Lyophilized preparation of pegylated thrombopoietic peptide, preparation method therefor, and use thereof

Abstract

Provided are a lyophilized preparation of a PEGylated thrombopoietic peptide, a preparation method therefor and a use thereof. The lyophilized preparation comprises a PEGylated thrombopoietic peptide, histidine, glycine, mannitol, and sucrose; the preparation method for the PEGylated thrombopoietic peptide comprises: conjugating a thrombopoietic peptide with polyethylene glycol to obtain a PEGylated thrombopoietic peptide, wherein the thrombopoietic peptide is prepared by using a fusion protein, an amino acid sequence of the fusion protein is as shown in Xm-(Y-Z)n, X is an insulin glargine mutant, Y is a protease cleavage site, Z is the thrombopoietic peptide, m is 1 or 2, and n is 1 or 2. The described fusion protein is efficiently expressed in the form of inclusion bodies in bacteria. The expressed fusion protein is cleaved by means of a specific protease to release a thrombopoietic peptide, which ensures the integrity of the thrombopoietic peptide and high cleavage efficiency. The provided PEGylated thrombopoietic peptide and the preparation thereof can effectively treat and prevent cancer treatment-induced thrombocytopenia, primary immune thrombocytopenia, chronic liver disease-associated thrombocytopenia, and hematopoietic acute radiation syndrome.

Description

Lyophilized formulations of PEGylated thrombopoietin, their preparation methods and uses Technical Field

[0001] This application relates to the field of biopharmaceuticals, specifically to a lyophilized formulation of a polyethylene glycol-modified thrombopoietin, its preparation method, and its uses. Background Technology

[0002] Thrombocytopenia is a common clinical condition, frequently seen in the treatment and prevention of cancer drug-induced thrombocytopenia (CTIT), primary immune thrombocytopenia (ITP), chronic liver disease-related thrombocytopenia syndrome, myelodysplastic syndrome (MDS), and acute radiation syndrome (ARS).

[0003] Drug-induced thrombocytopenic purpura (CTIT) is a common adverse reaction to antitumor drugs, with an incidence rate as high as 21.8% in patients undergoing antitumor therapy. The median time to the first drop in platelet count is 1–2 weeks after chemotherapy, although some drug-induced CTIT can also occur 2 weeks after chemotherapy. Most patients with CTIT also experience other cytopenias; the incidence of isolated thrombocytopenia in patients with solid tumors with thrombocytopenia is approximately 17.7%. Chemotherapy drugs that easily lead to CTIT include gemcitabine, topotecan, and temozolomide. Combination chemotherapy regimens are more likely to cause CTIT than single-agent chemotherapy. Among multi-drug combination regimens, GP (gemcitabine, cisplatin / carboplatin), EP (etoposide, cisplatin), CODOX-M / IVAC, GEMOX, ICE, and MAID regimens have a higher risk of CTIT. Thrombocytopenia usually begins around 5 days after chemotherapy, reaches its lowest value on days 7–14, then gradually rises, and the platelet count returns to baseline on days 28–35.

[0004] Acute radiation syndrome (ARS) is an acute illness caused by high-dose penetrating radiation irradiating the whole body in a short period of time. It leads to bone marrow suppression, resulting in an inability to produce a sufficient number of new white blood cells, red blood cells, and platelets. ARS often occurs in radiological accidents or nuclear weapon explosions.

[0005] Primary immune thrombocytopenia (ITP) is an acquired autoimmune bleeding disorder characterized by isolated, unexplained decreases in peripheral blood platelet counts. The clinical manifestations are highly variable, ranging from asymptomatic thrombocytopenia and mucosal bleeding to severe internal bleeding and even fatal intracranial hemorrhage. Elderly patients have a significantly higher risk of fatal bleeding than younger patients. Some patients experience fatigue and anxiety. The Chinese guidelines for the diagnosis and treatment of ITP in adults (2020 edition) recommend second-line treatment, including thrombopoietin-stimulating agents (TPAs). These agents typically take effect within 1-2 weeks, with an efficacy rate exceeding 60%. However, the efficacy is often not maintained after discontinuation, requiring individualized maintenance therapy. For patients who are unresponsive to or intolerant to one TPA, switching to another TPA or using sequential therapy may be beneficial.

[0006] Chronic liver disease-related thrombocytopenia is internationally defined as a platelet count below 150 × 10⁻⁶. 9 / L, in China, is usually defined as a platelet count below 100 × 10⁹ / L. 9 / L. Liver disease-related thrombocytopenia is a complex pathophysiological process involving multiple factors and mechanisms, including decreased platelet production, increased destruction, abnormal distribution, and increased consumption.

[0007] The main interventions for thrombocytopenia include platelet transfusion, splenic artery embolization and splenectomy, and thrombopoietin receptor agonists (TPO-RA). While platelet transfusion is the fastest way to increase platelet count, it only provides temporary relief, and the improvement is unpredictable and uncontrollable. In some patients with thrombocytopenia, repeated platelet transfusions may lead to alloimmunization-induced platelet transfusion ineffectiveness. Platelet transfusion also carries risks such as infection, fever, and non-hemolytic reactions. Furthermore, its short shelf life and limited clinical supply restrict its clinical application; it is recommended only for patients at risk of life-threatening bleeding or requiring emergency surgery. Currently, the TPO-RA drugs approved for marketing in China mainly include recombinant human thrombopoietin (rhTPO), oral small molecule TPO-RA drugs (eltrombopag, hetrombopag, avatrombopag tablets and rutrobopag), and peptide-like TPO-RA drugs (roprostine).

[0008] PEGylated thrombopoietin is based on the active peptide region structure of the marketed drug Romiplostim. It utilizes polyethylene glycol (PEG) to replace IgG1 Fc for long-acting fusion. One PEG molecule is coupled to one or two active peptides, forming a spatial conformation similar to that of Romiplostim containing homotetrameric TMPs. This preserves high affinity for the rhuMpl receptor, achieving long-acting action while avoiding Fc-mediated recycling in vivo via FcRn binding. Therefore, improving the preparation methods of thrombopoietin or developing formulations based on PEGylated thrombopoietin is essential. Summary of the Invention

[0009] This application aims to at least partially address one of the technical problems existing in the prior art. To this end, this application provides a lyophilized formulation of a polyethylene glycol-modified thrombopoietin, which effectively avoids charge heterogeneity and also has the advantages of high stability.

[0010] In a first aspect, this application proposes a fusion protein. According to embodiments of this application, the fusion protein has an amino acid sequence as shown in formula (I), Xm-(YZ)n (I);

[0011] Wherein, X is a glargine insulin mutant, and the amino acid sequence of the glargine insulin mutant is shown in any one of SEQ ID NO:1, 9 to 11;

[0012] Y represents the enzyme cleavage site;

[0013] Z is a thrombopoietin-promoting peptide;

[0014] m is 1 or 2;

[0015] n is 1 or 2;

[0016] The fusion protein is adapted to be cleaved by a cleavage enzyme to form the free thrombopoietin, which is not cleaved by the cleavage enzyme.

[0017] In a second aspect of this application, the use of the fusion protein described in the first aspect in the preparation of thrombopoietin or polyethylene glycol-modified thrombopoietin is proposed.

[0018] In a third aspect of this application, a method for preparing a thrombopoietin-promoting peptide is provided. According to an embodiment of this application, the method includes: obtaining inclusion bodies of bacterial cells, the inclusion bodies comprising the fusion protein described in the first aspect; and enzymatically digesting the fusion protein in the inclusion bodies to obtain the thrombopoietin-promoting peptide.

[0019] In a fourth aspect of this application, a method for preparing a polyethylene glycol-modified thrombopoietin is provided. According to an embodiment of this application, the method includes: coupling the thrombopoietin with polyethylene glycol to obtain the polyethylene glycol-modified thrombopoietin; the thrombopoietin is prepared using the fusion protein described in the first aspect, or prepared according to the method described in the third aspect.

[0020] In a fifth aspect, this application provides an injectable solution. According to an embodiment of this application, the injectable solution comprises: a polyethylene glycol-modified thrombopoietin, a buffer salt, and a protective agent.

[0021] In a sixth aspect of this application, a lyophilized formulation is provided. According to an embodiment of this application, the lyophilized formulation is obtained by lyophilizing the injection solution described in the fifth aspect.

[0022] In a seventh aspect of this application, the application discloses the use of a polyethylene glycol-modified thrombopoietin peptide, said use being for:

[0023] Treatment and / or prevention of disease, and / or

[0024] To prepare medicines for the treatment and / or prevention of diseases;

[0025] The diseases mentioned include treatment and prevention of tumor drug-induced thrombocytopenia, primary immune thrombocytopenia, chronic liver disease-related thrombocytopenia, and acute radiation syndrome-hematopoietic syndrome.

[0026] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0027] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0028] Figure 1 is a schematic diagram of the construction of the pET-30a-GI-NPC recombinant plasmid in Example 1 of this application.

[0029] Figure 2 shows a typical lysine endopeptidase cleavage pattern in Example 3 of this application;

[0030] Figure 3 is the HPLC detection chromatogram of the platelet-producing peptide after purification in Example 4 of this application;

[0031] Figure 4 is a typical spectrum of the addition reaction between the thrombopoietin-promoting peptide and the maleimide polyethylene glycol derivative in Example 5 of this application.

[0032] Figure 5 shows the HPLC detection chromatogram of the PEG20K-NPC stock solution in Example 6 of this application;

[0033] Figure 6 shows the related material spectra of the injection and lyophilized formulation in Example 7 of this application after 37°C for 1 month.

[0034] Figure 7 shows the charge heterogeneity detection spectra of prescriptions 1 and 2 in Example 9 of this application;

[0035] Figure 8 shows the dose-response curve of PN20 on platelets in normal mice in Example 13 of this application;

[0036] Figure 9 shows the dose-response curve of PN20 on platelets in a rat model of thrombocytopenia caused by chronic liver disease in Example 14 of this application;

[0037] Figure 10 shows the dose-response curve of the effect of PN20 on platelets in a mouse model of immune thrombocytopenia in Example 15 of this application;

[0038] Figure 11 shows the dose-response curve of the effect of PN20 on platelets in a mouse model of chemotherapy-induced thrombocytopenia in Example 16 of this application;

[0039] Figure 12 is a time-dependent graph of the measured platelet count after a single dose of PN20 to a healthy subject in Example 17 of this application.

