A composition containing recombinant protein of engineered bacteria and application thereof in preparation of medicine for preventing and / or treating plague

Abstract

The application provides a composition containing recombinant protein of an engineered bacterium and application of the composition in preparation of a medicine for preventing and / or treating plague, and comprises: a) 0.05-0.2 mass parts of the recombinant protein, wherein the recombinant protein is obtained after fermentation, cell disruption and ion exchange chromatography purification of the genetically engineered bacterium, and has biological activity of resisting plague-related pathogenic microorganisms. The application also provides a freeze-dried composition containing the recombinant protein, which is suitable for a preparation process of the recombinant protein medicine, and can improve the stability, targeting and action persistence of the preparation, and the safety of the preparation meets the regulatory requirements.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a composition containing engineered bacterial recombinant protein and its application in the preparation of drugs for the prevention and / or treatment of epidemics. Background Technology

[0002] Plagues are major infectious diseases that can spread widely in a short period of time and pose a serious threat to human health. They are characterized by high infectivity, rapid transmission, and high mortality rates, and have always been a major challenge to global public health. From a pathogenic perspective, plagues can be caused by a variety of pathogenic microorganisms, including bacteria and viruses. While bacterial plagues, such as plague caused by Yersinia pestis, can be partially controlled with antibiotics and traditional vaccines, problems remain, such as the increasing prevalence of antibiotic resistance, short-term immunity of traditional vaccines, and limited protective efficacy.

[0003] In recent years, recombinant protein drugs have shown great potential in the field of epidemic prevention and control due to their strong targeting and well-defined mechanisms of action. Recombinant proteins are typically produced through fermentation expression by genetically engineered bacteria, with the bacteria serving merely as the "fermentation tank" for protein synthesis. Ultimately, high-purity protein products must be obtained through lysis and purification. However, the development of recombinant protein drugs still faces several bottlenecks: proteins are prone to inactivation and aggregation during separation, purification, and formulation storage, resulting in insufficient stability; their short in vivo half-life necessitates frequent administration; and the residual endotoxins after fermentation by engineered bacteria must be strictly controlled, otherwise, safety risks may arise.

[0004] In existing technologies, some approaches attempt to directly formulate live engineered bacteria into drug delivery products, relying on the bacteria to continuously produce drugs in vivo. However, this route has significant drawbacks. Engineered bacteria often carry potent promoters and lysogenic cleavage genes, and the endotoxin background is difficult to completely eliminate. Whole-particle administration poses serious safety risks and is difficult to obtain regulatory approval. On the other hand, the bacteria are used as "drug production tools" rather than end products, which is completely inconsistent with the process chain of recombinant protein drugs, which is "centered on protein purification," and cannot meet the needs of industrial-scale production. Therefore, there is an urgent need to develop a recombinant protein lyophilized formulation adapted to the "engineered bacteria expression-lysis-purification" process to solve key issues such as protein stability, targeting, duration of action, and safety. Summary of the Invention

[0005] To overcome the deficiencies in the prior art, a composition containing engineered bacterial recombinant protein is provided, and its use in the preparation of medicaments for the prevention and / or treatment of epidemics is provided.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A composition containing recombinant protein from engineered bacteria, comprising: a) 0.05-0.2 parts by weight of recombinant protein, wherein the recombinant protein is obtained by fermentation expression, cell disruption, and ion exchange chromatography purification of genetically engineered bacteria, and has bioactivity against plague-related pathogenic microorganisms; b) 8-35 parts by weight of modified lyophilization protectant, wherein the modified lyophilization protectant is composed of trehalose and sucrose in a mass ratio of 1.5:1-3:1; c) 0-20 parts by weight of sustained-release microsphere carrier, wherein the sustained-release microsphere carrier is composed of polylactic acid-glycolic acid copolymer; d) 0-15 parts by weight of targeted modification excipient, wherein the targeted modification excipient is selected from folic acid-chitosan conjugate and / or RGD polypeptide-hyaluronic acid complex; wherein the lyophilized powder has a particle size distribution D50 of 30-60 µm, a rehydration time ≤3 min, and does not contain intact viable bacterial cells.

[0007] Preferably, the recombinant protein is one or more of a superantigen, cytokine, single-domain antibody, or nanobody.

[0008] Preferably, the fermentation stage of the genetically engineered bacteria uses the T7 promoter or the λPL promoter, and the bacterial cells are collected 2–6 h after induction of expression.

[0009] Preferably, the bacterial cell disruption is performed using a high-pressure homogenization process, and the endotoxin content of the purified recombinant protein is controlled to be <10 EU / mg protein.

[0010] Preferably, after purification by ion exchange chromatography, the purity of the recombinant protein is ≥95%, and it shows a single peak when detected by SEC-HPLC.

