A recombinant batroxobin, its pharmaceutical composition, preparation method and use

Abstract

This invention provides a recombinant batroxobin, its pharmaceutical composition, preparation method, and uses, belonging to the field of biopharmaceutical technology. The recombinant batroxobin exhibits fibrinogen-cleaving activity; the mannose number distribution of the N-glycosylated oligosaccharide chains of the recombinant batroxobin is between M9 and M15, with M9, M10, and M11 being the predominant glycoforms, and M10 being the most abundant single glycoform; in the N-glycosylated high-mannose oligosaccharide chains of the recombinant batroxobin, some oligosaccharide chains undergo phosphorylation modification; and the isoelectric point (pI) of the recombinant batroxobin is distributed in the pH range of 6.5 to 8.5. This recombinant batroxobin and its pharmaceutical composition can be used to prepare drugs for treating or preventing surgical bleeding, especially showing superior hemostatic effects compared to existing products in orthopedic surgical bleeding models.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, and more specifically, to a recombinant batroxobin, its pharmaceutical composition, preparation method, and uses. Background Technology

[0002] Batroxobin is a serine protease isolated from the venom of the Bothrops atrox snake, and it is widely used clinically due to its unique hemostatic mechanism. Unlike thrombin in the physiological coagulation process, batroxobin selectively cleaves the Aα chain of fibrinogen molecules, releasing fibrinoid A, which in turn promotes the formation and polymerization of fibrin I monomers into a non-crosslinked fibrin clot. This clot effectively stops bleeding while being easily degraded by the body's own fibrinolytic system, avoiding the risk of forming a stable thrombus.

[0003] Currently, commercially available batroxobin products, such as hemocoagulase, are all obtained through extraction and purification from natural snake venom. However, this production method has inherent drawbacks. First, the composition and yield of snake venom are affected by various uncontrollable factors such as the individual snake, its age, region, season, and diet, leading to large quality fluctuations between batches of raw materials and posing a significant challenge to controlling the uniformity of drug quality. Second, snake venom contains various biologically active impurity proteins, such as hemorrhagic toxins and neurotoxins, which are difficult to completely remove during purification processes, posing potential safety hazards. Finally, the supply of natural snake venom is limited by snake farming and venom collection, creating a supply chain bottleneck.

[0004] To overcome the aforementioned problems, researchers have attempted to produce batroxobin using gene recombination technology. For example, Chinese patent CN108738322B discloses a recombinant batroxobin expressed in Pichia pastoris, wherein the N-glycosylated oligosaccharide chain is mainly a glycoform containing 11 to 13 mannose molecules (M11 to M13), the isoelectric point pI is not less than 7.4, and this recombinant batroxobin is used to prepare external hemostatic pads or hemostatic powders.

[0005] However, existing technologies still have shortcomings. First, current recombinant batroxobin products are mainly developed as topical formulations, while there is still a huge clinical demand for batroxobin drug compositions that can be administered systemically via intravenous or intramuscular injection. Injectable formulations place much higher demands on purity, homogeneity, stability, and safety than topical formulations. Second, while existing technologies disclose recombinant batroxobins with specific glycosylation profiles and isoelectric point ranges, they do not reveal whether recombinant batroxobins with other specific post-translational modification characteristics are suitable for injection, nor do they provide a drug composition that meets injection requirements and achieves long-term stable storage. Therefore, developing a recombinant batroxobin and its drug composition with controllable quality, suitable for systemic administration, and with good stability remains a pressing technical problem to be solved in this field. Summary of the Invention

[0006] In view of this, the present invention aims to provide a recombinant batroxobin with specific physicochemical properties and post-translational modification features, as well as a pharmaceutical composition containing the same, a method for its preparation and its use, to solve the problems of limited sources of natural batroxobin, unstable quality and unsuitability of existing recombinant batroxobin for injection administration in the prior art.

[0007] To achieve the above objectives, the present invention provides a recombinant batroxobin enzyme with fibrinogen-cleaving activity. The N-glycosylated oligosaccharide chains of the recombinant batroxobin have a mannose number distribution between M9 and M15, with M9, M10, and M11 being the predominant types, and M10 having the highest relative abundance. In the N-glycosylated high-mannose oligosaccharide chains of the recombinant batroxobin, some oligosaccharide chains undergo phosphorylation modification. Furthermore, the isoelectric point (pI) of the recombinant batroxobin is distributed within the pH range of 6.5 to 8.5. This unique glycosylation profile, phosphorylation modification, and charge heterogeneity constitute the structural features that distinguish the recombinant batroxobin of the present invention from existing technologies, and lay the foundation for its excellent pharmaceutical properties.

[0008] Optionally, the recombinant batroxobin is produced using a Pichia pastoris expression system. Using Pichia pastoris as the host cell enables large-scale, high-density fermentation production of recombinant batroxobin, ensuring product supply and batch-to-batch consistency from the source.

[0009] The purity of the recombinant batroxobin is not less than 98%. High purity is an important guarantee for drug safety and efficacy, especially for injectable protein drugs, as it can significantly reduce the risk of immunogenicity and adverse reactions caused by impurity proteins.

[0010] Optionally, in the N-glycosylated high-mannose oligosaccharide chains, the relative abundance of phosphorylated oligosaccharide chains is 3% to 7% of the total N-glycosylated high-mannose oligosaccharide chains; The relative abundance was determined by 2-AB labeled N-glycan spectroscopy, using fluorescence detection and / or mass spectrometry, as a percentage of the sum of phosphorylated sugar peak areas to the total area of ​​high-mannose sugar chain peaks.

[0011] Another aspect of the present invention provides a pharmaceutical composition comprising recombinant batroxobin as described above. The pharmaceutical composition is suitable for parenteral administration and comprises pharmaceutically acceptable excipients, including buffers, protein stabilizers, isotonic modifiers, and excipients. This composition provides a stable chemical environment for the active ingredient, recombinant batroxobin, enabling its safe administration to patients via parenteral routes such as injection.