[0040] Figure 13 is a time-dependent graph showing the measured platelet count after a single dose of PN20 in a patient subject in Example 18 of this application. Detailed Implementation

[0041] The embodiments of this application are described in detail below. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0042] It should be noted that 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. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

[0043] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0044] To facilitate understanding of this application, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this application pertains.

[0045] For the purposes of this specification and the appended claims, unless otherwise indicated, all expressed quantities of ingredients, percentages or proportions of materials, figures for reaction conditions, and other numerical values ​​used in the specification and claims should be understood to be modified in all cases by the term "about". Therefore, unless indicated to the contrary, the numerical parameters listed in the following specification and the appended claims are approximate values ​​and may vary depending on the desired properties claimed in this application. In no way is there any attempt to limit the application of the equivalence principle to the scope of the claims, and each numerical parameter should be interpreted at least according to the number of significant figures reported and by applying conventional rounding techniques.

[0046] While the wide range of numerical ranges and parameters listed in this application are approximate, the numerical values ​​listed in the specific embodiments are reported as precisely as possible. However, any numerical value inherently contains a certain degree of error unavoidably arising from the standard deviation encountered in its respective experimental measurements. Furthermore, all ranges disclosed herein should be understood to include any and all subranges thereof. For example, the range “1 to 10” includes any and all subranges between the minimum value 1 and the maximum value 10 (inclusive).

[0047] The use of any and all examples or exemplary language (e.g., “such as”) provided herein is intended only to better illustrate the application and does not constitute a limitation on the scope of the application unless otherwise required. No language in the specification should be construed as indicating that any unclaimed element is necessary for the practice of this application. For the purposes of description and the appended claims, unless otherwise stated, all representations, quantities, percentages, etc., should be understood to be modified in all cases by the term “about.” Furthermore, all scopes include any combination of the maximum and minimum points disclosed and include any intermediate ranges therein, which may or may not be specifically enumerated herein.

[0048] As used herein, the terms “a,” “an,” or “at least one” are used interchangeably in this application and are defined as meaning “a” or “one or more.”

[0049] Unless otherwise stated, the terms “including,” “having,” “including,” and “containing” should be interpreted as open-ended terms (i.e., meaning “including but not limited to”).

[0050] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0051] In this document, the term "thrombopoietin" refers to the thrombopoietin to be produced in this application. It should be noted that the free thrombopoietin has the same amino acid sequence as the thrombopoietin. However, when the thrombopoietin forms a fusion protein with the insulin glargine mutant, the N-terminus of the thrombopoietin is linked to the C-terminus of the restriction enzyme site via a peptide bond, while the N-terminus of the free thrombopoietin is an amino group. Exemplarily, in the fusion protein of this application, the N-terminus of the thrombopoietin is linked to the C-terminus of the restriction enzyme site via a peptide bond.

[0052] In this article, the term "cleaving enzyme" generally refers to a class of enzymes that can specifically cleave protein peptide chains. These enzymes recognize specific amino acid sequences (i.e., cleavage sites) and cleave at the C-terminus of the corresponding amino acid sequence. For example, the cleaving enzyme can be recombinant bovine enterokinase, whose cleavage site is DDDDK. Recombinant bovine enterokinase can cleave proteins containing DDDDKX1X2X3X4…X n The protein is cleaved to obtain X1X2X3X4……X n Protein. For example, the cleavage enzyme may be a recombinant lysine endopeptidase, the cleavage site of which is K, and the recombinant lysine endopeptidase can cleave proteins containing KX1X2X3X4…X. n The protein is cleaved to obtain X1X2X3X4……X n protein.

[0053] In this document, the term "treatment" refers to the administration of a drug or compound to an individual to achieve a desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of a disease or its symptoms, and / or therapeutic in terms of partial or complete cure of a disease and / or adverse effects caused by the disease. As used herein, "treatment" encompasses diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of a condition in an individual who is susceptible but has not yet been diagnosed with the disease; (b) inhibition of disease, such as blocking disease progression; or (c) relief of disease, such as reducing symptoms associated with the disease. As used herein, "treatment" encompasses any administration of a drug or compound to an individual to treat, cure, relieve, improve, reduce, or inhibit the individual's disease, including but not limited to administration of a drug containing a compound described herein to an individual in need.

[0054] As used herein, the term “application” means administration by a physician, nurse, healthcare provider, patient, or any other individual, including self-application. This includes not only delivery into the body but also prescription, distribution, or any other means of facilitating delivery.

[0055] In this article, the term "patient" refers to a mammal that requires treatment for its symptoms or condition.

[0056] This application discloses a fusion protein and its preparation method and uses, the uses and preparation method of a polyethylene glycolated thrombopoietin, an injection solution and a lyophilized formulation and their uses, which will be described in detail below.

[0057] Fusion protein

[0058] In a first aspect, this application proposes a fusion protein. According to embodiments of this application, the fusion protein has an amino acid sequence as shown in formula (I), Xm-(YZ)n (I);

[0059] Wherein, X is a glargine insulin mutant, and the amino acid sequence of the glargine insulin mutant is shown in any one of SEQ ID NO:1, 9 to 11;

[0060] Y represents the enzyme cleavage site;

[0061] Z is a thrombopoietin-promoting peptide;

[0062] m is 1 or 2;

[0063] n is 1 or 2;

[0064] The fusion protein is adapted to be cleaved by a cleavage enzyme to form the free thrombopoietin, which is not cleaved by the cleavage enzyme.

[0065] The inventors of this application, through screening proteins at the X position in fusion proteins, unexpectedly discovered that using a glargine insulin mutant can significantly improve the efficient expression of the fusion protein. Specifically, compared to glargine insulin (amino acid sequences shown in SEQ ID NO:6 and SEQ ID NO:12), the glargine insulin mutant of this application mutates lysine to arginine, effectively avoiding lysine-specific recognition. Furthermore, the glargine insulin mutant of this application retains all cysteine ​​residues, allowing the expressed fusion protein to easily form inclusion bodies. Additionally, compared to glargine insulin, the glargine insulin mutant introduces a MATTSTATTR sequence, which fuses to the N-terminus of glargine insulin, contributing to an increased fusion protein expression yield. Therefore, the fusion protein of this application is expressed in bacterial cells as inclusion bodies, eliminating the need for transmembrane transport and secretion, and exhibiting high expression in host bacteria. Moreover, the thrombopoietin is not cleaved by cleavage enzymes, avoiding non-specific enzymatic cleavage and ensuring the integrity of the thrombopoietin.

[0066] In addition, the inventors of this application also tried various proteins at position X, such as maltose-binding protein, hirudin, and TrX. The results showed that the fusion proteins formed by the above proteins and thrombopoietin could not be expressed efficiently, resulting in low expression levels of the prepared fusion proteins and low yields of the subsequently produced thrombopoietin, which was not suitable for industrial production.

[0067] A chain: FVNQHLCGSHLVEALYLVCGERGFFYTPKTRR (SEQ ID NO: 6).

[0068] Chain B: GIVEQCCTSICSLYQLENYCG (SEQ ID NO: 12).

[0069] According to embodiments of this application, the fusion protein may further include at least one of the following technical features:

[0070] According to embodiments of this application, the cleavage enzyme is selected from at least one of recombinant lysine endopeptidase, enterokinase, recombinant lysine endopeptidase, and recombinant carboxypeptidase B.

[0071] According to an embodiment of this application, the amino acid sequence of the thrombopoietin from the N-terminus to the C-terminus is as follows:

[0072] Cys-(Gly)p-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-(Gly)q-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala;

[0073] Where p and q are each an independent integer between 5 and 10.

[0074] According to an embodiment of this application, m is 1.

[0075] According to an embodiment of this application, n is 1.

[0076] According to an embodiment of this application, the amino acid sequence of the thrombopoietin is shown in SEQ ID NO:2.

[0077] CGGGGGIEGPTLRQWLAARAGGGGGGGGIEGPTLRQWLAARA (SEQ ID NO: 2).

[0078] According to embodiments of this application, the cleavage enzyme is selected from recombinant lysine endopeptidase.

[0079] According to embodiments of this application, the amino acid sequence of the enzyme cleavage site is DDDDK or K.

[0080] According to an embodiment of this application, the structure of formula (I) is XYZ or X-(YZ)2.

[0081] In one specific embodiment, the structure of formula (I) is X+Y+Z, and its amino acid sequence is shown in SEQ ID NO:3, where X is a glargine insulin mutant, Y is the enterokinase recognition sequence DDDDK, and Z is a thrombopoietin peptide. Thus, the fusion protein can be expressed efficiently.

[0082] In another specific embodiment, the structure of formula (I) is X+(Y+Z)2, and its amino acid sequence is shown in SEQ ID NO:4, where X is a glargine insulin mutant, Y is the recognition sequence K for recombinant lysine endopeptidase and recombinant carboxypeptidase B, and Z is a thrombopoietin-promoting peptide. Thus, the fusion protein can be expressed efficiently.

[0083] In another specific embodiment, the structure of formula (I) is X+Y+Z, and its amino acid sequence is shown in SEQ ID NO:5, where X is a glargine insulin mutant, Y is the recombinant lysine endopeptidase recognition sequence K, and Z is a platelet-producing peptide. Thus, the fusion protein can be expressed efficiently. Furthermore, compared with other structures, after dissolution with urea, the recombinant lysine endopeptidase of this fusion protein remains active, exhibiting high cleavage efficiency.

[0084] According to embodiments of this application, the amino acid sequence of the fusion protein is shown in any one of SEQ ID NO:3 to 5.

[0085] use

[0086] In a second aspect of this application, the use of the fusion protein described in the first aspect in the preparation of thrombopoietin or polyethylene glycol-modified thrombopoietin is proposed. The fusion protein described in the first aspect of this application is expressed in bacterial cells as inclusion bodies, requiring no transmembrane transport or secretion, and can be efficiently expressed in host bacteria. When the expressed fusion protein is cleaved using a cleaving enzyme, the thrombopoietin is not cleaved by the cleaving enzyme, avoiding non-specific enzymatic cleavage, thus ensuring the integrity of the thrombopoietin and exhibiting high cleavage efficiency.

