target purity.

[0048] The target purity of hirudin in this invention is 98%. Hirudin with a purity greater than or equal to 98 has significant effects in anticoagulation, thrombolysis, improving microcirculation, and synergistic repair of blood vessels.

[0049] (4) Add a stabilizer to the obtained hirudin, pre-freeze to completely solidify the hirudin, and obtain the target hirudin freeze-dried formulation after drying.

[0050] By adding stabilizers, pre-freezing and freeze-drying, a freeze-dried preparation of hirudin with stable activity and easy storage and transportation can be prepared. In this embodiment, the stabilizers include mannitol at a volume content of 5-10% and sucrose at a volume content of 1-5%, with the stabilizer added at a ratio of 10%-30% of the hirudin's weight.

[0051] By pre-freezing, the liquid hirudin is converted into a solid state. During drying, the active ingredients will inevitably migrate with the moisture. In this embodiment, the pre-freezing temperature is -40°C and is maintained at this temperature for 2-4 hours to ensure that the hirudin is completely solidified.

[0052] The solidified hirudin was freeze-dried. First, it was dried at -20°C to remove more than 90% of the moisture. Then, it was dried a second time at 25°C. Finally, the complete drying of hirudin was confirmed by pressure rise test. The residual moisture of the resulting freeze-dried formulation was ≤3%, the reconstitution time was ≤30 seconds, and the specific activity was ≥20000 ATU / g. The activity loss was <5% after 24 months of storage at 4°C.

[0053] The highly active hirudin freeze-dried formulation prepared according to the above-described method can achieve an activity of 20,000 ATU / g, which is 10-20 times that of the natural extraction method, with batch-to-batch activity differences of <±5%. Because the preparation method of the highly active hirudin freeze-dried formulation of this invention utilizes genetic engineering and undergoes multi-step purification, it effectively eliminates the risk of allergies and activity interference caused by impurities. Furthermore, the large-scale fermentation production method is not constrained by natural resources, has a short production cycle, effectively reduces production costs, and ensures stable and controllable prices, making it suitable for large-scale commercial demand. This invention uses gene editing combined with fermentation technology to produce hirudin, resulting in low waste emissions, low carbon emissions, and renewable raw materials.

[0054] Examples of the above-mentioned highly active hirudin freeze-dried preparations preserved under different conditions were compared with hirudin extracted using natural extraction methods.

[0055] Example 1

[0056] Reconstitute the hirudin lyophilized preparation stored at 4℃ for 24 months: Under sterile conditions, draw 1ml of physiological saline and inject it into the lyophilized preparation bottle containing 10mg of hirudin lyophilized preparation. Gently tap the bottle to wet the hirudin lyophilized preparation with solvent, let it stand for 1 minute, and slowly rotate the bottle until the lyophilized powder is completely dissolved. Check the clarity of the solution and use it only after confirming that there are no particles.

[0057] Example 2

[0058] Reconstitute the hirudin lyophilized preparation stored at 25℃ for 24 months: Under sterile conditions, draw 1ml of physiological saline and inject it into the lyophilized preparation bottle containing 10mg of hirudin lyophilized preparation. Gently tap the bottle to wet the hirudin lyophilized preparation with solvent, let it stand for 1 minute, and slowly rotate the bottle until the lyophilized powder is completely dissolved. Check the clarity of the solution and use it only after confirming that there are no particles.

[0059] Example 3

[0060] For reconstitution of hirudin lyophilized preparations stored at 4℃ for less than 1 month: Under sterile conditions, draw 1ml of physiological saline and inject it into a lyophilized preparation bottle containing 10mg of hirudin lyophilized preparation. Gently tap the bottle to wet the hirudin lyophilized preparation with solvent, let it stand for 1 minute, and slowly rotate the bottle until the lyophilized powder is completely dissolved. Check the clarity of the solution and use it only after confirming that there are no particles.

[0061] Example 4

[0062] For reconstitution of hirudin lyophilized preparations stored at 25℃ for less than 1 month: Under sterile conditions, draw 1ml of physiological saline and inject it into a lyophilized preparation bottle containing 10mg of hirudin lyophilized preparation. Gently tap the bottle to wet the hirudin lyophilized preparation with solvent, let it stand for 1 minute, and slowly rotate the bottle until the lyophilized powder is completely dissolved. Check the clarity of the solution and use it only after confirming that there are no particles.

[0063] Comparative Examples

[0064] Hirudin is extracted using a natural extraction method: Fresh or dried leeches that have been killed are prepared as raw materials. The fresh or dried leeches are homogenized or pulverized, and then soaked in an appropriate amount of clean water for 1-3 hours. Impurities are removed by centrifugation, and the supernatant is collected. The supernatant is further purified by acid precipitation or other methods. The pH is adjusted to near neutral to ensure a suitable solution environment for subsequent processing. Then, the salt in the solution is removed and concentrated. Hirudin is further purified by high performance liquid chromatography (HPLC) or reversed-phase high performance liquid chromatography (RP-HPLC). Appropriate excipients are added to the hirudin and it is dried to finally obtain pure natural hirudin.

[0065] This invention uses the thrombin titration method to determine the specific activity of hirudin. Specifically, the activity unit (ATU) is calculated by measuring the ability of hirudin to neutralize thrombin. 1 g of hirudin neutralizes 5 g of thrombin (molar ratio 1:1) and is defined as 1 ATU. Hirudin sample is added to a test tube, followed by a buffer solution containing fibrinogen. The tube is incubated at 37°C for 5 minutes, and thrombin solution is added dropwise (5 μl per minute). The volume of thrombin consumed during coagulation is recorded.

