Use of cyclodextrin and derivative thereof as protective agent in preparation of TACI-fc fusion protein liquid pharmaceutical formulation

Abstract

Use of a cyclodextrin and a derivative thereof as a protective agent in the preparation of a TACI-Fc fusion protein liquid pharmaceutical formulation. Using the cyclodextrin as the protective agent can enhance the long-term storage stability of a TACI-Fc fusion protein, maintain good biological activity, improve the production efficiency of drugs, and greatly improve administration convenience.

Description

Use of cyclodextrin and its derivatives as protective agents in the preparation of TACI-Fc fusion protein liquid pharmaceutical formulations Technical Field

[0001] This invention relates to the use of cyclodextrin as a protective agent in the development of liquid pharmaceutical formulations of TACI-Fc fusion proteins, and belongs to the field of pharmaceutical formulation development for anti-autoimmune diseases. Background Technology

[0002] In recent years, the biopharmaceutical market has maintained a rapid growth trend. Fusion proteins and antibody drugs have played a significant role in oncology and autoimmune diseases. However, with in-depth pathological research and advancements in development technology, while benefiting patients, these drugs also face physical changes such as aggregation, denaturation, and precipitation, as well as chemical changes such as isomerization, deamidation, and oxidation. These changes can affect the safety and efficacy of the products. Therefore, stable formulations are needed to ensure that fusion proteins and antibody drugs retain the necessary biological activity for treatment before being used in patients. In contemporary drug development, excipients and protective agents are indispensable parts of biopharmaceutical products, playing a crucial role in the development of both small-molecule and large-molecule drug formulations. Commonly used protective agents include glycerol, sucrose, and trehalose.

[0003] Furthermore, the need to develop highly concentrated liquid formulations of antibody drugs for subcutaneous or intramuscular injection to improve ease of administration further increases this challenge, as high concentrations can significantly increase the viscosity of liquid formulations and the tendency for proteins to aggregate. Aggregates may contain protein degradation products and can lead to various side effects, such as triggering undesirable immune responses.

[0004] Compared to typical macromolecular formulations, antibody-like fusion proteins or antibody formulations possess unique characteristics. On one hand, the fragile stability and structural complexity of monoclonal antibody drugs present significant challenges in their manufacture and storage. Due to the heterogeneous structure of antibodies, particularly their complementarity-determining regions (CDRs) and Fc glycosylation, the development of each monoclonal antibody formulation must be case-by-case. Challenges exist in antibody conformation, colloidal, or chemical structures during formulation development, such as oxidation, isomerization, deamidation, aggregation, denaturation, and fragmentation. Antibody stereostructure can alter under varying temperature, humidity, pH, and stress conditions, particularly in hypervariable regions (HVRs). Poorly formulated products may exhibit reduced activity; more importantly, increased immunogenicity can endanger patients. Therefore, selecting optimal excipients to protect the antibody is crucial. (Reference 1: Monoclonal antibodies: formulations of marketed products and recent advances in novel delivery system, Yanan Cui et al., Drug Development and Industrial Pharmacy, Vol. 43, No. 4, pp. 519-530, 2017). On the other hand, fusion protein products are prone to aggregation and have poor stability. The adverse immune reactions or interference with the purification process caused by this have always been a problem that needs to be solved in this field. Moreover, the factors affecting the aggregation of fusion proteins are also quite complex, divided into external and internal factors. External factors mainly include temperature, physical pressure, and solvent factors (pH, ionic strength, concentration, metal ions, etc.); internal factors mainly include the structural characteristics of fusion proteins, sensitive residues, and unpaired cysteine ​​residues, etc. These factors can all affect the aggregation of fusion proteins, thereby affecting their stability and shelf life. Therefore, the selection of excipients for fusion proteins requires a lot of experimental exploration (Reference 2: Production Challenges for Complex Biologics: Fusion Proteins, Stefan R. Schmidt, American Pharmaceutical Review, pp. 1-5, 2017).

[0005] Sugars are frequently used as pharmaceutical excipients to stabilize proteins in liquid and lyophilized formulations. Sucrose and trehalose are natural osmolarity regulators used to stabilize microbial cell structures under harsh environmental conditions (such as high temperatures and low water levels). They are believed to stabilize proteins through preferential hydration at high concentrations in the liquid state, and through specific interactions with proteins and the formation of a highly viscous, glassy matrix in the solid state. It should be noted that some sugar excipients may be degraded or contain impurities, which could adversely affect protein stability.