[0087] Methods for preparing thrombopoietin

[0088] In a third aspect of this application, a method for preparing a thrombopoietin-promoting peptide is provided. According to an embodiment of this application, the method includes: obtaining inclusion bodies of bacterial cells, the inclusion bodies comprising the fusion protein described in the first aspect; and enzymatically digesting the fusion protein in the inclusion bodies to obtain the thrombopoietin-promoting peptide. The fusion protein described in the first aspect of this application is expressed in bacterial cells in the form of inclusion bodies, requiring no transmembrane transport or secretion, and can be efficiently expressed in host bacterial cells; the expressed fusion protein is cleaved using a cleaving enzyme, and the thrombopoietin-promoting peptide is not cleaved by the cleaving enzyme, thus avoiding non-specific enzymatic cleavage, ensuring the integrity of the thrombopoietin-promoting peptide, and exhibiting high cleavage efficiency.

[0089] According to embodiments of this application, the above method may further include at least one of the following technical features:

[0090] According to embodiments of this application, the bacterial cells are *Escherichia coli*. This allows for the acquisition of inclusion bodies with high expression levels, thereby enabling the production of large quantities of thrombopoietin-promoting peptides.

[0091] According to embodiments of this application, the enzymatic digestion is performed using a cleavage enzyme. Thus, a cleavage enzyme is selected based on the cleavage sites in the fusion protein, and the fusion protein is then enzymatically digested.

[0092] According to an embodiment of this application, the amino acid sequence of the cleavage site in the fusion protein is K, and the cleavage enzyme is selected from recombinant lysine endopeptidase. Therefore, a recombinant lysine endopeptidase capable of recognizing the cleavage site K is used for enzymatic digestion to obtain a thrombopoietin-promoting peptide.

[0093] According to embodiments of this application, in the enzymatic digestion system, the final concentration of the fusion protein is 5.0 mg / ml to 10.0 mg / ml, for example, 5.0 mg / ml, 5.1 mg / ml, 5.2 mg / ml, 5.3 mg / ml, 5.4 mg / ml, 5.5 mg / ml, 5.6 mg / ml, 5.7 mg / ml, 5.8 mg / ml, 5.9 mg / ml, 6.0 mg / ml, 6.1 mg / ml, 6.2 mg / ml, 6.3 mg / ml, 6.4 mg / ml, 6.5 mg / ml, 6.6 mg / ml, 6.7 mg / ml, 6.8 mg / ml, 6.9 mg / ml, 7.0 mg / ml, 7.1 mg / ml, 7.2 mg / ml, 7.3 mg / ml. 1, 7.4 mg / ml, 7.5 mg / ml, 7.6 mg / ml, 7.7 mg / ml, 7.8 mg / ml, 7.9 mg / ml, 8.0 mg / ml, 8.1 mg / ml, 8.2 mg / ml, 8.3 mg / ml, 8.4 mg / ml, 8.5 mg / ml, 8.6 mg / ml, 8.7 mg / ml, 8.8 mg / ml, 8.9 mg / ml, 9.0 mg / ml, 9.1 mg / ml, 9.2 mg / ml, 9.3 mg / ml, 9.4 mg / ml, 9.5 mg / ml, 9.6 mg / ml, 9.7 mg / ml, 9.8 mg / ml, 9.9 mg / ml, 10.0 mg / ml, and any two values ​​between them are considered as the range of values ​​between the endpoints.

[0094] According to embodiments of this application, the mass ratio of the fusion protein to the cleaving enzyme is 1:(1000-5000), for example 1:1000, 1:1250, 1:1500, 1:1750, 1:2000, 1:2500, 1:3000, 1:3500, 1:4000, 1:4500, 1:5000, and any two ratios thereof are used as a range between endpoints, such as 1:(1000-3000). This allows the cleaving enzyme (e.g., recombinant lysine endopeptidase) to adequately cleave the fusion protein.

[0095] According to an embodiment of this application, the temperature of the enzymatic digestion treatment is 25℃ to 35℃ (e.g., 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, and any two values ​​between them as endpoint values), and the time is 20h to 30h (e.g., 20h, 21h, 22h, 23h, 24h, 25h, 26h, 27h, 28h, 29h, 30h, and any two values ​​between them as endpoint values).

[0096] According to embodiments of this application, the fusion protein is denatured before the enzymatic digestion process. This denaturation process allows incorrect folding or faulty disulfide bonds in the fusion protein to be opened, facilitating the subsequent formation of a correct folded structure.

[0097] According to an embodiment of this application, the denaturation treatment is carried out under conditions of denaturant and detergent.

[0098] According to embodiments of this application, the denaturing agent includes urea at concentrations of 2 mol / L to 8 mol / L (e.g., 2 mol / L, 3 mol / L, 4 mol / L, 5 mol / L, 6 mol / L, 7 mol / L, 8 mol / L, and any two values ​​between them as endpoints), and urea at concentrations of 20 mM to 80 mM. Tris (e.g., 20 mol / L, 25 mol / L, 30 mol / L, 35 mol / L, 40 mol / L, 45 mol / L, 50 mol / L, 55 mol / L, 60 mol / L, 65 mol / L, 70 mol / L, 75 mol / L, 80 mol / L, and any two values ​​between them as endpoints), and the pH value of the denaturant is 8.5 to 9.5 (e.g., 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, and any two values ​​between them as endpoints).

[0099] According to embodiments of this application, the enzyme digestion product is subjected to reduction treatment. Thus, the reduction treatment allows the fusion protein to recover from its fully extended denatured state to its normal folded structure, thereby enabling the fusion protein to form correct disulfide bonds.

[0100] According to an embodiment of this application, the reduction process is carried out in a reducing agent.

[0101] According to embodiments of this application, the reducing agent is selected from at least one of TCEP, DTT, and β-mercaptoethanol.

[0102] According to embodiments of this application, the reducing agent is selected from TCEP. Since the glargine insulin mutant contains six cysteine ​​residues, and the first residue at the N-terminus of the thrombopoietin is cysteine, the seven cysteine ​​residues readily form disulfide bonds randomly, or form various disulfide bond products, leading to the loss of the thrombopoietin. Therefore, adding TCEP to the cleaved sample can improve the yield of the thrombopoietin.

[0103] According to embodiments of this application, the molar ratio of the thrombopoietin to the reducing agent is (1-3):1, for example 1.0:1, 1.5:1, 2.0:1, 2.5:1, 3.0:1, and any two ratios therebetween, as a range between endpoint values. This further allows the fusion protein to recover from its fully extended denatured state to its normal folded structure, facilitating the formation of correct disulfide bonds.

[0104] According to an embodiment of this application, the method further includes: purifying the reduction product.

[0105] According to an embodiment of this application, the purification process is performed using chromatographic chromatography.

[0106] Method for preparing PEGylated thrombopoietin

[0107] In a fourth aspect of this application, a method for preparing polyethylene glycol-modified thrombopoietin is provided. According to embodiments of this application, the method includes: coupling the thrombopoietin with polyethylene glycol to obtain the polyethylene glycol-modified thrombopoietin; the thrombopoietin is prepared using the fusion protein described in the first aspect, or prepared according to the method described in the third aspect. The fusion protein described in the first aspect of this application is expressed in bacterial cells as inclusion bodies, requiring no transmembrane transport or secretion, and can be efficiently expressed in host bacteria; the expressed fusion protein is cleaved using a cleaving enzyme, while the thrombopoietin is not cleaved by the cleaving enzyme, avoiding non-specific enzymatic cleavage, thus ensuring the integrity of the thrombopoietin and achieving high cleavage efficiency. Therefore, the above method has the advantages of simple preparation and high yield.

[0108] Furthermore, the PEGylated thrombopoietin prepared by the above method exhibits high rhuMpl (c-Mpl) receptor activity. According to literature reports and the general understanding of those skilled in the art, polyethylene glycol (PEG) modification of proteins and peptides leads to a significant decrease in activity. For example, PEG-rhEGF retains 60-70% of the biological activity of natural rhEGF (Effect of Molecular Size of PEGylated Recombinant Human Epidermal Growth Factor on the Biological Activity and Stability in Rat Wound Tissue); PEGylated tachyplesin I, compared to tachyplesin I, exhibits 64-fold and 32-fold weaker antibacterial activity against Escherichia coli and Staphylococcus epidermidis, respectively (Action mechanism of tachyplesin I and effects of PEGylation); the activities of PEGylated HR2 Peptides and PEGylated asparaginase also decrease to varying degrees with the number of PEG modifications and the molecular weight of PEG (Site-Specific PEGylation of HR2 Peptides: Effects of PEG Conjugation Position and Chain Length on HIV-1 Membrane Fusion Inhibition). and Proteolytic Degradation; WO 2011 / 003633 A1). However, unexpectedly, the activity of the polyethylene glycol thrombopoietin obtained in this application was enhanced by 15-20 times compared with the unmodified form. The possible reasons are as follows: (1) The polyethylene glycol thrombopoietin uses a unique modification site, with Cys introduced at the N-terminus as the modification site; (2) The unique tandem structure of the thrombopoietin; (3) The unique polyethylene glycol modification (1 polyethylene glycol modifies 2 thrombopoietins), forming a spatial homotetramer.

[0109] In this article, "polyglycolated platelet-forming peptide" and "polyglycolated platelet-forming peptide" are synonymous.

[0110] According to embodiments of this application, the above method may further include at least one of the following technical features:

[0111] According to embodiments of this application, the molar ratio of the thrombopoietin to polyethylene glycol is 1:(0.4 to 0.6), for example 1:0.4, 1:0.5, 1:0.6, and any two of these ratios are used as a range between endpoint values.

[0112] According to an embodiment of this application, the molar ratio of the thrombopoietin to polyethylene glycol is 1:(0.4-0.5).

[0113] According to an embodiment of this application, the coupling treatment is performed in a phosphate buffer solution with a pH of 5.5 to 7.5.

[0114] According to an embodiment of this application, the pH value of the phosphate buffer solution is 5.5 to 6.5, for example, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, and any two values ​​between them are used as the range between the endpoint values.

[0115] According to an embodiment of this application, prior to the coupling treatment, the thrombopoietin and TCEP are pre-mixed.

[0116] According to embodiments of this application, the molar ratio of the thrombopoietin to TCEP is 1:(0.5-2.0), for example 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2.0, and any two ratios therebetween are used as the range between the endpoint values.