[0012] Optionally, the buffer is a phosphate buffer, the protein stabilizer is human serum albumin, the isotonic adjuster is sodium chloride, and the excipient is mannitol. This specific combination of excipients has been shown to effectively protect the structure and bioactivity of recombinant batroxobin from significant alterations during freeze-drying and long-term storage.

[0013] Optionally, the pharmaceutical composition is an injection.

[0014] In another aspect, this invention provides a method for preparing the recombinant batroxobin as described above, comprising: solid-liquid separation of a fermentation broth to collect a solution containing the recombinant batroxobin; and purification of the solution, wherein the purification steps sequentially include: cation exchange chromatography, anion exchange chromatography, and molecular sieve chromatography. This multi-step chromatographic combination enables efficient separation based on various properties of proteins, such as charge and size, thereby effectively removing host proteins, DNA, endotoxins, and other process-related impurities. Furthermore, the specific purification sequence has been optimized to obtain high-purity recombinant batroxobin that meets pharmaceutical standards with high efficiency and recovery rate.

[0015] Optionally, the method further includes, before the solid-liquid separation step, a step of inducing Pichia pastoris host cells to express and secrete the recombinant batroxobin using methanol under fed-batch fermentation conditions where the cell wet weight reaches 150 g / L or higher. High-density fermentation technology can significantly increase the yield of the target protein per unit volume of fermentation broth, laying the foundation for industrial production.

[0016] The present invention also provides the use of recombinant batroxobin as described above in the preparation of medicaments for the treatment or prevention of bleeding.

[0017] Optionally, the bleeding is surgically induced; the surgery includes hard tissue surgery and / or soft tissue surgery; the hard tissue surgery includes bone repair surgery and dental restoration surgery; the soft tissue surgery includes organ repair surgery, organ resection surgery, and debridement surgery.

[0018] The present invention provides a recombinant batroxobin, its pharmaceutical composition, preparation method, and uses, which have the following beneficial effects: 1) This invention, by defining specific N-glycosylation profiles (M9 to M15 distribution, with M10 being the most abundant dominant glycoform) and phosphorylation modifications in recombinant batroxobin, combined with a charge heterogeneity range of pI 6.5-8.5, endows the product with a unique microstructural basis. This specific glycoform and modification combination effectively enhances the thermodynamic stability and substrate binding activity of the enzyme molecule in vivo, providing structural assurance for its potent hemostatic effect in complex bleeding environments (especially bone wounds). On the other hand, this precise and controllable "quality fingerprint" ensures highly uniform physicochemical properties of the product, guaranteeing batch-to-batch quality consistency from the source and solving the batch-to-batch variation problem that is difficult to overcome with natural extracts. In addition, the recombinant batroxobin of this invention has extremely high product purity (purity of not less than 98% in both reversed-phase and size exclusion chromatography, and a single main band in non-reducing electrophoresis), and the chromatograms between batches highly overlap. The high purity significantly reduces the risk of immunogenicity and adverse reactions caused by host protein impurities during injection administration, fully meeting the stringent safety standards for injectable formulations; while the high batch-to-batch reproducibility demonstrates the robustness of the manufacturing process of this invention, providing a solid guarantee for the safety and effectiveness of large-scale industrial production and clinical use.

[0019] 2) This invention provides a pharmaceutical composition comprising the above-mentioned recombinant batroxobin, which is suitable for parenteral administration, preferably as an injection, overcoming the limitation of existing recombinant batroxobins being limited to topical application. The excipient combination of phosphate buffer, human serum albumin, sodium chloride, and mannitol provides a stable chemical environment for the recombinant batroxobin, which is beneficial for maintaining its biological activity.

[0020] 3) This invention employs a multi-step purification process involving cation exchange chromatography, anion exchange chromatography, and molecular sieve chromatography, which effectively removes host proteins, DNA, endotoxins, and process-related impurities to obtain high-purity recombinant batroxobin. In a further preferred embodiment, combined with high-density Pichia pastoris fermentation, large-scale production of recombinant batroxobin can be achieved, avoiding batch variations and safety risks associated with natural extraction methods from the source. The resulting recombinant batroxobin has a purity of not less than 98%, meeting the stringent standards for injectable drugs.

[0021] 4) In clinically representative orthopedic surgical bleeding models (such as rabbit knee replacement surgery), the recombinant batroxobin pharmaceutical composition of the present invention showed a more significant hemostatic effect than commercially available natural batroxobin products (batroxine). This indicates that the unique structural features of the present invention may endow it with superior biological activity or in vivo pharmacokinetic properties, especially in handling complex bleeding situations such as diffuse oozing from bone wounds, thus possessing greater clinical application value.

[0022] Other features and advantages of the invention will become clear from the following detailed description of exemplary embodiments of the invention with reference to the accompanying drawings. Attached Figure Description

[0023] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments of the invention and, together with their description, serve to explain the principles of the invention.

[0024] Figure 1 The reverse-phase purity analysis chromatogram of the recombinant batroxobin provided in the embodiments of the present invention shows a purity of not less than 98%, high batch-to-batch consistency, and no incompletely glycosylated structures.

[0025] Figure 2 The chromatogram of recombinant batroxobin provided in this embodiment of the invention shows a purity of not less than 98%, high batch-to-batch consistency, and absence of high molecular weight impurities such as polymers.

[0026] Figure 3 The non-reducing SDS-PAGE electrophoresis image of the recombinant batroxobin provided in this embodiment of the invention has a purity of not less than 98%, high batch-to-batch consistency, and no product-related degradation impurities.