[0006] It has been reported that natural cyclodextrins (CD) and their inclusion complexes have poor solubility in water, but are cheaper and are generally preferred in solid dosage forms, while their value in injections is limited. (Reference 3: Cyclodextrins and Drug Membrane Permeation: Thermodynamic Considerations, Suppakan Sripetch et al., Journal of Pharmaceutical Sciences, Vol. 111, No. 9, pp. 2571-2580, 2022) Typical natural cyclodextrins are composed of 6, 7, and 8 pyranose units, respectively named alpha-cyclodextrin (α-CD), beta-cyclodextrin (β-CD), and gamma-cyclodextrin (γ-CD).

[0007] Currently, the use of human transmembrane activators targeting recombinant B lymphocyte stimulating factor (BLyS) and proliferation-inducing ligand (APRIL) and calmotropic cyclin ligand interacting factor (TACI) fragments to form fusion proteins with the Fc domain of human immunoglobulin IgG to treat various autoimmune diseases is supported by marketed drugs, products in multiple clinical stages, and clinical data. For example, telitacicept was the first TACI-Fc drug to receive market approval (amino acid sequence: SEQ ID NO:1), and it received full approval from the China National Medical Products Administration in November 2023 for the treatment of systemic lupus erythematosus. In addition, atacicept (amino acid sequence: SEQ ID NO:2; 92.5% sequence identity with SEQ ID NO:1) and Povetacicept (amino acid sequence: SEQ ID NO:3; 78.4% sequence identity with SEQ ID NO:1) are in multiple clinical trials.

[0008] Freeze-dried preparations greatly limit the speed of drug production and significantly increase the time patients spend seeking medical treatment and the burden on hospitals. Therefore, there is a great clinical need to improve the convenience of medication for patients.

[0009] Summary of the Invention

[0010] To address the aforementioned issues, this invention uses the TACI-Fc molecule shown in SEQ ID NO:1 as a representative of TACI-Fc drugs. Through extensive screening of excipients and their combinations, effective excipients for liquid formulation development were identified. Through numerous experiments, this invention surprisingly discovered that using cyclodextrin or cyclodextrin derivatives as protective agents can develop liquid drug formulations of TACI-Fc fusion proteins with excellent stability, capable of stable preservation for over 18 months under preservative-free aseptic conditions at 4°C. This invention demonstrates that the use of cyclodextrin or cyclodextrin derivatives can effectively improve the long-term stability of TACI-Fc fusion proteins, maintain good biological activity, and the developed liquid formulations can effectively increase drug production speed, reduce drug production costs, and significantly improve patient convenience, thereby better meeting clinical medication needs.

[0011] The present invention further discovers that when using cyclodextrin or cyclodextrin derivatives as protective agents, the developed specific formulations can still exhibit the aforementioned excellent stability even without the use of surfactant excipients in the formulation.

[0012] Specifically, the TACI-Fc fusion protein includes: (i) the extracellular region of TACI or a fragment thereof that binds to Blys and / or APRIL, and (ii) a fragment of the human immunoglobulin constant region.

[0013] More preferably, the protective agent is a cyclodextrin derivative; preferably, the cyclodextrin derivative is hydroxypropyl cyclodextrin; preferably, the hydroxypropyl cyclodextrin is selected from hydroxypropyl-α-cyclodextrin, hydroxypropyl-β-cyclodextrin or hydroxypropyl-γ-cyclodextrin; more preferably, the hydroxypropyl cyclodextrin is hydroxypropyl-β-cyclodextrin.

[0014] More preferably, the protective agent is cyclodextrin; preferably, the cyclodextrin is selected from α-cyclodextrin, β-cyclodextrin or γ-cyclodextrin; more preferably, the cyclodextrin is α-cyclodextrin.

[0015] More preferably, the amino acid sequence of the TACI-Fc fusion protein has 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or more than 99% identity with the sequence shown in SEQ ID NO:1.

[0016] More preferably, the amino acid sequence of the TACI-Fc fusion protein is shown in SEQ ID NO:1.

[0017] More preferably, the TACI-Fc fusion protein is teltascept.