[0066] Computational activity: Where U is ATU / g, C1 is thrombin concentration, V1 is the volume consumed, C2 is the sample concentration, and V2 is the sample volume.

[0067] The above experimental steps need to be repeated 3 times, and the average value should be taken to reduce error, compared with the activity assay.

[0068] The results are shown in the table below:

[0069]

[0070] The highly active hirudin prepared using the freeze-dried hirudin formulation method of this invention exhibits a significantly higher specific activity than fresh hirudin extracted using natural extraction methods, thanks to gene optimization and a highly efficient expression system. Natural extraction methods are prone to activity loss during the extraction process. Furthermore, low-temperature preservation is crucial for maintaining hirudin activity, while room temperature storage is only suitable for short-term temporary storage.

[0071] Application Examples: Application of Highly Active Hirudin Lyophilized Formulations in the Field of Medical Aesthetics

[0072] A skin protectant was prepared, comprising, by weight percentage, 40-60% hirudin lyophilized preparation and 40-60% hydroxypropyl cyclodextrin. The resulting product is a white to pale yellow powder, suitable for use in various cosmetics. The skin protectant was evaluated and found that, under normal, reasonable, and foreseeable use, its recommended addition level in cosmetics is ≤0.3%, and it will not pose any harm to human health. The core component of this skin protectant, hirudin, possesses highly active anticoagulant molecules. When applied to the skin surface, it can improve impaired microcirculation and effectively inhibit scar formation by reducing excessive collagen proliferation during the skin repair process. When applied to eye products, it can significantly reduce fine lines, dark circles, and puffiness; when applied to facial products, it can significantly reduce fine lines and brighten skin tone.

[0073] Therefore, the above-mentioned skin protectant can be applied to skin care products such as face masks and eye creams. Face masks made with this skin protectant formula can solve the problems of skin pigmentation and spots, and eye creams made with this skin protectant formula can target dark circles caused by staying up late and can save "stay-at-home face".

[0074] The above description is only a partial embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a highly active hirudin lyophilized formulation, characterized in that, Includes the following steps: (1) The hirudin gene was introduced into the chassis cells to construct a recombinant engineered strain; (2) The engineered strain is inoculated into the prepared culture medium and fermented at a set temperature. After the cell density reaches the target density, the expression is switched to methanol-induced expression and the fermentation broth is extracted after a preset induction time. (3) The obtained fermentation broth was concentrated by ultrafiltration and purified by chromatography to obtain a purified hirudin solution; (4) Add a stabilizer to the obtained hirudin solution, pre-freeze to completely solidify the hirudin solution, and obtain the target hirudin freeze-dried formulation after drying.

2. The method for preparing the highly active hirudin freeze-dried formulation according to claim 1, characterized in that, Step (1) includes: Obtain the hirudin gene and select a compatible vector based on the chassis cells to carry the hirudin gene; The vector carrying the hirudin gene was used to form a recombinant plasmid, which was then introduced into the chassis cells to screen and verify recombinant engineered strains that expressed hirudin protein.

3. The method for preparing the highly active hirudin freeze-dried formulation according to claim 2, characterized in that, In the recombinant plasmid formed by the vector carrying the hirudin gene, the hirudin gene is inserted into the vector by restriction endonuclease or Gibson assembly. The vector is then transformed into competent cells for plasmid amplification, and sequencing is used to verify the correctness of the inserted hirudin gene sequence.

4. The method for preparing the highly active hirudin freeze-dried formulation according to claim 3, characterized in that, The recombinant plasmid was introduced into chassis cells using electroporation or thermal shock methods. The chassis cells included at least one of yeast, Escherichia coli, Bacillus subtilis, and cyanobacteria.

5. The method for preparing the highly active hirudin lyophilized formulation according to claim 1, characterized in that, In step (2), the engineered strain is placed in a sterilized fermenter for fermentation. After sterilization, the fermenter is cooled to 25-28°C before the engineered strain is introduced. The engineered strain is introduced at 5%-10% of the fermenter volume. The fermentation continues until the cell density reaches OD200. 600 When the concentration is 20-30, switch to methanol-induced expression. During the methanol-induced phase, the residual methanol concentration is controlled to be 0.5-1% by feeding, and the dissolved oxygen is maintained at 20-30%.

6. The method for preparing the highly active hirudin lyophilized formulation according to claim 1, characterized in that, In step (3), the extracted fermentation broth is first centrifuged and filtered at 0.45 μm, then concentrated using an ultrafiltration membrane with a molecular weight cutoff of 3-10 kDa, and then purified in two steps by cation exchange chromatography and heparin affinity chromatography to obtain a hirudin solution with a purity greater than or equal to the target purity.

7. The method for preparing the highly active hirudin lyophilized formulation according to claim 1, characterized in that, The stabilizer comprises 5-10% mannitol by volume and 1-5% sucrose by volume.

8. The method for preparing the highly active hirudin lyophilized formulation according to claim 1, characterized in that, In step (4), the hirudin solution with added stabilizer is pre-frozen to -40°C, and then dried once at -20°C and twice at 25°C to obtain the freeze-dried formulation.

9. A highly active hirudin freeze-dried formulation, prepared by the method for preparing the highly active hirudin freeze-dried formulation according to any one of claims 1-8.

10. The application of a highly active hirudin freeze-dried formulation as described in claim 9 in medical aesthetic preparations.

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