[0027] Figure 4 The N-glycosylation analysis spectrum of the recombinant batroxobin provided in this embodiment of the invention shows the distribution of M9 to M15, with M9, M10 and M11 as the main peaks.

[0028] Figure 5 The complete molecular weight mass spectrometry distribution of recombinant batroxobin provided in this embodiment of the invention is shown. The complete molecular weight of recombinant batroxobin is distributed between 29222.75 Da and 31168.00 Da due to the molecular weight difference of its N-glycosylated oligosaccharide chains.

[0029] Figure 6 The image shows the molecular weight mass spectrum of the recombinant batroxobin after de-N-glycation provided in this embodiment of the invention. The theoretical molecular weight of the recombinant batroxobin polypeptide is 25492 Da. Due to the presence of two N-glycosylation sites, the molecular weight increases by 2 Da after de-N-glycation. The identification result is consistent with the theoretical result.

[0030] Figure 7 The capillary isoelectric focusing (cIEF) electrophoresis image of the recombinant batroxobin provided in the embodiments of the present invention shows that the isoelectric point pI is distributed in the pH range of 6.5-8.5.

[0031] Figure 8 These are photographs of the appearance of the pharmaceutical composition of the present invention and its clarified state after reconstitution.

[0032] Figure 9This is a long-term stability trend graph of the pharmaceutical composition of the present invention, showing the change in enzyme activity retention rate after the lyophilized injectable formulation of the present invention was stored at 5±3℃ for 30 months.

[0033] Figure 10 To explore the dosage of the pharmaceutical composition of the present invention in a CD-1 mouse tail-cutting hemorrhage model.

[0034] Figure 11 A bar chart comparing the bleeding time and total bleeding volume of the drug composition of the present invention with those of placebo and positive control group in a rat liver trauma hemorrhage model.

[0035] Figure 12 A bar chart comparing the bleeding time and total bleeding volume of the drug composition of this invention with placebo and positive control group in a rat model of caudal vertebral defect surgery bleeding.

[0036] Figure 13 A bar chart comparing the bleeding time and total bleeding volume of the pharmaceutical composition of this invention with placebo and positive control group in a rabbit knee replacement surgery bleeding model.

[0037] Figure 14 Standard curve for detecting the biological activity of recombinant batroxobin (substrate colorimetric method). Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0039] This invention provides a recombinant batroxobin with specific physicochemical properties and a stable pharmaceutical composition thereof, aiming to address the problems of batch-to-batch quality fluctuations, potential impurity risks, and supply chain bottlenecks associated with naturally derived batroxobin in existing technologies. It also fills the technological gap by providing a solution suitable for parenteral administration, addressing the limitation of existing recombinant products to topical application and their failure to meet the higher requirements of injectable administration. In the context of this invention, "recombinant batroxobin" refers to a protein obtained by expressing and producing the gene sequence encoding batroxobin protein in host cells through gene recombination technology. Compared to natural batroxobin extracted directly from snake venom, the recombinant production method ensures high batch-to-batch consistency of the product from the source and eliminates the risk of contamination by animal-derived pathogens. In this invention, several key structural features are used to define this recombinant batroxobin. Among them, "N-glycosylated oligosaccharide chain" refers to an oligosaccharide chain structure attached to a specific asparagine (N) residue side chain on the protein polypeptide chain during post-translational modification. This modification has a significant impact on protein folding, stability, solubility, and biological activity. "M9 to M15" describes a specific type of N-glycosylated oligosaccharide chain, namely a high-mannose N-glycosylated oligosaccharide chain, in which the oligosaccharide chain consists of 9 to 15 mannose residues and two core N-acetylglucosamine residues. The number after M represents the number of mannose residues. For example, "M10" refers to a high-mannose oligosaccharide chain containing 10 mannose residues. Furthermore, the recombinant batroxobinase described in this invention also features "phosphorylation modification," meaning that some mannose residues in its high-mannose oligosaccharide chain are covalently linked to phosphate groups. This phosphorylation modification imparts an additional negative charge to the protein molecule, which is one of the important factors affecting its charge heterogeneity. The "isoelectric point pI" refers to the pH value at which the net charge of a protein molecule is zero in a solution with a specific pH value. In this invention, the isoelectric point (pI) is not a single numerical value, but rather a range determined by high-resolution capillary isoelectric focusing electrophoresis (cIEF). This range reflects the protein charge heterogeneity caused by multiple factors, including glycosylation and phosphorylation modifications. This invention also relates to a "pharmaceutical composition" comprising the aforementioned recombinant batroxobin, which is a mixture of the active ingredient recombinant batroxobin and one or more pharmaceutically acceptable excipients. Specifically, this invention provides a "lyophilized formulation for injection," a sterile powder injection prepared by freeze-drying a solution containing the active ingredient, which requires reconstitution with a suitable solvent before use. This dosage form typically includes a "protein stabilizer" to protect the structural and functional stability of the active ingredient during the freeze-drying process and long-term storage.

[0040] The technical solution of the present invention will be described in detail below.

[0041] In one aspect of the invention, a recombinant batroxobin is provided, wherein the mannose number distribution of the N-glycosylated oligosaccharide chains of the recombinant batroxobin is between M9 and M15. This distribution range ensures the overall glycoform characteristics of the product. More importantly, within this distribution, M9, M10, and M11 are the predominant glycoforms, with M10 being the most abundant single glycoform. This glycosylation fingerprint pattern, dominated by M10, constitutes one of the core identification features of the product of the present invention, making it structurally significantly different from recombinant batroxobins in the prior art that are dominated by M11 to M13. This specific glycoform distribution is believed to potentially affect the protein's conformation, stability, and interaction with its target, thereby influencing its biological activity.