[0117] According to an embodiment of this application, the molar ratio of the thrombopoietin to TCEP is 1:(1.0 to 1.5).

[0118] According to embodiments of this application, the process further includes purifying the coupling treatment product.

[0119] According to an embodiment of this application, the purification process is performed using chromatographic chromatography.

[0120] According to embodiments of this application, the molecular weight of the polyethylene glycol is 5 to 50 kDa, for example, 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, and any two values ​​between them as the range between the endpoints.

[0121] According to embodiments of this application, the molecular weight of the polyethylene glycol is 15-25 kDa.

[0122] Injectable solutions and lyophilized preparations

[0123] In a fifth aspect, this application provides an injectable solution. According to embodiments of this application, the injectable solution comprises: a polyethylene glycol-modified thrombopoietin, a buffer salt, and a protective agent. By adding the above excipients, the injectable solution of this application effectively avoids the charge heterogeneity caused by ring-opening of maleimide in the polyethylene glycol-modified thrombopoietin, and also has the advantage of good storage stability.

[0124] According to embodiments of this application, the above-mentioned injection solution may further include at least one of the following technical features:

[0125] According to embodiments of this application, the polyethylene glycolated thrombopoietin is prepared using the fusion protein described in the first aspect, or prepared according to the method described in the fourth aspect.

[0126] According to an embodiment of this application, the final concentration of the PEGylated thrombopoietin, based on the total mass of the injection solution, is 0.2 mg / ml to 1.0 mg / ml, for example, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml, 1.0 mg / ml, and any two values ​​between them as endpoints.

[0127] According to an embodiment of this application, the pH value of the injection solution is 2.5 to 4.5, for example, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, and any two values ​​between them are used as the range between endpoint values.

[0128] According to an embodiment of this application, the final concentration of the buffer salt in the injection solution is 10 mmol / L to 100 mmol / L, for example, 10 mmol / L, 20 mmol / L, 30 mmol / L, 40 mmol / L, 50 mmol / L, 60 mmol / L, 70 mmol / L, 80 mmol / L, 90 mmol / L, 100 mmol / L, and any two values ​​between them as the range between the endpoints.

[0129] According to embodiments of this application, the buffer salt is selected from at least one of histidine, glycine, citrate-sodium citrate, or acetate-sodium acetate.

[0130] According to embodiments of this application, the buffer salt includes histidine and glycine.

[0131] According to an embodiment of this application, the buffer salt comprises 20 mmol / L glycine and 20 mmol / L histidine.

[0132] According to an embodiment of this application, the mass percentage of the protective agent is 1% to 10% based on the total mass of the injection solution, for example, 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, 10.0%, and any two values ​​between them as a range between endpoint values.

[0133] According to embodiments of this application, the protective agent accounts for 6% to 8% of the total mass of the injection solution, for example.

[0134] According to embodiments of this application, the protective agent is selected from at least one of sucrose, trehalose, and mannitol.

[0135] According to embodiments of this application, the protective agent is selected from sucrose and mannitol.

[0136] According to embodiments of this application, the mass ratio of sucrose to mannitol is 1:(1-3), for example 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2.0, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, 1:3.0, and any two of these ratios are used as a range of values ​​between endpoints.

[0137] In one optional embodiment of this application, the mass ratio of sucrose to mannitol is 1:2.

[0138] According to embodiments of this application, the injection solution further includes a pH adjuster.

[0139] According to an embodiment of this application, the injection solution comprises:

[0140] 0.2–1.0 mg / ml of pegylated thrombopoietin.

[0141] 2% (w / w) sucrose,

[0142] 4% (w / w) mannitol,

[0143] 20 mmol / L histidine

[0144] 20 mmol / L glycine

[0145] The pH value of the injection solution is 2.5 to 4.5.

[0146] In a sixth aspect, this application provides a lyophilized formulation. According to an embodiment of this application, the lyophilized formulation is obtained by lyophilizing the injection solution described in the fifth aspect. The lyophilized formulation of this application, through the addition of the aforementioned excipients, effectively avoids the problems of charge heterogeneity and poor storage stability caused by the ring-opening of maleimide in polyethylene glycol-modified thrombopoietin.

[0147] Uses and methods of treating diseases

[0148] In a seventh aspect of this application, the application discloses the use of a polyethylene glycol-modified thrombopoietin peptide, said use being for:

[0149] Treatment and / or prevention of disease, and / or

[0150] To prepare medicines for the treatment and / or prevention of diseases;

[0151] The diseases mentioned include treatment and prevention of tumor drug-induced thrombocytopenia, primary immune thrombocytopenia, chronic liver disease-related thrombocytopenia, and acute radiation syndrome-hematopoietic syndrome.

[0152] In an eighth aspect of this application, a method for treating and / or preventing a disease is proposed. According to an embodiment of this application, the method includes administering to a subject a pharmaceutically acceptable dose of a PEGylated thrombopoietin, wherein the disease includes the treatment and prevention of oncology drug-induced thrombocytopenic purpura, primary immune thrombocytopenic purpura, chronic liver disease-related thrombocytopenic purpura, and acute radiation syndrome-hematopoietic syndrome.

[0153] In this document, the term "treatment and prevention of oncology drug-related thrombocytopenia" refers to thrombocytopenia caused by oncology drugs used in the treatment and prevention of cancer. Oncology drugs include, but are not limited to, chemotherapy drugs (chemotherapy alone), radiotherapy, and biological drugs (e.g., monoclonal antibodies, multispecific antibodies, CAR-immune cells, TCR-immune cells, ADC drugs), etc., without special restrictions, and are all within the scope of protection of this application. The term "acute radiation syndrome hematopoietic syndrome" refers to bone marrow suppression caused by high-dose penetrating radiation irradiating the whole body within a short period of time during radiotherapy, chemotherapy, radiological accidents, and nuclear warfare, resulting in an inability to produce a sufficient number of new white blood cells, red blood cells, and platelets.

[0154] According to embodiments of this application, the above-described uses and methods may further include at least one of the following technical features:

[0155] According to embodiments of this application, the polyethylene glycolated thrombopoietin is prepared using the fusion protein described in the first aspect, or prepared according to the method described in the fourth aspect.

[0156] According to embodiments of this application, the polyethylene glycolated thrombopoietin is provided as a lyophilized formulation.

[0157] In this document, the term "lyophilized formulation" refers to an injection solution obtained by dissolving the lyophilized formulation in a reconstitution buffer. The reconstitution buffer includes, but is not limited to, physiological saline and sterile water for injection; the specific type is not limited and is within the scope of protection of this application.

[0158] According to embodiments of this application, the effective dose of the PEGylated thrombopoietin is 6 μg to 100 μg, for example, 6 μg, 7 μg, 8 μg, 9 μg, 10 μg, 11 μg, 12 μg, 13 μg, 14 μg, 15 μg, 16 μg, 17 μg, 18 μg, 19 μg, 20 μg, 21 μg, 22 μg, 23 μg, 24 μg. 25μg, 26μg, 27μg, 28μg, 29μg, 30μg, 31μg, 32μg, 33μg, 34μg, 35μg, 36μg, 37μg, 38 μg, 39μg, 40μg, 41μg, 42μg, 43μg, 44μg, 45μg, 46μg, 47μg, 48μg, 49μg, 50μg, 51μg ,52μg,53μg,54μg,55μg,56μg,57μg,58μg,59μg,60μg,61μg,62μg,63μg,64μg,6 5μg, 66μg, 67μg, 68μg, 69μg, 70μg, 71μg, 72μg, 73μg, 74μg, 75μg, 76μg, 77μg, 78μ g, 79μg, 80μg, 81μg, 82μg, 83μg, 84μg, 85μg, 86μg, 87μg, 88μg, 89μg, 90μg, 91μg, 92μg, 93μg, 94μg, 95μg, 96μg, 97μg, 98μg, 99μg, 100μg, and any two values ​​between them are considered as the range of values ​​between the endpoints.

[0159] According to embodiments of this application, the PEGylated thrombopoietin is administered only once per chemotherapy cycle, or once a week for patients with chronic liver disease before surgery, to treat primary immune thrombocytopenia.

[0160] In an optional embodiment of this application, the pegylated thrombopoietin has the efficacy of increasing and maintaining the peripheral blood platelet count in patients with drug-related thrombocytopenic purpura (DTCP) for the treatment and prevention of tumors with only one dose per chemotherapy cycle until the end of chemotherapy. Specifically:

[0161] (1) Polyethylene glycol-modified thrombopoietin can significantly alleviate the peripheral blood platelet count in BALB / c mice induced by whole-body irradiation with 60Co (2.5Gy) combined with intraperitoneal injection of carboplatin (50mg / kg), and can alleviate the decrease in platelet count, red blood cell count and hemoglobin content caused by modeling in three consecutive radiotherapy and chemotherapy cycles.

[0162] (2) In a phase I clinical study of pegylated thrombopoietin in healthy Chinese subjects, the peripheral blood platelet count was significantly increased after a single subcutaneous administration of PN20 in the 0.05–0.2 μg / kg dose groups. The increase was greater with increasing dosage. Peripheral blood platelet counts began to rise in all dose groups from day 3 to day 5 after administration, reached their highest value on day 11 to day 13, began to decline on day 15, and returned to normal on day 21. The "Chinese Expert Consensus on the Diagnosis and Treatment of Chemotherapy-Induced Thrombocytopenia" and the "Chinese Expert Consensus on the Diagnosis and Treatment of Chemotherapy-Related Thrombocytopenia" report that in patients with chemotherapy-induced thrombocytopenia, platelet counts begin to decline 3 to 5 days after chemotherapy. Endogenous TPO slowly increases, and it takes at least 5 to 7 days for TPO to raise peripheral blood platelet counts. Normal platelet lifespan is 8 to 10 days. Patients with chemotherapy-induced thrombocytopenia (CIT) generally begin to experience thrombocytopenia on day 7 of chemotherapy, reaching its lowest point on day 14, and recovering to baseline levels between days 28 and 35. This is complementary to the pattern of pegylated thrombopoietin (PTP) increasing peripheral blood platelet counts in healthy subjects, suggesting that this product may improve the low platelet count in CIT patients, raise and maintain platelet counts up to day 21 after chemotherapy, while avoiding the risk of high platelet counts due to natural platelet recovery after chemotherapy.

