Liquid pharmaceutical formulations of TACI-Fc fusion proteins

Abstract

The invention relates to a liquid medicine preparation, in particular to a liquid medicine preparation of TACI-FC fusion protein, and belongs to the field of anti-autoimmune disease medicines. The pharmaceutical preparation can improve the long-term storage stability of the TACI-Fc fusion protein, maintain good biological activity, improve the drug production efficiency and greatly improve the administration convenience.

Description

TACI-Fc fusion protein liquid pharmaceutical preparation Technical Field

[0001] The present invention relates to a liquid pharmaceutical preparation of TACI-Fc fusion protein, belonging to the field of anti-autoimmune disease drugs. Background Art

[0002] In recent years, the biopharmaceutical market has maintained a high growth trend. Fusion proteins and antibody drugs have played a huge role in tumors and autoimmune diseases. With the in-depth research of pathology and the development of development technology, while benefiting patients, they are also facing physical changes such as aggregation, denaturation, precipitation, and chemical changes such as isomerization, deamidation, and oxidation. These changes will affect the safety and effectiveness of the products. Therefore, stable formulations are needed to ensure that fusion proteins and antibody drugs still have the biological activity required for treatment before being used in patients. From 1986 to February 2021, 126 commercially available antibodies were approved worldwide, including 10 antibody-drug conjugates, 16 biosimilars, and 3 fusion proteins. There are a total of 136 commercial antibodies, including 36 lyophilized powders and 100 liquid preparations. (Reference 1: Robert G. Strickley, et.al. A review of formulations of commercially available antibodies. Journal of Pharmaceutical Sciences. 2021 (110), 2590-2608.)

[0003] Although freeze-dried antibody drug preparations have the advantages of being easy to store for a long time and having stable activity during transportation, freeze-dried dosage forms often become a bottleneck step in drug production speed due to the freeze-drying process, and greatly increase the cost of drug manufacturing. In addition, freeze-dried antibody drug preparations need to be re-dissolved before use, and in order to ensure the re-dissolution effect, the re-dissolution time is usually longer to avoid aggregation or changes in the structure of the active ingredient after re-dissolution, etc. Therefore, patients need to wait longer in the hospital, which also increases the pressure on the hospital. Therefore, the development of stable liquid preparations is increasingly becoming a better choice for antibody drug preparations.

[0004] However, compared to lyophilized formulations, developing antibody liquid formulations that are stable for long-term storage and transportation remains a significant challenge for the industry. Antibody liquid formulations can lose their biological activity during storage due to chemical and physical instabilities. Various chemical factors, such as deamidation, racemization, hydrolysis, oxidation, β-elimination, or disulfide exchange, as well as physical factors, such as antibody denaturation, aggregation, precipitation, or adsorption, can cause instability of the active ingredient.

[0005] Developing highly concentrated liquid formulations of antibody drugs for subcutaneous or intramuscular administration further increases the challenge because at high concentrations, the viscosity of the liquid formulation and the tendency of the protein to form aggregates can increase significantly. Aggregates can contain protein degradation products and can lead to various side effects, such as triggering undesirable immune responses.

[0006] Compared with general macromolecular preparations, antibody-like structural fusion proteins or antibody-like preparations have their own uniqueness. On the one hand, due to the fragile stability and structural complexity of monoclonal antibody drugs, the manufacture and storage of such drugs are extremely challenging. Due to the heterogeneous structure of antibodies, especially the complementary determining region (CDR) and Fc glycosylation, each formulation development of different monoclonal antibodies needs to be carried out on a case-by-case basis. When developing antibody preparations, there are challenges in antibody conformation, colloid or chemical structure, such as oxidation, isomerization, deamidation, aggregation, denaturation and breakage. When exposed to different temperature and humidity, pH and stress conditions, the three-dimensional structure of the antibody may change, especially in the hypervariable region (HVR). Undesirable products may show reduced activity, and more importantly, increased immunogenicity can endanger patients. Therefore, it is crucial to choose the best excipients to protect the antibody. (Reference 2: 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 caused or the interference with the purification process have always been a difficult problem to be solved in this field. Moreover, the factors affecting the aggregation of fusion proteins are also relatively complex and can be divided into external factors and internal factors. External factors mainly include temperature, physical pressure and solvent factors (pH, ionic strength, concentration, metal ions, etc.); internal factors mainly include fusion protein structural characteristics, sensitive residues and unpaired cysteines; these factors can 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 3: Production Challenges for Complex Biologics: Fusion Proteins, Stefan R. Schmidt, American Pharmaceutical Review, pp. 1-5, 2017).