[0018] More preferably, the concentration range of the TACI-Fc fusion protein is 40 mg / ml-240 mg / ml; preferably, the concentration range of the TACI-Fc fusion protein is 40 mg / ml-120 mg / ml; preferably, the concentration range of the TACI-Fc fusion protein is 60 mg / ml-100 mg / ml; preferably, the concentration range of the TACI-Fc fusion protein is 80 mg / ml-120 mg / ml; preferably, the concentration range of the TACI-Fc fusion protein is 80 mg / ml-100 mg / ml.

[0019] More preferably, the concentration of the TACI-Fc fusion protein is approximately 40 mg / ml, 50 mg / ml, 60 mg / ml, 70 mg / ml, 80 mg / ml, 90 mg / ml, 100 mg / ml, 110 mg / ml, 120 mg / ml, 130 mg / ml, 140 mg / ml, 150 mg / ml, 160 mg / ml, 170 mg / ml, 180 mg / ml, 190 mg / ml, 200 mg / ml, 210 mg / ml, 220 mg / ml, 230 mg / ml, or 240 mg / ml.

[0020] More preferably, the concentration range of the protective agent is 50 mmol / L-200 mmol / L; preferably, the concentration range of the protective agent is 80 mmol / L-200 mmol / L; preferably, the concentration range of the protective agent is 100 mmol / L-200 mmol / L; preferably, the concentration range of the protective agent is 100 mmol / L-160 mmol / L.

[0021] More preferably, the concentration of the protective agent is approximately 100 mmol / L, 110 mmol / L, 120 mmol / L, 130 mmol / L, 140 mmol / L, 150 mmol / L, 160 mmol / L, 170 mmol / L, 180 mmol / L, 190 mmol / L, or 200 mmol / L.

[0022] More preferably, the formulation further includes a buffer and / or a pH adjuster.

[0023] More preferably, the buffer is selected from histidine, histidine hydrochloride, phosphate, citrate, tromethamine, or succinic acid; preferably, the buffer is succinic acid; preferably, the concentration of the buffer is 5 mmol / L-20 mmol / L; preferably, the concentration of the buffer is 5 mmol / L-15 mmol / L.

[0024] More preferably, the concentration of the buffer is approximately 5 mmol / L, 6 mmol / L, 7 mmol / L, 8 mmol / L, 9 mmol / L, 10 mmol / L, 11 mmol / L, 12 mmol / L, 13 mmol / L, 14 mmol / L, 15 mmol / L, or 20 mmol / L.

[0025] More preferably, the pH adjuster is an inorganic base.

[0026] More preferably, the pH adjuster is selected from sodium hydroxide.

[0027] More preferably, the pH of the liquid formulation is 4.0-6.5; more preferably, the pH of the liquid formulation is 4.5-5.4; more preferably, the pH of the liquid formulation is 4.8-5.2.

[0028] More preferably, the pH of the liquid formulation is approximately 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5; preferably, the pH of the liquid formulation is approximately 5.1.

[0029] More preferably, the liquid formulation comprises TACI-Fc fusion protein, hydroxypropyl-β-cyclodextrin, and / or succinic acid; preferably, the formulation comprises approximately 80 mg / mL of TACI-Fc fusion protein, approximately 150 mmol / L of hydroxypropyl-β-cyclodextrin, and / or approximately 10 mmol / L of succinic acid; preferably, the formulation comprises 80 mg / mL of TACI-Fc fusion protein, 150 mmol / L of hydroxypropyl-β-cyclodextrin, and 10 mmol / L of succinic acid; more preferably, the pH of the liquid formulation is approximately 5.1.

[0030] Furthermore, the method for detecting the content of the aforementioned fusion protein is as follows: ultraviolet-visible spectrophotometry. Based on the fact that proteins have maximum ultraviolet absorption at 280 nm, the absorbance value of the teltascip sample at this wavelength is measured. After correcting for the absorbance at 320 nm, the absorbance value at 280 nm is found to be directly proportional to the protein concentration. The protein concentration is calculated using Lambert-Beer's law, thus determining the protein content. The formula for calculating protein content is as follows:

[0031] In the formula, ε is the extinction coefficient of teltacil, with the unit being (mg / ml)⁻¹·cm⁻¹;

[0032] A280 is the average absorbance of the sample solution at 280 nm;

[0033] A280 (correction) is the average absorbance of the sample solution at 280 nm after correction. Detailed Implementation

[0034] Unless otherwise defined, all technical terms used herein have the same meaning as understood by one of ordinary skill in the art. For definitions and terminology in this field, professionals may refer to Current Protocols in Molecular Biology (Ausubel).