[0042] Furthermore, in the N-glycosylated high-mannose oligosaccharide chains of the recombinant batroxobinase, some oligosaccharide chains undergo phosphorylation modification. This feature introduces a negative charge into the protein molecule, increasing its charge heterogeneity. The presence of phosphorylation modification is not only an important structural feature of the product of this invention, but also contrasts with recombinant batroxobinases in the prior art that do not mention this modification. This modification, together with the glycosylation distribution, shapes the protein's unique surface charge and three-dimensional structure, which may have a positive impact on its pharmacokinetic behavior and efficacy in vivo.

[0043] In this invention, the phosphorylated glycoform refers to a glycoform in which a phosphate group is covalently linked to a high-mannose N-glycosylated oligosaccharide chain, including monophosphorylated and diphosphorylated glycoforms. The phosphorylated glycoforms include, but are not limited to, M10-Phosphorylate, M11-Phosphorylate, and their isomers, such as M11-Phosphorylate_iso1, M11-Phosphorylate_iso2, etc.; when M9-Phosphorylate or other high-mannose phosphorylated glycoforms are present, they are also included. The relative abundance of the phosphorylated oligosaccharide chain to the total N-glycosylated high-mannose oligosaccharide chain is determined by 2-AB labeled N-glycosylation mass spectrometry, expressed as the percentage of the sum of the peak areas of all phosphorylated glycoforms to the total peak area of ​​the high-mannose glycoform. Preferably, the relative abundance is 3% to 7%. In a specific embodiment, the sum of the peak areas of the phosphorylated sugar forms includes at least the sum of the peak areas of M10-Phosphorylate, M11-Phosphorylate and their isomers; if M9-Phosphorylate and / or other monophosphorylated or diphosphorylated high-mannose sugar forms are detected, their peak areas are also included in the sum of the peak areas of the phosphorylated sugar forms.

[0044] Furthermore, the isoelectric point (pI) of the recombinant batroxobin is distributed within the pH range of 6.5 to 8.5. This broad isoelectric point (pI) range was determined by high-resolution capillary isoelectric focusing electrophoresis, which accurately reflects the protein charge heterogeneity resulting from different glycoforms (M9 to M15) and phosphorylation modifications. This isoelectric point (pI) range covers acidic, neutral, and basic regions, including components with an isoelectric point (pI) below 7.4, which clearly distinguishes it from products in the prior art that are limited to an isoelectric point (pI) of not less than 7.4. By controlling the isoelectric point (pI) within this specific range, the present invention achieves a recombinant batroxobin with controllable charge heterogeneity, which is crucial for ensuring batch-to-batch quality homogeneity and bioequivalence of the drug.

[0045] In summary, the three technical features of M10-dominant glycosylation profile, the presence of phosphorylation modification, and the isoelectric point (pI) distribution range of pH 6.5-8.5 work synergistically to construct the unique and identifiable physicochemical "fingerprint" of the recombinant batroxobin of this invention. This unique structural combination has been proven to be closely related to the technical effects subsequently observed in specific disease models, thus endowing the recombinant batroxobin with a structural basis and functional advantages that distinguish it from existing technology products.

[0046] Based on the above scheme, as a preferred embodiment, the recombinant batroxobin is produced by a Pichia pastoris expression system. Pichia pastoris, as a eukaryotic expression system, is capable of correctly folding and performing post-translational modifications, including N-glycosylation, on the expressed protein. Using the Pichia pastoris system, particularly through a methanol-induced expression strategy, high-level secretory expression of recombinant batroxobin can be achieved, and the resulting N-glycosylation type is primarily high-mannose, providing a basis for obtaining the M9 to M15 glycoform distribution defined in this invention. Production using this system is not only technologically mature and cost-controllable, but also easily scalable for industrial production, thus solving the supply chain bottleneck problem of naturally sourced batroxobin. In another preferred embodiment, the purity of the recombinant batroxobin is not less than 98%. For protein drugs intended for parenteral administration, high purity is a fundamental requirement to ensure their safety and efficacy. A purity of not less than 98% means that the content of host cell proteins, host cell DNA, process-related impurities (such as chromatography media shedding), and product-related impurities (such as aggregates and degradation fragments) in the product is controlled at extremely low levels. This greatly reduces the risk of triggering unexpected immune responses or producing toxic side effects, ensuring the safety of patients taking the medication.

[0047] In another aspect of the invention, a pharmaceutical composition is provided comprising recombinant batroxobin as defined in any of the preceding claims. This pharmaceutical composition is suitable for parenteral administration, such as intravenous or intramuscular injection. To achieve parenteral administration, the composition further comprises pharmaceutically acceptable excipients, including buffers, protein stabilizers, isotonic modifiers, and excipients. The buffers are used to maintain the stability of the pH of the composition to ensure the activity and structural integrity of the proteins; the protein stabilizers are used to prevent denaturation, aggregation, or adsorption of the proteins during preparation, storage, and use; the isotonic modifiers are used to adjust the osmotic pressure of the composition to make it isotonic with human body fluids to reduce irritation and pain during injection; and the excipients act as a skeletal support and protectant in the lyophilized formulation for injection.

[0048] In a preferred embodiment, the pharmaceutical composition is an injection; preferably, a lyophilized formulation for injection is used. Converting a liquid formulation into a solid formulation through freeze-drying significantly improves the long-term storage stability of protein drugs. In the dry state, the mobility of protein molecules is greatly reduced, and water-mediated degradation pathways (such as deamidation and oxidation) are effectively inhibited. This design allows the product to be stably stored at 5±3℃ for more than 30 months, far exceeding the shelf life of conventional liquid formulations, thereby reducing the requirements for stringent cold chain transportation and storage conditions, reducing storage and transportation costs, and extending the effective shelf life of the product. In a more specific embodiment, the excipient components of the lyophilized formulation for injection are defined. The buffer is a phosphate buffer, such as a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer system, whose buffering range is close to physiological pH and has good biocompatibility. The protein stabilizer is human serum albumin, a highly efficient protein protectant that effectively reduces the adsorption loss of batroxobin on the container surface and inhibits its aggregation in solution or during freeze-drying. The isotonic regulator is sodium chloride, which is the most commonly used osmotic pressure regulator in clinical practice. The excipient is mannitol, a commonly used lyophilization protectant and skeleton forming agent. It can form a good microcrystalline structure during lyophilization, protect proteins from damage caused by freezing and drying stress, and form an injectable lyophilized formulation with excellent appearance and rapid reconstitution.