[0011] Preferably, the modified lyophilization protectant further comprises 0.5–2% poloxamer 188 for inhibiting recombinant protein aggregation.

[0012] Preferably, the sustained-release microspheres encapsulate the recombinant protein using a w / o / w double emulsion method, with an encapsulation rate ≥80% and a cumulative release amount ≤60% in vitro over 72 hours.

[0013] Preferably, the targeted modification excipient is combined with the surface of the sustained-release microspheres through electrostatic adsorption or chemical coupling.

[0014] Preferably, after the formulation is placed at 40°C and 75% relative humidity for 1 month, the recombinant protein activity retention rate is ≥90%.

[0015] The present invention also provides an application of the composition for the preparation of drugs for the prevention and / or treatment of plague-related diseases such as plague, anthrax, and secondary bacterial infections of highly pathogenic influenza.

[0016] Preparation of each component of the composition and the preparation process of the composition (I) Preparation of recombinant proteins Escherichia coli strains were revived and amplified. They were fermented in LB medium at 37°C for 4-6 hours, and induced with IPTG at 30-34°C for 2-6 hours. The bacterial cells were collected after centrifugation at 10,000 rpm. After preliminary washing, the cells were homogenized and centrifuged at 10,000 rpm. The supernatant was collected, and the target protein was crudely extracted. The collected protein underwent two ion exchange cycles and buffer replacement before sample collection. The purity and content of the sample solution were determined, and recombinant protein solutions were prepared.

[0017] (II) Preparation of modified lyophilization protectant Mix trehalose and sucrose in a mass ratio of 1.5:1–3:1. If poloxamer 188 needs to be added, add it at 0.5–2% of the total mass. Stir and dissolve in deionized water to prepare a 10–20% protective agent solution. Filter and sterilize before use.

[0018] (III) Preparation of targeted modification excipients Folic acid-chitosan conjugate: The preparation steps are as follows, with the conjugate linked by amide bonds: First, dissolve chitosan in a 1% (w / v) acetic acid solution to prepare a chitosan solution with a concentration of 2–5 mg / mL. Second, mix folic acid and N-hydroxysuccinimide at a molar ratio of 1:1.2–1:1.8, add N,N-dicyclohexylcarbodiimide as a condensing agent, and activate the reaction at 25–35°C for 4–8 h to obtain an activated folic acid solution. Third, add the activated folic acid solution dropwise to the chitosan solution, with a molar ratio of folic acid to chitosan amino groups of 1:1–1:3, and react under light-protected conditions for 18–30 h. Fourth, transfer the reaction solution to a dialysis bag with a molecular weight cutoff of 8000–14000, dialyze with deionized water for 24–48 h, and freeze-dry to obtain the folic acid-chitosan conjugate.

[0019] RGD peptide-hyaluronic acid complex: linked by a thioether bond, prepared as follows: Step A, dissolve hyaluronic acid in phosphate buffer at pH 7.2–7.6 to prepare a hyaluronic acid solution with a concentration of 3–8 mg / mL; Step B, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxythiosuccinimide to the hyaluronic acid solution to activate the carboxyl groups for 30–60 minutes; Step C, add thiolated RGD peptide, with a molar ratio of thiolated RGD peptide to activated hyaluronic acid carboxyl groups of 1.8:1–3.2:1, and react under nitrogen protection for 12–24 h; Step D, purify the product by ultrafiltration centrifugation at a centrifugation force of 3000–5000×g, with a molecular weight cutoff of 10000 in the ultrafiltration tube, centrifuge 3–5 times, 15–25 minutes each time, and collect the complex.

[0020] (iv) Preparation of sustained-release microsphere carriers Preparation of polylactic acid-glycolic acid copolymer: Stage 1, lactic acid monomer and glycolic acid monomer are mixed in a molar ratio of 75:25–50:50, and 0.05–0.2% by mass of stannous octoate is added as a catalyst; Stage 2, melt polycondensation reaction is carried out at 160–190℃ and pressure <100 Pa for 8–16 h; Stage 3, the reaction product is dissolved in dichloromethane and purified by precipitation with 3–5 times the volume of methanol, and the precipitation is repeated 2–3 times; Stage 4, the product is dried in a vacuum drying oven at 40–50℃ to constant weight to obtain polylactic acid-glycolic acid copolymer; Preparation of sustained-release microspheres: Recombinant protein was encapsulated using a w / o / w double emulsion method. Polylactic acid-glycolic acid copolymer was dissolved in dichloromethane (concentration 5–10%), and the recombinant protein solution was added as the inner aqueous phase. The mixture was ultrasonically emulsified to form a primary emulsion. The primary emulsion was added to the outer aqueous phase containing emulsifier and stirred to form a double emulsion. After the organic solvent was evaporated, the microspheres were collected by centrifugation, washed, and freeze-dried for later use.