[0035] The amino acid three-letter codes and single-letter codes used in this invention are as described in J. biol. chem, 243, p3558 (1968).

[0036] In this invention, the term "TACI" stands for transmembrane activator and CAML interactor, a member of the tumor necrosis factor receptor superfamily. The term "BLys" refers to B lymphocyte stimulator, a member of the TNF ligand superfamily existing in both membrane-bound and soluble forms. It is specifically expressed on the surface of bone marrow cells and selectively stimulates B lymphocyte proliferation and immunoglobulin production. The term "APRIL" (a proliferation-inducing ligand) is a tumor necrosis factor (TNF) analog that stimulates the proliferation of primitive B cells and T cells in the body, promotes B cell accumulation, and increases spleen content. APRIL specifically binds to TACI and BCMA, preventing APRIL from binding to B cells and inhibiting the APRIL-induced primitive B cell proliferation response. Furthermore, APRIL competitively binds to receptors (BCMA and TACI) with BLys.

[0037] The term "TACI-Fc fusion protein" as used in this invention refers to a transmembrane activator, calcium regulator and cyclic protein ligand interactor (TACI)-immunoglobulin fusion protein (i.e., TACI-Fc fusion protein). The TACI-immunoglobulin fusion protein provided by this invention comprises: (i) the extracellular region of TACI or a fragment thereof binding to Blys and / or APRIL; and (ii) a fragment of the human immunoglobulin constant region.

[0038] The term “TACI extracellular domain or a fragment thereof binding to Blys and / or APRIL” can be specifically referred to in U.S. Patent Nos. 5,969,102, 6,316,222 and 6,500,428 and U.S. Patent Applications Nos. 09 / 569,245 and 09 / 627,206 (the contents of which are incorporated herein by reference) as the extracellular domain of TACI and specific fragments of the extracellular domain of TACI that can interact with TACI ligands, or the amino acid fragment of the extracellular domain of TACI disclosed in Chinese Patent Publication No. CN101323643A, which consists of amino acids 13-118 of the extracellular domain of TACI.

[0039] In the term "human immunoglobulin constant region fragment," the immunoglobulin portion is preferably IgG1, which may include a heavy chain constant region, such as the human heavy chain constant region. The preferred "human immunoglobulin constant region fragment" of this invention is an amino acid fragment containing a partial hinge region domain, a CH2 domain, and a CH3 domain.

[0040] The term "cyclodextrin derivative" in this invention refers to a compound obtained by replacing any one or more groups and / or atoms in a cyclodextrin molecule with any one or more arbitrary substituents, wherein the arbitrary substituents may be selected, for example, from methyl, ethyl, hydroxypropyl, hydroxyethyl, such as hydroxypropyl-β-cyclodextrin or hydroxypropyl-γ-cyclodextrin, which are cyclodextrin derivatives obtained by introducing hydroxypropyl groups onto β-CD or γ-CD molecules.

[0041] The term "treatment" as used in this invention relates to a given disease or condition, including but not limited to: suppressing the disease or condition, such as preventing the development of the disease or condition; alleviating the disease or condition, such as causing the disease or condition to subside; or alleviating the symptoms caused by the disease or condition, such as alleviating, preventing or treating the symptoms of the disease or condition.

[0042] The term "Telitacicept" (or "Telitacicept," which can be used interchangeably in this invention) is a TACI-Fc fusion protein with the amino acid sequence shown in SEQ ID NO:1, or see https: / / extranet.who.int / soinn / mod / page / view.php?id=137&inn_n=10932. Its preparation can be carried out in accordance with the contents disclosed in the patents with publication numbers CN101323643A or CN113613675A.

[0043] The term "about" in this invention is used to indicate that a numerical value includes inherent error variations in the device or the method used to determine the value, or variations that exist between the measured samples. Unless otherwise stated or clearly apparent from the context, the term "about" means within 10% of the reported value (unless the number would exceed 100% of the possible value or be less than 0%). When used in conjunction with a numerical range or series, unless otherwise stated, the term "about" applies to each value listed at the end of the range or in the series.

[0044] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention.

[0045] Example 1: Screening Experiment of Protective Agents

[0046] Based on the preliminary screening of protectants, methionine, sucrose, and hydroxypropyl-β-cyclodextrin were further selected as candidate protectants to screen for the best protectant that can be used in TACI-Fc formulations.