[0049] Furthermore, the injectable lyophilized formulation is prepared via a freeze-drying process comprising pre-freezing, primary drying, and secondary drying steps. This is a carefully optimized three-step process. The pre-freezing step rapidly cools the drug solution below the eutectic point, solidifying the water into fine ice crystals, laying the foundation for subsequent sublimation. The primary drying (main drying) step removes the solid ice crystals by directly sublimating them into water vapor under low temperature and high vacuum conditions, removing the vast majority of the water. The secondary drying (desorption drying) step removes residual water bound to the product under slightly higher temperature and higher vacuum conditions. By precisely controlling the temperature, pressure, and time parameters of each step, this freeze-drying process maximizes the preservation of the bioactivity and structural integrity of recombinant batroxobin, ultimately yielding a high-quality injectable lyophilized formulation.

[0050] In another embodiment of the invention, the pharmaceutical composition can also be directly prepared as a liquid injection. Recombinant batroxobin, phosphate buffer, human serum albumin, sodium chloride, and mannitol are dissolved in water for injection according to the above-described dosage. After adjusting the volume, the solution is filtered through a 0.22 μm filter membrane for sterilization and aseptically filled into vials or pre-filled syringes to obtain the pharmaceutical composition in liquid injection form. This liquid injection is also suitable for parenteral administration.

[0051] In another aspect of the invention, a method for preparing the recombinant batroxobin is provided. The method includes a purification step of a solution containing the recombinant batroxobin, the purification step comprising: a cation exchange chromatography step, an anion exchange chromatography step, and a molecular sieve chromatography step. This multi-step chromatographic combination strategy is key to achieving high product purity. Cation exchange chromatography is typically used as a capture step, utilizing the positively charged property of batroxobin at a specific pH to separate it from most negatively charged or neutral host protein impurities. Anion exchange chromatography, as an intermediate purification step, further removes impurities with different charge properties from the target protein. Molecular sieve chromatography, as the final purification step, effectively removes product aggregates and degradation fragments based on differences in molecular size, while simultaneously performing buffer replacement. As a preferred embodiment, the purification steps are defined as sequentially performing the cation exchange chromatography step, anion exchange chromatography step, and molecular sieve chromatography step. This specific purification sequence has been optimized to achieve excellent purification efficiency and product yield. First, cation exchange chromatography is used for capture and preliminary purification, which can rapidly concentrate the target product and remove a large number of impurities. Then, anion exchange chromatography is used to further refine the separation of impurities with similar properties in either cross-flow or binding mode. Finally, molecular sieve chromatography is used to remove product-related impurities such as aggregates, ensuring the homogeneity and safety of the final product. This orderly purification process ensures that a high-quality recombinant batroxobin stock solution with a purity of not less than 98% can be obtained consistently.

[0052] In another preferred embodiment, the method further includes, prior to the purification step, inducing Pichia pastoris host cells to express and secrete the recombinant batroxobin using methanol under high-density fed-batch fermentation conditions. High-density fermentation technology, by precisely controlling the feeding rate of carbon sources such as glycerol in the culture medium, allows yeast cells to grow to extremely high cell densities (e.g., wet weight exceeding 150 g / L), followed by the addition of methanol to induce the expression of the target protein. This strategy significantly increases the yield of recombinant batroxobin per unit volume of fermentation broth, resulting in a substantial improvement in the overall production volume productivity and economic benefits, laying a solid foundation for subsequent large-scale industrial production.

[0053] In another aspect of the invention, the use of the recombinant batroxobin in the preparation of a medicament for treating or preventing surgical bleeding is disclosed. As a thrombin-like enzyme, batroxobin's core mechanism of action involves specifically cleaving the fibrinogen Aα chain to release fibrin peptide A, thereby initiating the polymerization of fibrin monomers to form a fibrin clot, achieving local or systemic hemostasis. Therefore, the recombinant batroxobin with specific structural characteristics provided by the present invention can be developed into an effective hemostatic agent for various clinical scenarios requiring bleeding control. Further, the use is specifically limited to treating or preventing bleeding caused by orthopedic surgery. Orthopedic surgeries, especially those involving bone cutting and extensive soft tissue dissection, are often accompanied by diffuse wound bleeding, particularly from cancellous bone, which is difficult to control effectively with conventional hemostatic methods. Using the recombinant batroxobin of the present invention in such surgeries can effectively reduce intraoperative and postoperative bleeding through its systemic procoagulant effect, which has significant clinical value in improving surgical visibility, reducing transfusion requirements, and preventing postoperative hematoma and other complications. As a more specific application scenario, the orthopedic surgery is limited to joint replacement surgery or spinal fusion surgery. Both of these surgeries are major orthopedic procedures with significant surgical trauma and a high risk of bleeding, making them important application areas for hemostatic drugs. Experimental data show that the recombinant batroxobin of this invention exhibits superior hemostatic effects compared to existing marketed drugs in animal models simulating these two types of surgeries. This superior effect is closely related to the unique structural features of the recombinant batroxobin of this invention, such as its unique M10-dominant glycoform, phosphorylation modification, and specific isoelectric point range, highlighting the unique advantages and application potential of this invention in the field of hemostasis for high-end and complex orthopedic surgeries.