[0163] (3) The results of the Phase Ib clinical study of PN20 in chemotherapy-induced thrombocytopenia showed that the platelet count of all subjects in the 0.2 μg / kg and 0.5 μg / kg dose groups increased from baseline and the subjects were safe. The platelet count of the 0.5 μg / kg dose group showed a significant change from baseline, indicating preliminary efficacy.

[0164] In one optional embodiment of this application, the PEGylated thrombopoietin has the characteristic of treating chronic liver disease-related thrombocytopenia (CLD), including the ability to improve peripheral blood platelet counts with only a single dose before elective surgery in patients with CLD. Specifically, the PEGylated thrombopoietin has a significant therapeutic effect on thrombocytopenia in rat models of chronic liver disease-induced thrombocytopenia, and the magnitude and duration of PLT elevation are dose-related. After a single administration of different doses of PEGylated thrombopoietin, PLT began to increase 3 days after administration, with the peak PLT occurring on the 9th day after administration; the duration of PLT was approximately 8 days, and the improvement of thrombocytopenia response in model animals by PEGylated thrombopoietin showed a clear dose-related effect. Based on clinical experience with similar drugs, it is expected that PEGylated thrombopoietin can be administered subcutaneously before surgery to improve platelet levels in CLD patients and provide a longer time window for scheduling elective surgery, thereby providing patients with a more effective and convenient alternative to preoperative platelet correction.

[0165] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0166] Example 1: Construction of expression vector

[0167] 1. Based on the amino acid sequence of the fusion protein (as shown in SEQ ID NO:5), the design was optimized according to the E. coli codon. An Nde I restriction site was designed at the 5' end and a BamHI restriction site was designed at the 3' end as insertion sites. The nucleotide sequence was artificially synthesized into the whole genome and inserted into the pUC-57 vector, named pUC-57-GI-NPC.

[0168] Using pUC-57-GI-NPC plasmid as a template, the target fragment was recovered by double digestion with NdeI and BamHI restriction enzymes. This fragment was then ligated with a fragment recovered from plasmid pET-30a (purchased from Novagen) by NdeI and BamHI restriction enzyme digestion using T4 DNA ligase. The ligation was performed on this fragment and transformed into the Top10 cloning host strain of *E. coli*. The recombinant plasmid pET-30a-GI-NPC was screened using enzyme digestion and PCR verification (see Figure 1). This plasmid was then transformed into the expression host strain BL21(DE3) (Novagen). After expression screening, the recombinant expression strain, pET-30a-GI-NPC / BL21(DE3), was obtained, expressing fusion protein 1.

[0169] 2. Construction of other expression vectors

[0170] Different fusion proteins were constructed following step 1 of this embodiment to investigate their expression levels. The amino acid sequences of other fusion proteins are shown below:

[0171] Maltose-binding protein (MBP), lysine (restriction site) and thrombopoietin (amino acid sequence as shown in SEQ ID NO:2) are fused and expressed, and their amino acid sequence is shown in SEQ ID NO:7, namely pET-30a-MBP-NPC / BL21(DE3) recombinant engineered bacteria, expressing fusion protein 2;

[0172] Hirudin, lysine (enzyme cleavage site), and thrombopoietin (amino acid sequence as shown in SEQ ID NO:2) were fused and expressed, and their amino acid sequence is shown in SEQ ID NO:8, namely pET-30a-Hirudin-NPC / BL21(DE3) recombinant engineered bacteria, expressing fusion protein 3;

[0173] The direct expression of the thrombopoietin (amino acid sequence as shown in SEQ ID NO:2, i.e. without X and restriction enzyme sites) was achieved by the pET-30a-NPC / BL21(DE3) recombinant engineered bacteria expressing the thrombopoietin.

[0174] Example 2: Fermentation culture of fusion protein and preparation of thrombopoietin-promoting peptide

[0175] 1. The pET-30a-GI-NPC / BL21(DE3) recombinant engineered bacteria, pET-30a-MBP-NPC / BL21(DE3) recombinant engineered bacteria, pET-30a-Hirudin-NPC / BL21(DE3) recombinant engineered bacteria, and pET-30a-NPC / BL21(DE3) recombinant engineered bacteria that showed the best expression in Example 1 were streaked onto LB agar plates containing kanamycin and incubated overnight at 37°C. Bacterial growth was picked from the overnight kanamycin-containing LB agar plates and inoculated into test tubes containing LB liquid medium (10g tryptone, 5g yeast extract, 10g NaCl, diluted to 1000ml with water, autoclaved at 121°C for 30min). The culture was incubated at 37°C for 12 hours, and then transferred at a ratio of 1% to 200ml of LB medium in a 1000ml Erlenmeyer flask and incubated overnight at 37°C to obtain the seed culture for the next incubation. The seed culture from the upper tank was inoculated at a ratio of 5% into a 30L fermenter containing YT culture medium (4g / L tryptone, 3g / L yeast extract, 30g / L Na2HPO4·12H2O, 3g / L KH2PO4, 1g / L NaCl, 1g / L MgSO4·7H2O, 8g / L glucose, autoclaved at 121℃ for 30min). The fermentation was carried out at 30℃. Throughout the fermentation process, dissolved oxygen was maintained above 30% by adjusting the rotation speed, aeration rate, and pure oxygen supply. The pH was adjusted using 28% ammonia and maintained at 7.0. (OD of the bacterial culture...) 600 When the concentration reaches 10-14, add IPTG (isopropyl-β-D-thiogalactoside) to a final concentration of 0.2 mM, continue culturing for 4 hours, then stop fermentation. Centrifuge at 8000 rpm for 10 minutes, discard the supernatant, collect the cells, and store them in a -20℃ refrigerator for later use.

[0176] 2. Collect the bacterial cells expressing different fusion proteins or thrombopoietin from step 2 of this embodiment. Take a certain amount of bacterial cells and resuspend them at a mass ratio of bacterial cells to buffer (100 mmol / L Tris, 5 mmol / L EDTA, pH 9.0–9.5) of 1:20. Homogenize at 500–700 Ba 3–5 times and centrifuge to collect the precipitate. Then, disperse and dissolve the bacterial cells at a mass ratio of bacterial cells to buffer (50 mm Tris, 6 mol / L urea, pH 8.5–9.5) of 1:20 and centrifuge to collect the supernatant. Digest the inclusion body solution obtained from a certain amount of bacterial cells with a protease lysine endopeptidase (Lys-C) to fusion protein mass ratio of 1:1000 and a protein concentration of 10 mg / ml to obtain crude thrombopoietin. The protein content results are shown in the table below (protein content was determined by HPLC). The yield of crude thrombopoietin is as follows:

[0177] Example 3: Comparison of platelet-producing peptide digestion conditions

[0178] 1. Take 20g of the bacterial cells preserved in Example 2, and resuspend them at a mass ratio of 1:20 of bacterial cells to buffer solution (100mmol / L Tris, 5mmol / L EDTA, pH 9.0-9.5). Homogenize at 500-700 Ba 3-5 times, centrifuge and collect the precipitate. Then disperse and dissolve the bacterial cells at a mass ratio of 1:20 of bacterial cells to buffer solution (50mm Tris, 6mol / L urea, pH 8.5-9.5), centrifuge and collect the supernatant.

[0179] The supernatant was mixed with different concentrations of fusion protein and different amounts of protease, and then subjected to enzymatic digestion to obtain the content of thrombopoietin. The concentration of fusion protein, amount of protease, digestion time, and content of thrombopoietin during the enzymatic digestion process are shown in Table 1. The results showed that the content of thrombopoietin was higher when the mass ratio of protease to fusion protein was 1:1000–1:5000.

[0180] Table 1

[0181] 2. Take 500g of the bacterial cells preserved in Example 2, and resuspend them at a mass ratio of bacterial cells to buffer (100mmol / L Tris, 5mmol / L EDTA, pH 9.0–9.5) of 1:20. Homogenize at 500–700 Ba 3–5 times, and centrifuge to collect the precipitate. Then, disperse and dissolve the precipitate at a mass ratio of bacterial cells to buffer (50mm Tris, 6mol / L urea, pH 8.5–9.5) of 1:20, and centrifuge to collect the supernatant. Finally, digest all the inclusion body lysate obtained from 500g of bacterial cells with a protease to fusion protein mass ratio of 1:2000 and a protein concentration of 10mg / ml to obtain 5g of crude thrombopoietin. The typical cleavage chromatogram is shown in HPLC Figure 2 (NPC is thrombopoietin, GI-K is glargine insulin mutant and lysine).

[0182] Example 4: Platelet-Promoting Peptide Ion Chromatography

[0183] 1. Take 430 mg of the crude thrombopoietin obtained in step 2 of Example 3. Sample 1 was not treated with a reducing agent. Sample 2 was treated with TCEP (tris(2-formylethyl)phosphine hydrochloride) at a mass ratio of 2:1, and the mixture was stirred for 30 minutes. Sample 3 was treated with TCEP at a mass ratio of 1:1, and the mixture was stirred for 30 minutes. Then, the pH of samples 1, 2, and 3 was adjusted to 9.0–9.5. The Q anion exchange chromatography column was washed and equilibrated using standard methods, and the breakthrough samples were collected. The breakthrough sample results are shown in Table 2.

[0184] Table 2

[0185] 2. Take 5g of crude thrombopoietin (NPC) (preparation method see step 2 of Example 3), add it to the mixture at a mass ratio of NPC to TCEP of 2:1, stir for 1 hour, adjust the pH to 6.5-7.0, centrifuge and collect the supernatant. Adjust the pH of the supernatant to 3.0-3.5, and perform Source 30S chromatography with a loading of no more than 10mg / ml. Solution A is 20mmol / L HAC, 30% ACN, and solution B is 20mmol / L HAC, 30% ACN, 0.3mol / L NaCl. Elute with a linear gradient of 0-50% B for 10CV. Collect the UV absorption peak to obtain 3g of high-purity NPC stock solution. The HPLC detection chromatogram is shown in Figure 3.