[0047] 1.1 Experimental Methods: TACI-Fc protein (SEQ ID NO:1) was dialyzed and concentrated into the formulation in Table 1. Then, the volume was adjusted, filtered, and dispensed. The prepared samples were stored at 40℃ and 25℃ respectively. SEC purity was detected at 3d, 7d, and 14d at 40℃, and at 1m and 3m at 25℃.

[0048] Table 1 Protectant Formulation Information

[0049] Table 2. Results of Purity (SEC-Purity) at 40℃ (%)

[0050] Table 3. Results of Purity (SEC-Purity) (%) at 25℃

[0051] 1.2 Experimental Results: See Tables 2 and 3. The experimental results show that:

[0052] 1) By comparing the SEC results of samples 1, 2, and 3, it can be seen that the higher the pH, the more aggregates there are and the less degradation occurs, but the increase in aggregates is much greater than the decrease in degradation.

[0053] 2) A comparison of 2 and 4 shows that methionine has a better protective effect than sucrose.

[0054] 3) Comparison of No. 2, 4, and 6 shows that hydroxypropyl-β-cyclodextrin has the best protective effect, which is superior to methionine and sucrose protectants.

[0055] 4) Comparing samples 5, 6, and 7, it can be seen that as the protein concentration increases, the purity decreases sequentially.

[0056] Conclusion: In summary, the protective effects of the three protectants, from highest to lowest, are hydroxypropyl-β-cyclodextrin, methionine, and sucrose, indicating that hydroxypropyl-β-cyclodextrin is the most effective protectant.

[0057] Example 2: Experiment to investigate the concentration of hydroxypropyl-β-cyclodextrin

[0058] This embodiment focuses on investigating the effect of hydroxypropyl-β-cyclodextrin concentration on protein stability.

[0059] 2.1 Experimental Methods: TACI-Fc (SEQ ID NO:1) was dialyzed and concentrated into the formulation in Table 4. Then, the volume was adjusted, filtered, and dispensed. The prepared sample was then stored at 40°C. SEC purity, nrCE-SDS results, and osmotic pressure were measured at 3d and 7d time points.

[0060] Table 4 Formula Information

[0061] Table 5. Results of purity (SEC-purity) at 40℃ (%)

[0062] Table 6 nrCE-SDS Results

[0063] Table 7 Osmotic pressure (mOsm / kg) results

[0064] 2.2 Experimental Results: See Tables 5-7. The experimental results show that the purity decreases sequentially with the decrease of hydroxypropyl-β-cyclodextrin concentration. The results in Table 7 show that isotonic effect can be achieved when the concentration of hydroxypropyl-β-cyclodextrin is 140 mmol / L. The concentration of hydroxypropyl-β-cyclodextrin will be increased in the future to explore other possible solution osmotic pressures. The concentration of hydroxypropyl-β-cyclodextrin will be further set at 150 mmol / L, and the osmotic pressure will be explored through experiments.

[0065] Example 3: Investigation of Osmotic Pressure

[0066] The experiment was conducted in two parallel groups, A and B, as shown in Table 8 for specific formulation information.

[0067] Table 8 Formula Information

[0068] Table 9. Osmotic pressure (mOsm / kg) and pH results

[0069] 3.1 Experimental Results: See Table 9. The results show that when the concentration of TACI-Fc (SEQ ID NO:1) is 80 mg / mL, the osmotic pressure of both groups A and B is around 330; when the concentration of TACI-Fc (SEQ ID NO:1) is 120 mg / mL, the osmotic pressure of both groups A and B is around 400. The osmotic pressure measured before and after filtration is not significantly different, indicating that filtration has no effect on osmotic pressure, and the pH is within the range of 5.1 ± 0.1.

[0070] 3.2 Experimental Conclusions: In summary, the exemplary formulation with better preliminary results is composed of: 10 mmol / L succinic acid, 150 mmol / L hydroxypropyl-β-cyclodextrin, protein concentration of 80 mg / mL, and pH adjusted to 5.1 ± 0.1 with sodium hydroxide.

[0071] Example 4: Performance test of other cyclodextrin derivatives as protective agents

[0072] In Example 3, it was found that hydroxypropyl-β-cyclodextrin had a better protective effect on TACI-Fc protein than sucrose. This example focuses on the effect of hydroxypropyl-γ-cyclodextrin, obtained by introducing hydroxypropyl, as a protective agent on the stability of the formulation.