[0054] To more clearly illustrate the technical solution of the present invention, a detailed description is provided below in conjunction with specific embodiments.

[0055] The following examples describe the preparation and characterization of the recombinant batroxobin stock solution of the present invention. First, the expression engineered strain was constructed. The batroxobin encoding gene, optimized for codon preference in *Pichia pastoris*, was cloned into the pPIC9k expression vector, and subsequently transformed into the *Pichia pastoris* GS115 host strain via electroporation. Using screening plates containing up to 4.0 mg / mL of G418 antibiotic, a high-copy-integration positive recombinant was screened. This recombinant exhibited the highest batroxobin secretion capacity in a shake-flask level methanol-induced expression assay. Subsequently, this #7 clone was used for high-density fermentation in a 50 L fermenter. The fermentation process includes: primary seed culture, where the strain is amplified in the culture medium to an OD600 of not less than 5; secondary seed culture, further amplifying to an OD600 of not less than 4; then transferred to a fermenter for batch glycerol culture until the cell wet weight reaches above 80 g / L; next, a glycerol feeding stage is initiated, where glycerol is fed continuously to further increase the cell wet weight to above 150 g / L; finally, a methanol-induced expression stage is initiated, with continuous methanol feeding for at least 48 hours until the final cell wet weight reaches above 300 g / L. After fermentation, the fermentation supernatant containing recombinant batroxobin is collected by centrifugation. The collected fermentation supernatant is purified through a series of chromatographic steps. First, the supernatant is loaded onto an SP Fast Flow cation exchange chromatography column and eluted using a linear gradient of 50-200 mM sodium chloride to collect the target protein peak. Next, the collected fraction is loaded onto a Q High Performance anion exchange chromatography column and impurities are further removed using a 50-300 mM sodium chloride gradient. Finally, the sample was purified and buffer replaced using a Superdex 75 molecular sieve chromatography column, with the final buffer system being a 20 mmol / L phosphate buffer solution (pH 6.8 ± 0.2). This optimized three-step chromatography method yielded a high-purity recombinant batroxobin stock solution.

[0056] The obtained stock solution underwent comprehensive quality characterization. For example... Figure 1 As shown, the reversed-phase high-performance liquid chromatography (RP-HPLC) analysis results indicate that its purity is not less than 98%. Figure 2 As shown, the size exclusion high-performance liquid chromatography (SEC-HPLC) analysis results indicate that the purity of the main peak is not less than 98%, indicating that the aggregate content in the sample is extremely low. Figure 3 As shown, in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) pattern under non-reducing conditions, the sample appeared as a single, clear band, further confirming its high purity and molecular weight uniformity. The N-glycosylation structure of the original solution was precisely analyzed. Figure 4As shown in Table 1, the N-glycosylated oligosaccharide chains released by the enzyme were detected by Q-TOF mass spectrometry. The results showed that all oligosaccharide chains were high-mannose, with mannose content ranging from M9 to M15. The relative abundance (normalized response value) of each glycotype is as follows: M9 13.28%, M10 28.10%, M11 24.20%, M12 10.64%, M13 12.57%, M14 9.03%, and M15 2.18%. The data clearly indicate that M9, M10, and M11 are the main glycotypes, with M10 being the most abundant single glycotype at 28.10%. Simultaneously, mass spectrometry analysis also confirmed phosphorylation modification on some oligosaccharide chains, accounting for 3.28%.

[0057] Table 1 Figure 5 and Figure 6 The high mannose distribution in the N-glycan spectrum of this invention was further confirmed by mass spectrometry molecular weight analysis. Figure 7 As shown, the sample was analyzed using capillary isoelectric focusing (cIEF) electrophoresis. The results showed that the isoelectric point (pI) of the recombinant batroxobin exhibited a distribution range, with the main peaks distributed between pH 6.851 and 8.007. This measured pI distribution range falls entirely within the pH range of 6.5 to 8.5 required by this invention, reflecting the controllable charge heterogeneity of the product. The above preparation method and characterization results fully support the technical solution of this invention regarding recombinant batroxobin and its preparation method.

[0058] Based on the recombinant batroxobin stock solution prepared above, a lyophilized formulation for injection was further prepared. In a specific pharmaceutical composition example, the formulation per 1000 vials is as follows: 1000.0 batroxobin units (BU) of recombinant batroxobin active ingredient, 3.18 g of phosphate buffer (composed of NaH2PO4 and Na2HPO4) (pH 7.4 ± 0.2 after preparation), 0.5 g of human serum albumin, 5.84 g of sodium chloride, and 20 g of mannitol. All the above components were dissolved in water for injection and the volume was adjusted to 0.5 L. The preparation process includes: thoroughly mixing the above excipients with the recombinant batroxobin stock solution, and then filtering the mixture using a sterile filter membrane with a pore size of 0.22 μm to ensure the sterility of the pharmaceutical composition. The sterile solution was accurately dispensed into 2 mL vials, with 0.5 mL dispensed into each vial. The dispensed vials were then sent to a freeze dryer for freeze drying. The lyophilization process is set as follows: first, pre-freeze at a temperature not exceeding -50°C for at least 5 hours; then, enter the first drying stage, drying at a plate temperature not exceeding 0°C and a vacuum degree not exceeding 100 Pa for at least 8 hours; finally, perform a second drying, drying at a plate temperature not exceeding 30°C and a vacuum degree not exceeding 30 Pa for at least 6 hours. After lyophilization, nitrogen gas is introduced into the vials under aseptic conditions and the vials are immediately sealed. The final lyophilized formulation for injection, such as... Figure 8 As shown, the substance appears as a loose, white or off-white mass with an intact structure. When used, adding 1 mL of water for injection to the vial results in complete reconstitution within 30 seconds, forming a clear, colorless solution free of insoluble matter, indicating excellent reconstitution properties. Long-term stability studies were conducted on this lyophilized injectable formulation. Figure 9 As shown, the results indicate that after 30 months of storage under refrigeration conditions of 5±3℃, the enzyme activity retention rate is still higher than 90%, and the reconstitution time is less than 20 seconds (far superior to the quality standard of no more than 60 seconds), proving that the formulation and freeze-drying process can effectively ensure the stability of recombinant batroxobin during long-term storage.