[0186] Example 5: Coupling of thrombopoietin with polyethylene glycol

[0187] 1. Take the platelet-producing peptide stock solution obtained in step 2 of Example 4, and mix the platelet-producing peptide with TCEP at a molar ratio of 1:1. Add phosphate buffer solutions with different pH values ​​(pH values ​​of 5.5, 6.5, and 7.5) at an initial concentration of 0.2 mol / L to the platelet-producing peptide mixture to make the final concentration of phosphate buffer solution 30 mmol / L. Dissolve bi-terminated maleimide polyethylene glycol (20 kDa) and perform a coupling reaction at a molar ratio of platelet-producing peptide to polyethylene glycol of 1:0.4. Under different pH values ​​(5.5–7.5), stir at room temperature for 1 hour to obtain polyethylene glycol-modified platelet-producing peptide.

[0188] The effect of different pH values ​​on coupling efficiency is shown in Table 3. The results showed that when the pH value of the coupling reaction was 5.5 to 6.5, the PEGylated thrombopoietin had fewer impurities B and C.

[0189] Table 3

[0190] 2. Study on the relationship between TCEP and coupling reaction efficiency

[0191] The platelet-producing peptide stock solution obtained in step 2 of Example 4 was divided into three equal parts, with or without TCEP, or with different amounts of TCEP. Then, 0.2 mol / L phosphate buffer (pH 6.0) was added to the platelet-producing peptide stock solution or mixture to bring the final concentration of the phosphate buffer to 30 mmol / L. Bis-terminated maleimide polyethylene glycol (20 kDa) was dissolved and coupled at a molar ratio of 1:0.5 to the platelet-producing peptide. The reaction was carried out at room temperature for 1 hour at pH 6.0 with stirring to obtain PEGylated platelet-producing peptide. The content of the platelet-producing peptide, the TCEP feed ratio (molar ratio of TCEP to platelet-producing peptide), the yield of the target product, and the impurity content are shown in Table 4.

[0192] The results showed that the addition of TCEP could effectively suppress the generation of impurities B and C.

[0193] Table 4

[0194] 3. Coupling reaction efficiency with different feed ratios

[0195] The thrombopoietin stock solution obtained in step 2 of Example 4 was mixed with TCEP at a molar ratio of 1:1. Phosphate buffer (pH 6.0) with an initial concentration of 0.2 mol / L was added to the thrombopoietin mixture to bring the final concentration of the phosphate buffer to 30 mmol / L. Bis-terminated maleimide polyethylene glycol (20 kDa) was dissolved, and a coupling reaction was carried out according to different molar ratios (feed ratios) of thrombopoietin to polyethylene glycol. The reaction was carried out at room temperature and stirred for 1 hour at pH 6.0 to obtain polyethylene glycol-modified thrombopoietin. The content of thrombopoietin, polyethylene glycol feed ratio, target product yield, and impurity content are shown in Table 5.

[0196] Table 5

[0197] 4. Take 2g of the thrombopoietin obtained in step 2 of Example 4, mix the thrombopoietin with TCEP at a molar ratio of 1:1, and add 0.2mol / L phosphate buffer (pH 6.0) to the thrombopoietin mixture to make the final concentration of phosphate buffer 30mmol / L. Dissolve the bi-terminated maleimide polyethylene glycol (20kDa) and add it at a molar ratio of 1:0.5 to thrombopoietin for coupling reaction. Stir the reaction at room temperature for 1 hour at pH 6.0, and then terminate the reaction to obtain 1.6g of the modified product (PEG20K-NPC). The HPLC chromatogram is shown in Figure 4.

[0198] Example 6: Purification of PEGylated thrombopoietin

[0199] Take 1g of polyethylene glycolated thrombopoietin (preparation method see step 4 in Example 5), and perform Source 30S chromatography with a loading of no more than 5mg / ml. Solution A is 20mmol / L HAC; solution B is 20mmol / L HAC, 0.3mol / L NaCl, elute with a 0-30% B gradient for 10CV, collect the UV absorption peak, and obtain 0.6g of high-purity sample.

[0200] Take an ultrafiltration column (Minikros Sampler (5kDa)) and prepare an ultrafiltration buffer (20 mmol / L His, pH 5.0–5.5). For the first ultrafiltration, concentrate the 0.6 g sample concentration to 3–5 mg / ml, add buffer to dilute 4 times, and continue ultrafiltration to 3–5 mg / ml. Repeat 3–5 times to obtain the stock solution, which is PEGylated thrombopoietin PN20. The HPLC detection chromatogram is shown in Figure 5.

[0201] PN20-Mo is a single-end coupling product of NPC and PEG. PN20-Mo is also generated during the preparation of PN20. High-purity PN20-Mo can be effectively separated and obtained by the Source30S chromatography method in Example 4.

[0202] Example 7: Screening of pegylated thrombopoietin formulations

[0203] (1) Prescription design

[0204] According to Table 6 of the formulation design, the prescribed amounts of histidine, glycine, mannitol, and sucrose were added to the polyethylene glycolated thrombopoietin PN20 stock solution prepared in Example 6. The pH value was adjusted to 4.0 using pH adjusters (sodium hydroxide and hydrochloric acid). After aseptic filtration through a 0.22 μm microporous filter, a portion was filled into vials, capped, and labeled to obtain the polyethylene glycolated thrombopoietin injection. The other portion was filled into vials, freeze-dried, capped, and labeled to obtain the polyethylene glycolated thrombopoietin lyophilized preparation.

[0205] Table 6: Formulation Table of Polyethylene Glycolized Thromboplastin Injection and Lyophilized Preparation

[0206] (2) Examination conditions, examination time and testing indicators

[0207] Examination conditions 1: 37℃, duration: 1 month;

[0208] Observation conditions 2: 4℃, duration 1 month;

[0209] Detection indicator: related substances.

[0210] (3) The results are shown in Table 7:

[0211] Table 7: Results of the two dosage forms

[0212] Results analysis: The related substance chromatograms of the injection and lyophilized formulations after 1 month at 37℃ are shown in Figure 6; no significant difference in stability was observed between the two dosage forms after 1 month at 4℃; after 1 month at 37℃, related substance 1 was detected in 5.5% and related substance 2 was detected in 1.46% of the injection, while related substance 1 was not detected in the lyophilized formulation and related substance 2 was detected in 1.43% of the lyophilized formulation. Under the same conditions, the lyophilized formulation was more stable than the injection.

[0213] Example 8: Screening of pegylated thrombopoietin formulations with different pH buffers

[0214] (1) Prescription design

[0215] According to Table 8 of the formulation design, the prescribed amounts of histidine, mannitol, sucrose, glycine, citric acid, sodium citrate, acetic acid, and sodium acetate were added to the polyethylene glycolated thrombopoietin PN20 stock solution prepared in Example 6. The pH was adjusted to the required value with pH adjusters (sodium hydroxide and hydrochloric acid). The solution was then aseptically filtered through a 0.22 μm microporous filter and filled into vials. After freeze-drying, capping, and labeling, the polyethylene glycolated thrombopoietin lyophilized formulation was obtained.

[0216] Table 8: Screening prescriptions for pegylated thrombopoietin-promoting peptides with different pH buffers

[0217] (2) Sample investigation

[0218] (3) Results of the investigation

[0219] The results of the investigation are shown in Table 9:

[0220] Table 9: Statistical analysis of screening results for PEGylated thrombopoietin-promoting peptides with different pH buffers

[0221] The pH buffers were glycine-histidine (Formula 1), citric acid-sodium citrate (Formula 2), acetate-sodium acetate (Formula 3), and histidine (Formula 4). No related substances were detected in any of these buffers after one month of testing at 4°C. After one month of testing at 37°C, the maximum related substance concentration (MRC) was 2.28% for the histidine-glycine buffer and 3.16% for the total related substances; the maximum MRC concentration (MRC) was 4.61% for the citric acid-sodium citrate buffer and 6.31% for the total related substances; the maximum MRC concentration (MRC) was 3.22% for the acetate-sodium acetate buffer and 5.13% for the total related substances; and the maximum MRC concentration (MRC) was 2.92% for the histidine buffer and 4.44% for the total related substances.

[0222] Example 9: Screening of different pH formulations of pegylated thrombopoietin-promoting peptides

[0223] (1) Prescription design

[0224] According to Table 10 of the formulation design, the prescribed amounts of histidine, glycine, mannitol, and sucrose were added to the polyethylene glycolated thrombopoietin PN20 stock solution prepared in Example 6, and the pH was adjusted to the required value with pH adjusters (sodium hydroxide and hydrochloric acid). The solution was then aseptically filtered through a 0.22 μm microporous filter, filled into vials, and freeze-dried, capped, and labeled to obtain the polyethylene glycolated thrombopoietin lyophilized formulation.

[0225] Table 10: Prescriptions of PEGylated thrombopoietin at different pH values

[0226] (2) Sample investigation

[0227] (3) Results of the investigation

[0228] The results of charge heterogeneity for prescriptions 1 and 2 are shown in Table 11:

[0229] Table 11: Results of charge heterogeneity detection for prescriptions 1 and 2

[0230] The charge heterogeneity detection spectra of prescriptions 1 and 2 are shown in Figure 7. The formulation showed an acidic variation peak under the condition of weakly alkaline pH (pH 7.5), and no acidic or alkaline variation peaks were observed under the condition of weakly acidic pH (pH 4.0).

[0231] This product contains an imide group. Under weakly alkaline conditions, the CN bond of the imide will break, resulting in the ring-opening of maleimide in this product, thus causing charge heterogeneity. Controlling the pH of the solution to be weakly acidic or acidic can effectively avoid the formation of charge heterogeneous impurities caused by the ring-opening of maleimide.

[0232] The results of the examination of prescriptions 3, 4, and 5 are shown in Table 12:

[0233] Table 12: Screening and Testing Results of pH Ranges for Prescriptions 3, 4, and 5

[0234] When tested at 4℃ for 15 and 30 days, no related substances were detected in any of the three batches of samples; when tested at 37℃ for 15 and 30 days, related substances 1 and 2 were detected in all three batches of samples.

[0235] Example 10: Screening of Protective Agent Dosage for Pegylated Thromboplastin-Promoting Peptide Formulations

[0236] (1) Prescription design

[0237] According to the formulation design table 13, the prescribed amounts of histidine, mannitol, sucrose, and glycine were added to the polyethylene glycolated platelet-producing peptide PN20 stock solution prepared in Example 6. The pH was adjusted to the required value with pH adjusters (sodium hydroxide and hydrochloric acid). The solution was aseptically filtered through a 0.22 μm microporous filter, filled into vials, and then freeze-dried, capped, and labeled to obtain the polyethylene glycolated platelet-producing lyophilized formulation.