[0073] 4.1 Experimental Methods: TACI-Fc protein (SEQ ID NO:1) was dialyzed and concentrated into the formulation in Table 10. Then, the volume was adjusted, filtered, and dispensed. The prepared samples were stored at 25℃ and 40℃ respectively. At 40℃, SEC purity, capillary electrophoresis (nrCE-SDS) changes, protein cell activity, and binding activity were detected at 1 and 2 weeks.

[0074] Table 10 Formula Information

[0075] Table 11 Results of Visible Foreign Objects

[0076] Table 12 Results of Purity (SEC-Purity) (%) at 40℃

[0077] Table 13 Results of Purity (SEC-Purity) (%) at 25℃

[0078] Table 14 Results of nrCE-SDS (%) at 40℃

[0079] Table 15 Results of nrCE-SDS (%) at 25℃

[0080] Table 16 Results of Activity (%) at 40℃

[0081] Table 17 Results of Activity (%) at 25℃

[0082] 4.2 Experimental Results: See Tables 11-17, where,

[0083] 1) The results in Table 11 show that after being placed at 25℃ for 4 weeks and at 40℃ for 2 weeks, the visible foreign matter in the purity samples of hydroxypropyl-β-cyclodextrin and hydroxypropyl-γ-cyclodextrin did not change.

[0084] 2) The test results in Tables 12 and 13 show that after being placed at 25℃ and 40℃ for 2 weeks, the purity changes of hydroxypropyl-β-cyclodextrin and hydroxypropyl-γ-cyclodextrin SEC are basically the same, and they have the same protective effect.

[0085] 3) The results in Tables 14 and 15 show that after being placed at 25℃ and 40℃ for 2 weeks, the purity changes of hydroxypropyl-β-cyclodextrin and hydroxypropyl-γ-cyclodextrin CE-SDS are basically the same.

[0086] 4) The results in Tables 16 and 17 show that after being placed at 25°C and 40°C for 2 weeks, there was no significant difference in activity between hydroxypropyl-β-cyclodextrin and hydroxypropyl-γ-cyclodextrin.

[0087] 4.3 Experimental conclusion: Hydroxypropyl-γ-cyclodextrin has the same protective effect as hydroxypropyl-β-cyclodextrin.

[0088] Example 5: Performance test of cyclodextrin as a protective agent

[0089] This embodiment uses α-cyclodextrin as an example to explore the effect of cyclodextrin protectants on formulation stability.

[0090] 5.1 Experimental methods: TACI-Fc protein (SEQ ID NO:1) was dialyzed and concentrated into the formula in Table 18. After volume adjustment, filtration and aliquoting, the prepared samples were stored at 25℃ and 40℃ respectively. SEC purity and visible foreign matter were detected at 1w and 2w time points at 40℃ and 2w time points at 25℃.

[0091] Table 18 Formula Information

[0092] Table 19 Results of Visible Foreign Bodies

[0093] Table 20 Results of Purity (SEC-Purity) at 40℃ (%)

[0094] Table 21 Results of Purity (SEC-Purity) at 25℃ (%)

[0095] 5.2 Experimental Results:

[0096] 1) The results in Table 19 show that after being placed at 25℃ for 2 weeks and at 40℃ for 2 weeks, the visible foreign matter in the purity samples of α-cyclodextrin and hydroxypropyl-β-cyclodextrin did not change.

[0097] 2) The experimental results in Tables 20 and 21 show that after being placed at 25℃ and 40℃ for 2 weeks, the difference in SEC purity between α-cyclodextrin and hydroxypropyl-β-cyclodextrin is not significant, and the protective effect of α-cyclodextrin is slightly better than that of hydroxypropyl-β-cyclodextrin.

[0098] 5.3 Experimental conclusion: α-Cyclodextrin has the same protective effect as hydroxypropyl-β-cyclodextrin.

[0099] Example 6: pH Exploratory Experiment

[0100] This embodiment focuses on exploring the effect of different pH values ​​on the stability of TACI-Fc protein.