[0021] (v) Preparation process of lyophilized powder composition The recombinant protein solution is mixed with the modified lyophilization protectant, sustained-release microsphere carrier (if used), and targeted modification excipient (if used) in proportion, stirred evenly, and then subjected to a three-stage freeze-drying process: the first stage freezing temperature is -15 to -25℃, the second stage is -35 to -45℃, and the third stage is -50 to -60℃, finally obtaining a lyophilized powder composition.

[0022] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: This invention provides a lyophilized composition containing recombinant protein, which is adapted to the preparation process of recombinant protein drugs, while improving the stability, targeting and duration of action of the formulation, and ensuring that the safety meets regulatory requirements. Detailed Implementation

[0023] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] In the specific embodiments of this application, the sources of various main raw materials are briefly described as follows: Trehalose: Zhengzhou Yukong Biotechnology Co., Ltd. CAS No. 99-20-7 Sucrose: Hubei Weishi Chemical Reagent Co., Ltd., CAS No. 57-50-1 Folic acid: Hubei Weishi Chemical Reagent Co., Ltd. CAS No. 59-30-3 N-Hydroxysuccinimide: Thermo Fisher China, CAS No. 6066-82-6 N,N-Dicyclohexylcarbodiimide: Wuhan Kemic Biomedical Technology Co., Ltd., CAS No. 538-75-0 Chitosan: Shenzhen Zhongfayuan Biotechnology Co., Ltd., CAS No. 9012-76-4 Thiol-modified RGD peptide: Xi'an Qiyue Biotechnology Co., Ltd., product name: Thiol-RGD peptide cyclic peptide modification-thiol. Hyaluronic acid: Xi'an Qiyue Biotechnology Co., Ltd. CAS No. 9004-61-9 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride: Wuhan Kemike Biomedical Technology Co., Ltd. CAS No. 25952-53-8 N-Hydroxythiosuccinimide: Thermo Fisher Scientific China CAS No. 106627-54-7 Lactic acid monomers and glycolic acid monomers: Shenzhen Polymer Biochemical Technology Co., Ltd. Stannous octoate: Air Products, model number T-9 catalyst, CAS number 301-10-0 The engineered bacteria is Escherichia coli.

[0025] The *Escherichia coli* described is accessed under CCTCC NO: M 20252770. The *Escherichia coli* used in this application is *Escherichia coli* XH-0118, which was deposited on December 3, 2025, at the China Center for Type Culture Collection (CCTCC) in accordance with the Budapest Treaty on the International Recognition of Microbial Deposits for Patent Proceedings. The address of CCTCC is: Wuhan University, Wuhan, Hubei Province, China, 430072, China. The viability of the deposit was confirmed on December 10, 2025. This deposit will be extended upon request before its expiration, meeting the requirements for microbial deposit in patent proceedings.

[0026] The technical solution of this application is as follows:

[0027] Example 1 Superantigen high-dose lyophilized powder; Recombinant protein: Superantigen (SAg) was expressed using our own E. coli Shenshi XH-0118 (CCTCC M20252770), and the bacteria were harvested after induction with the T7 promoter for 4 h; High-pressure homogenization (100MPa, 4 times) + lysozyme (3 mg / g cells) was used to break the bacteria; After two-step ion exchange, an SAg solution with a purity of 97.1% and an endotoxin of 5.2 EU / mg was obtained.

[0028] Modified freeze-drying protectant: Trehalose:sucrose = 1.5:1, plus 1% poloxamer 188, 15% aqueous solution.

[0029] Sustained-release microspheres: PLGA (75:25), w / o / w reemulsification, encapsulation efficiency 82.3%.

[0030] Targeted excipients: 6 parts folic acid-chitosan + 5 parts RGD-HA.

[0031] Formula: SAg 0.2 parts + Protectant 22 parts + Microspheres 8 parts + Targeted excipients (total 11 parts) → Three-stage freeze drying (-25 ℃ → -40 ℃ → -50 ℃).

[0032] Example 2 Low-dose lyophilized superantigen powder; Except for changing the amount of SAg to 0.05 parts, the amount of protective agent to 28 parts, and the amount of microspheres to 15 parts, the rest of the process is the same as in Example 1.

[0033] The final product had a purity of 96.8%, an endotoxin content of 6.1 EU / mg, and an encapsulation rate of 81.5%.

[0034] Example 3 Recombinant protein preparation: Superantigen dual-targeting enhancement group (validating excipient synergy) Based on Example 1, the folic acid-chitosan was increased to 8 parts and RGD-HA was increased to 6 parts, while the rest remained unchanged.

[0035] The targeting efficiency increased from 78.5% to 85.2%, and the release half-life increased from 14.5 to 16.8 days, demonstrating that the dual-targeting excipient has additional benefits for the superantigen.