[0238] Table 13: Screening Prescription Table for Dosage of Pegylated Platelet-Promoting Peptides and Excipients

[0239] (2) Sample investigation

[0240] (3) Results of the investigation

[0241] The results of appearance, osmolality, and related substance detection are shown in Table 14:

[0242] Table 14: Statistical analysis of appearance and related substances in the screening results of PEGylated thrombopoietin protective agents

[0243] Example 11: Effects of pegylated thrombopoietin on ECGs in Baf-3-rhuMpl receptor cells 50 Measurement

[0244] Baf-3-rhuMpl receptor cells were constructed by recombinantly transfecting Baf-3 cells with the rhuMpl (c-Mpl) receptor. The bioactivity of the thrombopoietin NPC, the polyethylene glycol monomodifier PN20-mo (one PEG-conjugated NPC peptide) prepared in Example 6, the PEGylated thrombopoietin PN20 (one PEG-conjugated two NPC peptides) prepared in Example 6, and Romiplostim on Baf-3-rhuMpl receptor cells were evaluated.

[0245] The results of the PN20, PN20-Mo, and NPC receptor cell activity experiments are shown in the table below:

[0246] The results of PN20, Romiplostim, and NPC receptor cell activity assays are shown in the table below:

[0247] In vitro cell viability assays showed that NPC, PN20-mo, PN20, and Romiplostim EC were effective against cytotoxicity. 50 They are 3.046×10 -4 1.857×10 -5 1.529×10 -5 2.128×10 -5 PN20 and Romiplostim at the same molar concentrations on EC5 cells of Baf-3-rhuMpl receptor cells. 50 Consistently, the specific activity of the polyethylene glycol monomodifier PN20-mo against Baf-3-rhuMpl receptor cells was lower than that of PN20, while the specific activity of PN20 against Baf-3-rhuMpl receptor cells was significantly higher than that of recombinant thrombopoietin NPC.

[0248] Example 12: Determination of the binding affinity between pegylated thrombopoietin and rhuMpl receptor

[0249] The pharmacological action of thrombopoietin (NPC) is to specifically bind to the rhuMpl (c-Mpl) receptor, thereby activating signaling pathways such as JAK-STAT, regulating megakaryocyte development and platelet production, and thus increasing platelet count. Based on the ability of anti-rhuMpl antibodies to bind to the rhuMpl receptor, anti-rhuMpl antibodies (manufacturer: Abcam, catalog number AB109003) were coated onto an ELISA plate, and then the rhuMpl receptor (synthesized by Shanghai Jikai Gene, NCBI serial number NM_005373) was used. Different concentrations of PN20 ligand (preparation method see Example 6) and a certain concentration of Bio-NPC ligand were used to simultaneously compete for binding to the rhuMpl receptor. The affinity of PN20, NPC, Romiplostim with the c-Mpl receptor was determined using the Biotin-Avidin-HRP system. The OD value was determined using experimental results on the Y-axis, and the molar concentrations of PN20, NPC, and Romiplostim were determined on the X-axis. Curve fitting was performed using Origin Pro 2017 parametric regression software to calculate the EC50 of the sample. 50 This allows us to determine the biological activity at the same molar concentration.

[0250] The statistical results (molar concentration) of the affinity binding experiments of Romiplostim and PN20 receptors are shown in the table below:

[0251] Molar concentration-based rhuMpl receptor binding assays showed that Romiplostim, NPC, and PN20 have an IC50 affinity for the rhuMpl receptor. 50 They are 5.51×10 -3 6.85×10 -2 5.77×10 -3 PN20 and Romiplostim have comparable binding affinity to the rhuMpl receptor.

[0252] Example 13: Dose-response test of platelet count in normal mice

[0253] The dose-response relationship of different doses (1–81 μg / kg) of lyophilized formulations of polyethylene glycol-induced platelet-producing peptide PN20 (preparation method as described in Example 7) on platelet-raising effects in normal BALB / c mice was studied. Dose-response curves were plotted, and the median effective dose (ED) was calculated. 50The equivalence between PEGylated thrombopoietin and Romiplostim was calculated. In normal mice, PEGylated thrombopoietin induced a dose-dependent increase in platelet count in the range of 1–81 μg / kg. The platelet count in each dose group reached its peak on day 6 (D6) after administration, and then the platelet count in each group gradually decreased, returning to the pre-administration level on day 12.

[0254] The logarithm of platelet count on day 6 was plotted on the ordinate, and the logarithm of the dose + 1 of PEGylated thrombopoietin in each group was plotted on the abscissa (the normal control group was calculated with a PN20 dose of 0). A linear regression was performed using Origin 8.0 software, and the results are shown in Figure 8. Based on the fitted linear equation, the dose-effect ratio of 30 μg / kg Romiplostim to PEGylated thrombopoietin was calculated to be 8.72:1. The ED of the relative maximum effect value of PEGylated thrombopoietin observed in this experiment was... 50 It is 21.9 μg / kg.

[0255] Example 14: Efficacy test of a rat model of thrombocytopenia induced by chronic liver disease

[0256] SD rats were induced to develop chronic liver disease thrombocytopenia by intraperitoneal injection of thioacetamide (TAA). The rats were administered 200 mg / kg TAA twice weekly for two weeks, after which the TAA dose was increased to 300 mg / kg to continue modeling. After eight weeks of model induction, the rat model was established. Lyophilized PEGylated thrombopoietin PN20 (preparation method see Example 7) was administered subcutaneously at doses of 10 μg / kg, 30 μg / kg, and 90 μg / kg, respectively. The positive control drug, avatrombopag maleate, was administered by gavage at a dose of 2 mg / kg once daily for 17 consecutive days. During the treatment period, all participating animals (excluding the normal control group) were induced to develop the model by intraperitoneal injection of 300 mg / kg TAA twice weekly. The dose-response curve of platelet function in the rat model is shown in Figure 9.

[0257] The results showed that pegylated thrombopoietin (PEGylated thrombopoietin) had a significant therapeutic effect on thrombocytopenia in model animals, and the magnitude and duration of PLT elevation were dose-related. After a single administration of different doses of PEGylated thrombopoietin, the PLT levels in the blood of model animals began to show an upward trend on day 1 after administration, and were significantly higher than those in the model control group on day 3 (P<0.05). The peak PLT count in the low-dose group occurred on day 3 after administration, while the peak PLT counts in the medium-dose and high-dose groups occurred on days 9 and 10, respectively. The duration of action of PEGylated thrombopoietin in the low- and medium-dose groups was 8 days, while the duration of action in the high-dose group was greater than or equal to 12 days. PEGylated thrombopoietin showed a significant dose-related effect on improving thrombocytopenia in model animals. The positive control drug avatrambopag maleate had no therapeutic effect on thrombocytopenia in the model animals (the reason may be related to species differences in TPO receptors; avatrambopag only has a platelet-raising effect in humans and chimpanzees, but not in mice, rats, beagles, and monkeys). During the treatment period, the blood PLT counts in the positive control group at each time point were comparable to those in the model control group, and there was no trend of improvement compared to the model control group at the same time point.

[0258] Example 15: Efficacy test of a mouse model of immune thrombocytopenic purpura

[0259] BALB / c mice were induced to develop thrombocytopenia by continuous intraperitoneal injection of CD41 antibody (2 μg / day) to simulate the ITP process. This resulted in a significant decrease in peripheral blood platelet counts starting from day 3 and continuing until the end of the experiment on day 15. The dose-response curves of platelet action in the mouse model after intraperitoneal injection of a lyophilized formulation of pegylated thrombopoietin PN20 (preparation method as described in Example 7, injection doses of 10 μg / kg, 30 μg / kg, and 90 μg / kg for each group, respectively) or Romiplostim on days 1 (D1) and 7 (D7) are shown in Figure 10.

[0260] The results showed that pegylated thrombopoietin could alleviate the thrombocytopenia induced by CD41 antibody to some extent, and both administrations showed a dose-dependent increase in platelet count on day 5 after administration. On day 5 after the first administration, 100 μg / kg Romiplostim and 10 μg / kg pegylated thrombopoietin had comparable therapeutic effects; on day 5 after the second administration, 100 μg / kg Romiplostim and 90 μg / kg pegylated thrombopoietin had comparable therapeutic effects. Furthermore, pegylated thrombopoietin or Romiplostim alleviated the leukopenia during the trial, but had no significant effect on changes in peripheral blood erythrocyte count and hemoglobin levels.

[0261] Example 16: Efficacy test of a mouse model of thrombocytopenia induced by radiotherapy and chemotherapy

[0262] use 60 Whole-body irradiation with Co (2.5 Gy) combined with intraperitoneal injection of carboplatin (50 mg / kg) induced a decrease in peripheral blood platelet count in BALB / c mice, simulating the thrombocytopenia process induced by radiotherapy and chemotherapy. The therapeutic effect of pegylated thrombopoietin on radiotherapy- and carboplatin-induced thrombocytopenia in mice over three experimental cycles was observed and compared with Romiplostim. The day after modeling, mice were treated with 100 μg / kg of Romiplostim or 10 μg / kg, 30 μg / kg, or 90 μg / kg of a lyophilized formulation of pegylated thrombopoietin PN20 (preparation method see Example 7). The dose-response curves of platelet action in the mouse model are shown in Figure 11.

[0263] The results showed that treatment significantly alleviated the decrease in platelet count, red blood cell count, and hemoglobin content caused by the model. The platelet-raising effect of 90 μg / kg PEGylated thrombopoietin was similar to that of 100 μg / kg Romiplostim.

[0264] Example 17: A Phase I clinical study evaluating the safety, tolerability, pharmacokinetics, pharmacodynamic characteristics, and C-QTc of pegylated thrombopoietin in healthy Chinese subjects.