[0101] 6.1 Experimental Methods: TACI-Fc protein (SEQ ID NO:1) was dialyzed into 10 mmol / L succinic acid. The protein was concentrated to 40 mg / mL and then quantified. Stock solution was added and the concentration was further increased to the target concentration. The final formulation information is shown in Table 22. The prepared samples were then stored at 25℃ and 4℃. At 25℃, SEC purity was detected at 1, 2, 3, and 4 weeks. At 4℃, SEC purity was detected at 1, 2, 3, and 4 minutes.

[0102] Table 22 pH Exploratory Experiment Recipe Information

[0103] Table 23. Purity at 25℃ (SEC-HPLC method) (%) Results

[0104] Table 24. Purity at 4℃ (SEC-HPLC method) (%) Results

[0105] 6.2 Experimental Results: As shown in Tables 23 and 24, the results show that 1) there is no significant difference in protein purity (SEC-HPLC method) between pH 4.5 and 5.4. As the pH increases, the number of aggregates increases and the degradation decreases. However, the increase in aggregates is greater than the decrease in degradation, resulting in a downward trend in the main peak. To balance the degree of aggregates and degradation, and considering that a lower pH may cause pain during injection of liquid formulations, pH 4.8-5.2 can be further optimized.

[0106] Example 7: Long-term stability test

[0107] This embodiment focuses on examining the long-term and accelerated stability of the exemplary prescription concentrate dispensed into pre-filled syringes.

[0108] 7.1 Experimental Methods: TACI-Fc (SEQ ID NO:1) was dialyzed into the following formulations. After dialyzing, the samples were concentrated, diluted to a fixed volume, filtered, and dispensed. The specific formulation information is shown in Table 25. The long-term stability of the samples under different temperature conditions was investigated according to the temperature and time parameters in Table 26.

[0109] Table 25 Formulation Information

[0110] Table 26 Time Points for Long-Term Stability Assessment

[0111] 7.2 Experimental Results:

[0112] 1) According to SEC purity data, after TACI-Fc (SEQ ID NO:1) was placed at -20 to 4℃ for 24 months, the polymer content increased from 1.3% to 2.88%, and the purity decreased from 98.6% to 94.7%, both of which were within the quality standard range.

[0113] 2) No increase in particulate matter (visible foreign matter and insoluble microparticles) was observed, and all results were within acceptable limits;

[0114] 3) Other product quality attributes, including peptide map, appearance, osmotic pressure, pH, CE-SDS (r / nr), RP-HPLC, activity, protein content, bacterial endotoxin, and container seal (CCI), showed almost no changes, and all results were within acceptable ranges.

[0115] 7.3 Experimental Conclusion: Through rigorous long-term stability evaluation, it was found that the formulation involved in this invention, when filled into pre-filled auto-injectors (PFS) and stored at -20 to 4°C for extended periods, exhibits good stability and meets all requirements of the stability quality standards.

[0116] By using SEQ ID NO:1 as an exemplary representative of the TACI-Fc fusion protein, the above experiments advantageously demonstrate the significant advantages of cyclodextrin and its derivatives as protective agents in the preparation of liquid pharmaceutical formulations of the TACI-Fc fusion protein.

[0117] The spirit of the present invention has been described in detail above through preferred embodiments. Those skilled in the art will understand that any modifications, equivalent variations, and alterations made to the above embodiments based on the technical essence of the present invention fall within the protection scope of the present invention.

Claims

1. The use of cyclodextrin and its derivatives as protective agents in the preparation of TACI-Fc fusion protein liquid pharmaceutical formulations, wherein the TACI-Fc fusion protein comprises: (i) the extracellular region of TACI or fragments thereof that bind Blys and / or APRIL, and (ii) fragments of the human immunoglobulin constant region.

2. Use according to claim 1, characterized in that, The protective agent is a cyclodextrin derivative; preferably, the cyclodextrin derivative is hydroxypropyl cyclodextrin; preferably, the hydroxypropyl cyclodextrin is selected from hydroxypropyl-α-cyclodextrin, hydroxypropyl-β-cyclodextrin or hydroxypropyl-γ-cyclodextrin; more preferably, the hydroxypropyl cyclodextrin is hydroxypropyl-β-cyclodextrin.

3. Use according to claim 1, characterized in that, The protective agent is cyclodextrin; preferably, the cyclodextrin is selected from α-cyclodextrin, β-cyclodextrin or γ-cyclodextrin; more preferably, the cyclodextrin is α-cyclodextrin.