[0059] In this invention, the enzyme activity (batroxobin units, BU) of recombinant batroxobin was determined using a substrate colorimetric method. The specific method is as follows: Based on the mechanism of action of batroxobin, it can cleave peptide bonds formed by arginine residues in proteins or polypeptides. Using S-2238 (HD-Phe-Pip-Arg-pNA·2HCl) as a substrate, batroxobin cleaves the arginine residues, releasing p-nitroaniline (pNA), which exhibits a characteristic absorption signal at 405 nm. Excess substrate results in a linear relationship between the biological activity of batroxobin and the absorption signal. A standard curve was plotted with the batroxobin potency (BU) of the international reference standard as the x-axis and the absorbance at 405 nm as the y-axis (e.g., ...). Figure 14The specific activity (BU / mg) of the sample is calculated by reading the corresponding potency from the standard curve based on the absorbance of the sample. Each determination should include both a standard and a blank control, and data processing should be performed using the bioassay statistical methods outlined in the relevant general chapters of the Pharmacopoeia of the People's Republic of China.

[0060] To verify the in vivo hemostatic effect of the pharmaceutical composition of this invention and to compare it with existing drugs, four pharmacodynamic evaluation experiments were conducted. First, the dosage of the pharmaceutical composition of this invention was explored using a CD-1 mouse tail-cutting hemorrhage model. Two hundred and twenty rats (half male and half female) were selected and randomly divided into 11 groups of ten rats each: a placebo group (administered with an equal volume of physiological saline), a positive control group (administered with three doses: low (0.5 U / kg), medium (1.0 U / kg), and high (2.0 U / kg) doses of the commercially available natural batroxobin preparation "Batroxotin"), and seven dose groups of the recombinant batroxobin pharmaceutical composition of this invention, ranging from low (0.113 BU / kg) to high (3.6 BU / kg). The bleeding time of each group was recorded and statistically analyzed. Figure 10 As shown, the effective dose range of the pharmaceutical composition of the present invention is from 0.113 BU / kg to 0.45 BU / kg, and the optimal dose for the positive control is 0.5 U / kg (in another test, it was confirmed that the hemostatic time was longer at 0.25 U / kg than at 0.5 U / kg, which is not shown here). Figure 10 The statistical significance markers were: compared with the placebo group (Saline), p < 0.05, p < 0.01, p < 0.001.

[0061] Based on the effective dose range determined through exploration, comparisons were made with placebo and positive control in three different bleeding models.

[0062] The first method is a rat liver trauma hemorrhage model, which effectively simulates liver hemorrhage caused by clinical trauma or surgery. Seventy-two healthy SD rats (half male, half female) were randomly divided into six groups of 12 rats each: a sham-operated group, a placebo group (administered with an equal volume of saline), a positive control group (administered the optimal dose of batroxobin), and three dose groups (low, medium, and high) of the recombinant batroxobin drug composition of this invention. The low-dose group received 0.057 BU / kg body weight, the medium-dose group received 0.114 BU / kg body weight, and the high-dose group received 0.228 BU / kg body weight. All drugs were administered intravenously 15 minutes before surgery. The results were recorded and the bleeding time and total postoperative blood loss were statistically analyzed for each group. Figure 11 As shown, compared with placebo, the optimal dose of the pharmaceutical composition of the present invention (0.228 BU / kg) significantly reduced bleeding time and bleeding volume more than the positive control. Figure 11The statistical significance markers were: compared with the placebo group, p < 0.05, p < 0.01, p < 0.001.

[0063] The second method is a rat caudal vertebral defect hemorrhage model, which can effectively simulate the complex wound bleeding situation during clinical spinal surgery. Seventy-two healthy SD rats, half male and half female, were randomly divided into six groups of 12 rats each: a sham-operated group, a placebo group (administered with an equal volume of saline), a positive control group (administered the optimal dose of batroxobin), and three dose groups (low, medium, and high doses) of the recombinant batroxobin drug composition of this invention. The low-dose group received 0.057 BU / kg body weight, the medium-dose group received 0.114 BU / kg body weight, and the high-dose group received 0.228 BU / kg body weight. All drugs were administered intravenously 15 minutes before surgery. The experimental results were recorded and the bleeding time and total postoperative blood loss were statistically analyzed for each group. Figure 12 As shown, compared with placebo, the optimal dose of the pharmaceutical composition of the present invention (0.228 BU / kg) significantly reduced bleeding time and bleeding volume more than the positive control. Figure 12 The statistical significance markers were: compared with the placebo group, p < 0.05, p < 0.01, p < 0.001.