[0265] This clinical study enrolled 27 healthy subjects, divided into an experimental group (A) and a placebo group (P). The placebo group had 6 subjects. Within the experimental group, there were 2 subjects in the 0.05 μg / kg dose group (2A); 8 subjects in the 0.1 μg / kg dose group (8A / 2P); 8 subjects in the 0.15 μg / kg dose group (8A / 2P); and 3 subjects in the 0.2 μg / kg dose group (3A / 2P). All subjects completed the 28-day safety observation period after administration. Results showed that a single subcutaneous administration of the lyophilized PEGylated thrombopoietin PN20 formulation (preparation method see Example 7) in the 0.05–0.2 μg / kg dose groups significantly increased platelet count (PLT), with the increase increasing with increasing dosage. Figure 12 shows the time-dependent platelet count measurements after a single PN20 administration in healthy subjects. The pharmacodynamic parameters of platelet count after a single dose of pegylated thrombopoietin in healthy subjects are shown in the table below:

[0266] Results showed that platelet counts (PLTs) began to increase in all dose groups from days 3 to 5 after administration, reaching their peak on days 11 to 13, and began to decline on day 15. Peripheral blood platelet counts returned to normal on day 21. The platelet count in the 0.2 μg / kg dose group increased more than two-fold from baseline. All dose groups were well-tolerated and no unexpected adverse events occurred. No clinically significant changes in the QTc interval were delayed in any dose group.

[0267] Example 18: Evaluation of single-arm, open-label, single-dose, and multiple-dose escalation studies of pegylated thrombopoietin for injection in patients with chemotherapy-induced thrombocytopenia (CIT).

[0268] In this embodiment, a lyophilized formulation of pegylated thrombopoietin PN20 (preparation method see Example 7) was used to treat multiple clinical patients with chemotherapy-induced thrombocytopenia. The results are shown in Figure 13. The results showed that the platelet counts of all subjects in the 0.2 μg / kg and 0.5 μg / kg dose groups increased from baseline, and the subjects showed good safety. The platelet count change from baseline was more significant in the 0.5 μg / kg dose group, showing preliminary efficacy.

[0269] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0270] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A fusion protein, characterized in that, The fusion protein has an amino acid sequence as shown in formula (I). Xm-(YZ)n(I); Wherein, X is a glargine insulin mutant, and the amino acid sequence of the glargine insulin mutant is shown in any one of SEQ ID NO:1, 9 to 11; Y represents the enzyme cleavage site; Z is a thrombopoietin-promoting peptide; m is 1 or 2; n is 1 or 2; The fusion protein is adapted to be cleaved by a cleavage enzyme to form the free thrombopoietin, which is not cleaved by the cleavage enzyme.

2. The fusion protein according to claim 1, characterized in that, The fusion protein has at least one of the following characteristics: i) The cleavage enzyme is selected from at least one of recombinant lysine endopeptidase, enterokinase, recombinant lysine endopeptidase and recombinant carboxypeptidase B; ii) The amino acid sequence of the thrombopoietin from the N-terminus to the C-terminus is as follows: Cys-(Gly)p-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-(Gly)q-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala; Where p and q are each an independent integer between 5 and 10; iii) m is 1; iv)n is 1.

3. The fusion protein according to claim 1, characterized in that, The amino acid sequence of the thrombopoietin peptide is shown in SEQ ID NO:2; And / or, the cleavage enzyme is selected from recombinant lysine endopeptidase; And / or, the amino acid sequence of the enzyme cleavage site is DDDDK or K; And / or, the structure of the formula (I) is XYZ or X-(YZ)2.

4. The fusion protein according to claim 1, characterized in that, The amino acid sequence of the fusion protein is shown in any one of SEQ ID NO:3 to 5.

5. Use of the fusion protein according to any one of claims 1 to 4 in the preparation of thrombopoietin or polyethylene glycol-modified thrombopoietin.

6. A method for preparing thrombopoietin, characterized in that, include: Obtaining inclusion bodies of bacterial cells, wherein the inclusion bodies comprise the fusion protein according to any one of claims 1 to 4; The fusion protein in the inclusion body was subjected to enzymatic digestion to obtain the thrombopoietin-promoting peptide.

7. The method according to claim 6, characterized in that, The method has at least one of the following features: i) The enzyme digestion treatment is performed using a digestion enzyme; ii) Prior to the enzyme digestion treatment, the fusion protein is denatured. iii) Reduce the enzyme digestion product.

8. The method according to claim 6, characterized in that, The amino acid sequence of the cleavage site in the fusion protein is K, and the cleavage enzyme is selected from recombinant lysine endopeptidase.

9. The method according to claim 6, characterized in that, In the enzyme digestion system, the final concentration of the fusion protein is 5 mg / ml to 10 mg / ml.

10. The method according to claim 6, characterized in that, The mass ratio of the fusion protein to the cleavage enzyme is 1:(1000-5000).

11. The method according to claim 6, characterized in that, The enzyme digestion treatment was performed at a temperature of 25℃ to 35℃ for 8 hours to 24 hours.

12. The method according to claim 7, characterized in that, The denaturation treatment was carried out under conditions of denaturant and detergent.

13. The method according to claim 12, characterized in that, The denaturing agent comprises 2 mol / L to 8 mol / L urea and 20 mM to 80 mM Tris, and the pH value of the denaturing agent is 8.5 to 9.

5.

14. The method according to claim 7, characterized in that, The reduction process is carried out in a reducing agent.

15. The method according to claim 14, characterized in that, The reducing agent is selected from at least one of TCEP, DTT, and β-mercaptoethanol, more preferably TCEP.

16. The method according to claim 14, characterized in that, The molar ratio of the thrombopoietin to the reducing agent is (1-3):

1.

17. The method according to claim 7, characterized in that, The method further includes: purifying the reduction product.

18. The method according to claim 17, characterized in that, The purification process was performed using chromatographic chromatography.

19. A method for preparing polyethylene glycol-modified thrombopoietin, characterized in that, include: The thrombopoietin was coupled with polyethylene glycol to obtain the polyethylene glycolated thrombopoietin. The thrombopoietin is prepared using the fusion protein according to any one of claims 1 to 4, or prepared according to the method according to any one of claims 6 to 18.

20. The method according to claim 19, characterized in that, The method includes at least one of the following features: a) The molar ratio of the thrombopoietin to polyethylene glycol is 1:(0.4-0.6); b) The coupling treatment is performed in a phosphate buffer solution with a pH of 5.5 to 7.5; c) Prior to the coupling treatment, the thrombopoietin and TCEP are pre-mixed and treated. d) Further includes purifying the coupling treatment product; e) The molecular weight of the polyethylene glycol is 5 to 50 kDa.

21. The method according to claim 20, characterized in that, The molar ratio of the thrombopoietin to polyethylene glycol is 1:(0.4-0.5).

22. The method according to claim 20, characterized in that, The pH value of the phosphate buffer solution is 5.5 to 6.

5.

23. The method according to claim 20, characterized in that, The molar ratio of the thrombopoietin to TCEP is 1:(0.5-2.0).

24. The method according to claim 20, characterized in that, The molar ratio of the thrombopoietin to TCEP is 1:(1.0-1.5).

25. The method according to claim 20, characterized in that, The purification process was performed using chromatographic chromatography.

26. The method according to claim 20, characterized in that, The molecular weight of the polyethylene glycol is 15-25 kDa.

27. An injection solution, characterized in that, include: Polyglycolated thrombopoietin, buffer salts, and protectants.

28. The injection solution according to claim 27, characterized in that, The injection solution has at least one of the following characteristics: a) The PEGylated thrombopoietin is prepared using the fusion protein according to any one of claims 1 to 4, or prepared according to the method according to any one of claims 19 to 26; b) The final concentration of the PEGylated thrombopoietin is 0.2 mg / ml to 1.0 mg / ml, based on the total mass of the injection solution; c) The pH value of the injection solution is 2.5–4.5; d) The buffer salt is selected from at least one of histidine, glycine, citrate-sodium citrate, or acetate-sodium acetate; e) The final concentration of the buffer salt in the injection solution is 10–100 mmol / L; f) The protective agent is selected from at least one of sucrose, trehalose, and mannitol; g) The protective agent accounts for 1% to 10% of the total mass of the injection solution.

29. The injection solution according to claim 28, characterized in that, The buffer salts include histidine and glycine.

30. The injection solution according to claim 28, characterized in that, The buffer salt comprises 20 mmol / L glycine and 20 mmol / L histidine.

31. The injection solution according to claim 28, characterized in that, The protective agent comprises 6% to 8% of the total mass of the injection solution.

32. The injection solution according to claim 28, characterized in that, The protective agent is selected from sucrose and mannitol.

33. The injection solution according to claim 32, characterized in that, The mass ratio of sucrose to mannitol is 1:(1-2).

34. The injection solution according to claim 27 or 28, characterized in that, The injection solution further includes a pH adjuster.

35. The injection solution according to claim 27 or 28, characterized in that, The injection solution comprises: 0.2–1.0 mg / ml of pegylated thrombopoietin. 2% (w / w) sucrose, 4% (w / w) mannitol, 20 mmol / L histidine 20 mmol / L glycine The pH value of the injection solution is 2.5 to 4.

5.

36. A lyophilized formulation, characterized in that, The lyophilized formulation is obtained by lyophilizing the injection solution according to any one of claims 27 to 35.

37. Use of the polyethylene glycol-modified thrombopoietin, the injection solution according to any one of claims 27-35, or the lyophilized formulation according to claim 36, wherein the use is for: Treatment and / or prevention of disease, and / or To prepare medicines for the treatment and / or prevention of diseases; in, The diseases mentioned include treatment and prevention of cancer drug-induced thrombocytopenic purpura, primary immune thrombocytopenic purpura, chronic liver disease-related thrombocytopenic purpura, and acute radiation syndrome-hematopoietic syndrome.

38. The use according to claim 37, characterized in that, The PEGylated thrombopoietin is prepared using the fusion protein according to any one of claims 1 to 4, or according to the method described in any one of claims 19 to 26.

39. The use according to claim 37, characterized in that, The PEGylated thrombopoietin is provided as a lyophilized formulation.

40. The use according to claim 37, characterized in that, The effective dose of the PEGylated thrombopoietin is 6 μg to 100 μg.

41. The use according to claim 37, characterized in that, The PEGylated thrombopoietin is administered once per chemotherapy cycle, or once per week for patients with chronic liver disease before surgery, to treat primary immune thrombocytopenia, and to treat and prevent acute radiation syndrome and hematopoietic syndrome.

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