4. Use according to any one of claims 1 to 3, characterized in that, The amino acid sequence of the TACI-Fc fusion protein has more than 78% identity with the sequence shown in SEQ ID NO:

1.

5. Use according to claim 4, characterized in that, The amino acid sequence of the TACI-Fc fusion protein is shown in SEQ ID NO:

1.

6. Use according to claim 5, characterized in that, The TACI-Fc fusion protein is teltascept.

7. Use according to any one of claims 1 to 6, characterized in that, The concentration range of the TACI-Fc fusion protein is 40 mg / ml to 240 mg / ml; preferably, the concentration range of the TACI-Fc fusion protein is 40 mg / ml to 120 mg / ml; preferably, the concentration range of the TACI-Fc fusion protein is 60 mg / ml to 100 mg / ml; preferably, the concentration range of the TACI-Fc fusion protein is 80 mg / ml to 120 mg / ml; preferably, the concentration range of the TACI-Fc fusion protein is 80 mg / ml to 100 mg / ml.

8. Use according to claim 7, characterized in that, The concentration of the TACI-Fc fusion protein is approximately 40 mg / ml, 50 mg / ml, 60 mg / ml, 70 mg / ml, 80 mg / ml, 90 mg / ml, 100 mg / ml, 110 mg / ml, 120 mg / ml, 130 mg / ml, 140 mg / ml, 150 mg / ml, 160 mg / ml, 170 mg / ml, 180 mg / ml, 190 mg / ml, 200 mg / ml, 210 mg / ml, 220 mg / ml, 230 mg / ml, or 240 mg / ml.

9. Use according to any one of claims 1 to 8, characterized in that, The concentration range of the protective agent is 50 mmol / L-200 mmol / L; preferably, the concentration range of the protective agent is 80 mmol / L-200 mmol / L; preferably, the concentration range of the protective agent is 100 mmol / L-200 mmol / L; preferably, the concentration range of the protective agent is 100 mmol / L-160 mmol / L.

10. Use according to any one of claims 1 to 8, characterized in that, The concentration of the protective agent is approximately 100 mmol / L, 110 mmol / L, 120 mmol / L, 130 mmol / L, 140 mmol / L, 150 mmol / L, 160 mmol / L, 170 mmol / L, 180 mmol / L, 190 mmol / L, or 200 mmol / L.

11. The use according to any one of claims 1-10, characterized in that, The formulation does not contain surfactants.

12. The use according to any one of claims 1-11, characterized in that, The formulation further includes buffers and / or pH adjusters.

13. The use according to claim 12, characterized in that, The buffer is selected from histidine, histidine hydrochloride, phosphate, citrate, tromethamine, or succinic acid; preferably, the buffer is succinic acid; preferably, the concentration of the buffer is 5 mmol / L-20 mmol / L; preferably, the concentration of the buffer is 5 mmol / L-15 mmol / L.

14. The use according to claim 13, characterized in that, The concentration of the buffer is approximately 5 mmol / L, 6 mmol / L, 7 mmol / L, 8 mmol / L, 9 mmol / L, 10 mmol / L, 11 mmol / L, 12 mmol / L, 13 mmol / L, 14 mmol / L, 15 mmol / L, or 20 mmol / L.

15. The use according to any one of claims 12-14, characterized in that, The pH adjuster is an inorganic base.

16. The use according to claim 15, characterized in that, The pH adjuster is selected from sodium hydroxide.

17. The use according to claim 16, characterized in that, The pH of the liquid formulation is 4.0-6.5; preferably, the pH of the liquid formulation is 4.5-5.4; preferably, the pH of the liquid formulation is 4.8-5.

2.

18. The use according to claim 17, characterized in that, The pH of the liquid formulation is approximately 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5; preferably, the pH of the liquid formulation is approximately 5.

1.

19. The use according to claim 18, characterized in that, The liquid formulation comprises TACI-Fc fusion protein, hydroxypropyl-β-cyclodextrin, and / or succinic acid; preferably, the formulation comprises approximately 80 mg / mL of TACI-Fc fusion protein, approximately 150 mmol / L of hydroxypropyl-β-cyclodextrin, and / or approximately 10 mmol / L of succinic acid; more preferably, the formulation comprises 80 mg / mL of TACI-Fc fusion protein, 150 mmol / L of hydroxypropyl-β-cyclodextrin, and 10 mmol / L of succinic acid; further preferably, the pH of the liquid formulation is approximately 5.1.

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