[0064] The third rabbit knee replacement surgery bleeding model effectively simulates complex wound bleeding during clinical joint replacement surgery. Forty healthy New Zealand white rabbits (half male and half female) were randomly divided into five groups of eight each: a placebo group (administered with an equal volume of saline), a positive control group (administered with the optimal dose of batroxobin), and three dose groups (low, medium, and high) of the recombinant batroxobin drug composition of this invention. The low-dose group received 0.03 BU / kg body weight, the medium-dose group received 0.06 BU / kg body weight, and the high-dose group received 0.12 BU / kg body weight. All drugs were administered intravenously 15 minutes before surgery. The bleeding time and total postoperative blood loss were recorded and statistically analyzed for each group. Figure 13As shown, the mean bleeding time in the placebo group was 12.48 ± 3.67 minutes, and the total postoperative blood loss was 20802.5 ± 5135.1 mg. The mean bleeding time in the positive control group (administered 0.25 U / kg batroxobin) was significantly shortened to 7.44 ± 2.21 minutes (compared to the placebo group, P < 0.001), and the total postoperative blood loss was also significantly reduced to 12464.4 ± 4648.9 mg (compared to the placebo group, P < 0.001), demonstrating its definite hemostatic effect. The high-dose recombinant batroxobin group of this invention (administered 0.12 BU / kg) showed even better hemostatic effect. The mean bleeding time in this group was only 4.55 ± 1.29 minutes, which was not only significantly better than the placebo group (P < 0.001), but also statistically significant compared with the batroxobin positive control group (P < 0.05). Similarly, regarding total postoperative blood loss, the average blood loss in the high-dose group of this invention was 5955.8 ± 3043.0 mg, which was not only much lower than that in the placebo group (P < 0.001), but also significantly lower than that in the positive control group (P < 0.01). Figure 13 The statistical significance markers were: compared with the placebo group, p < 0.05, p < 0.01, p < 0.001; compared with the positive control group (batroxobin), #p < 0.05; compared with the low-dose test substance group, Δp < 0.05, ΔΔp < 0.01.

[0065] Based on the pharmacodynamic results of the rat liver trauma hemorrhage model, rat caudal vertebral defect hemorrhage model, and rabbit knee replacement surgery hemorrhage model, the recombinant batroxobin pharmaceutical composition of the present invention, when administered intravenously, significantly reduces bleeding and shortens hemostasis time in both hard tissue surgeries (such as bone repair surgery and dental restoration, see the rat caudal vertebral defect model and rabbit knee replacement model) and soft tissue surgeries (such as organ repair surgery, organ resection, and debridement, see the rat liver trauma model), confirming its use in the preparation of drugs for the treatment or prevention of surgical bleeding. Furthermore, the recombinant batroxobin pharmaceutical composition of the present invention, after reconstitution, is also suitable for intramuscular injection or direct topical application to wounds, such as through local infusion, spraying, application, or rinsing, for the prevention or treatment of surgical bleeding.

[0066] The above experimental results clearly demonstrate that the recombinant batroxobin provided by this invention, with its specific structural characteristics (M10-dominant glycoform, phosphorylation modification, and specific isoelectric point pI range), exhibits significantly superior hemostatic efficacy compared to the widely used gold standard drug—naturally derived batroxobin—in simulated clinical surgical models, particularly in stringent bleeding models of major orthopedic surgeries. This advantage is not simply equivalent or non-inferior, but statistically superior, fully demonstrating the inventiveness and clinical application value of this invention and suggesting that its unique molecular structure may endow it with stronger biological activity in the bone tissue wound microenvironment.

[0067] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A recombinant batroxobin, characterized in that, The recombinant batroxobin has fibrinogen cleavage activity; The mannose number distribution of the N-glycosylated oligosaccharide chains of the recombinant batroxobin is between M9 and M15, wherein M9, M10 and M11 are the main types of the N-glycosylated oligosaccharide chains, with the M10 glycotype having the highest relative abundance. In the N-glycosylated high-mannose oligosaccharide chains of the recombinant batroxobinase, some oligosaccharide chains undergo phosphorylation modification; and The isoelectric point (pI) of the recombinant batroxobin is distributed in the pH range of 6.5 to 8.

5.

2. The recombinant batroxobin according to claim 1, characterized in that, The recombinant batroxobin was produced using a Pichia pastoris expression system; The purity of the recombinant batroxobin is not less than 98%.

3. The recombinant batroxobin according to claim 1 or 2, characterized in that, In the N-glycosylated high-mannose oligosaccharide chains, the relative abundance of phosphorylated oligosaccharide chains is 3% to 7% of the total N-glycosylated high-mannose oligosaccharide chains; The relative abundance was determined by 2-AB labeled N-glycan spectroscopy, using fluorescence detection and / or mass spectrometry, as a percentage of the sum of phosphorylated sugar peak areas to the total area of ​​high-mannose sugar chain peaks.

4. A pharmaceutical composition comprising the recombinant batroxobin as described in any one of claims 1-3, characterized in that, The pharmaceutical composition is suitable for parenteral administration and contains pharmaceutically acceptable excipients, including buffers, protein stabilizers, isotonic modifiers, and excipients.

5. The pharmaceutical composition according to claim 4, characterized in that, The buffer is a phosphate buffer, the protein stabilizer is human serum albumin, the isotonic regulator is sodium chloride, and the excipient is mannitol.

6. The pharmaceutical composition according to claim 4, characterized in that, The pharmaceutical composition is an injectable preparation.

7. A method for preparing recombinant batroxobin as described in any one of claims 1-3, characterized in that, include: The fermentation broth was subjected to solid-liquid separation, and the solution containing recombinant batroxobin was collected. The solution is purified, and the purification steps include, in sequence, a cation exchange chromatography step, an anion exchange chromatography step, and a molecular sieve chromatography step.

8. The method for preparing recombinant batroxobin according to claim 7, characterized in that, The method further includes, prior to the solid-liquid separation step: The step involves inducing Pichia pastoris host cells to express and secrete the recombinant batroxobin under fed-batch fermentation conditions where the cell wet weight reaches 150 g / L or more.

9. Use of the recombinant batroxobin as described in any one of claims 1-3 in the preparation of a medicament for the treatment or prevention of bleeding.

10. The use according to claim 9, characterized in that, The bleeding was caused by the surgery; The surgery includes hard tissue surgery and / or soft tissue surgery; The hard tissue surgeries include bone repair surgery and dental restoration surgery; The soft tissue surgeries include organ repair, organ resection, and debridement.

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