[0036] Proportional Design Comparative Example 1: Anti-Plague Single-Domain Antibody Group Replace SAg with "anti-plague single-domain antibody", and the rest of the formulation is completely consistent with Example 1.

[0037] Results: The activity recovery rate was 90.1%, the retention rate after 1 month of storage was 84.3%, the targeting efficiency was 46.3%, and the release half-life was 8.8 days, all of which were significantly lower than those of SAg (p<0.01).

[0038] Comparative Example 2: Cytokine Group SAg was replaced with "cytokines," and the rest was the same as in Example 1. Results: The activity recovery rate was 89.4%, the storage retention rate was 83.7%, the targeting efficiency was 48.1%, and the release half-life was 9.2 days, which were still significantly lower than SAg.

[0039] Comparative Example 3: Blank vector (without protein), containing only protectant, microspheres, and targeting excipients, confirming that the protein itself is the source of activity.

[0040] Comparative Example 4 used a superantigen without targeting excipients, omitting folic acid-chitosan and RGD-HA, and otherwise remained the same as in Example 1. The targeting efficiency decreased to 45.6%, indicating that superantigens still require synergistic excipients to maximize their advantages.

[0041] Comparative Example 5 used a superantigen but without sustained-release microspheres (PLGA microspheres omitted), otherwise the same as in Example 1. The release half-life was shortened to 5.1 days, demonstrating that the microspheres are crucial for long-lasting effects.

[0042] Comparative Example 6, single-stage freeze-drying (-50 °C one-step method), used the same formulation as Example 1, but employed single-stage freeze-drying. The activity retention rate decreased to 79.5%, the particle size distribution broadened (D50 51 µm), and the rehydration time was 63 s, all inferior to three-stage freeze-drying.

[0043] Table 1. Comparison of the superantigen formulation with other proteins (mean ± SD, n=3)

[0044]

[0045] Conclusion: Under the same formulation platform, the superantigen exhibits superior activity, stability, targeting ability, and half-life compared to anti-plague single-domain antibodies and cytokines (p < 0.01). Targeting excipients and sustained-release microspheres significantly enhance the superantigen's efficacy; both are indispensable.

[0046] The three-stage freeze-drying process can preserve the activity of superantigens to the maximum extent.

[0047] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles 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 composition containing recombinant protein from engineered bacteria, characterized in that, The product comprises: a) 0.05–0.2 parts by weight of a recombinant protein, which is obtained by fermentation expression, cell disruption, and ion exchange chromatography purification of genetically engineered bacteria, and has bioactivity against plague-related pathogenic microorganisms; b) 8–35 parts by weight of a modified lyophilization protectant, which is composed of trehalose and sucrose in a mass ratio of 1.5:1–3:1; c) 0–20 parts by weight of a sustained-release microsphere carrier, which is composed of polylactic acid-glycolic acid copolymer; d) 0–15 parts by weight of a targeted modification excipient, which is selected from folic acid-chitosan conjugate and / or RGD peptide-hyaluronic acid complex; wherein the lyophilized powder has a particle size distribution D50 of 30–60 µm, a rehydration time ≤3 min, and does not contain intact live bacteria.

2. The composition according to claim 1, characterized in that, The recombinant protein is one or more of a superantigen, cytokine, single-domain antibody, or nanobody.

3. The composition according to claim 1, characterized in that, The fermentation stage of the genetically engineered bacteria uses the T7 promoter or the λPL promoter, and the bacterial cells are collected 2–6 h after induction of expression.

4. The composition according to claim 1, characterized in that, The bacterial cells were lysed using a high-pressure homogenization process, and the endotoxin content of the purified recombinant protein was controlled to be < 10 EU / mg protein.

5. The composition according to claim 1, characterized in that, After two ion exchange chromatography analyses, the purity of the recombinant protein was ≥95%, and it showed a single peak as detected by SEC-HPLC.

6. The composition according to claim 1, characterized in that, The modified lyophilization protectant further comprises 0.5–2% poloxamer 188 for inhibiting recombinant protein aggregation.

7. The composition according to claim 1, characterized in that, The sustained-release microspheres encapsulate recombinant proteins using a w / o / w double emulsion method, achieving an encapsulation rate of ≥80% and a cumulative release of ≤60% in vitro over 72 hours.

8. The composition according to claim 1, characterized in that, The targeted modification excipients are bound to the surface of the sustained-release microspheres through electrostatic adsorption or chemical coupling.

9. The composition according to claim 1, characterized in that, After the formulation was placed at 40°C and 75% relative humidity for one month, the recombinant protein activity retention rate was ≥90%.

10. An application of the composition according to any one of claims 1-9, characterized in that, Drugs used to prepare for the prevention and / or treatment of plague-related diseases such as plague, anthrax, and secondary bacterial infections of highly pathogenic influenza.

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