[0007] Tetasip is a first-of-its-kind recombinant TACI-Fc fusion protein for B-cell-related autoimmune diseases. It targets and neutralizes BLyS and APRIL, two key cell signaling molecules in the B-cell pathway. It is an antibody-like fusion protein for the treatment of human autoimmune diseases, consisting of a truncated TACI and an immunoglobulin Fc sequence optimized to reduce ADCC and CDC effects. It exhibits excellent biological activity and safety and has been approved for marketing by the China National Medical Products Administration for the treatment of systemic lupus erythematosus. Tetasip for injection is a lyophilized formulation, which significantly limits drug production speed and significantly increases the time patients spend seeking medical treatment and the burden on hospitals. Therefore, there is an urgent need to develop a liquid formulation.

[0008] Summary of the Invention

[0009] To address the above-mentioned problems, the present invention provides a liquid pharmaceutical formulation of a TACI-Fc fusion protein, which can effectively improve the long-term stability of the TACI-Fc fusion protein, maintain good biological activity, and effectively increase the speed of drug production, reduce drug production costs, and significantly improve the convenience of patient use, thereby better meeting clinical drug needs.

[0010] The present invention provides a TACI-Fc liquid formulation with excellent stability, capable of being stored stably for over 18 months under preservative-free, sterile conditions at 4°C. The present inventors also surprisingly discovered that the developed formulation can achieve these requirements without the use of a surfactant.

[0011] Specifically, the present invention provides a TACI-Fc fusion protein liquid preparation, which includes a TACI-Fc fusion protein, a protective agent, a buffer and a pH adjuster, wherein: the TACI-Fc fusion protein includes: (i) a TACI extracellular region or a fragment thereof that binds to Blys and / or APRIL, and (ii) a human immunoglobulin constant region fragment; the protective agent is hydroxypropyl-β-cyclodextrin; the buffer is succinic acid; and the pH of the liquid preparation is 5.0-5.2.

[0012] Furthermore, the concentration range of the TACI-Fc fusion protein is 40 mg / ml-240 mg / ml.

[0013] Furthermore, the concentration of the TACI-Fc fusion protein is about 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.

[0014] Furthermore, the fusion protein content was determined by UV-visible spectrophotometry, where the absorbance of the tetasip sample at 280 nm was measured, based on the maximum UV absorption of the protein at that wavelength. After correcting the absorbance at 320 nm, the absorbance at 280 nm was proportional to the protein concentration. The protein concentration was calculated according to the Lambert-Beer law to determine the protein content. The protein content calculation formula is as follows:

[0015] Where, ε is the extinction coefficient of tadalafil, unit is (mg / ml) -1 cm -1 ;

[0016] A280 is the average absorbance of the sample solution at 280 nm;

[0017] A280 (corrected) is the average corrected absorbance of the sample solution at 280 nm.

[0018] Furthermore, the concentration range of the protective agent is 100mmol / L-200mmol / L.

[0019] Furthermore, the concentration of the protective agent is about 100mmol / L, 110mmol / L, 120mmol / L, 130mmol / L, 140mmol / L, 150mmol / L, 160mmol / L, 170mmol / L, 180mmol / L, 190mmol / L, or 200mmol / L.

[0020] Furthermore, the concentration range of the buffer is 5 mmol / L-15 mmol / L.

[0021] Furthermore, the concentration of the buffer is about 5mmol / L, 6mmol / L, 7mmol / L, 8mmol / L, 9mmol / L, 10mmol / L, 11mmol / L, 12mmol / L, 13mmol / L, 14mmol / L, or 15mmol / L.

[0022] Furthermore, the pH regulator is an alkaline inorganic salt, more preferably sodium hydroxide.

[0023] Furthermore, the pH of the liquid preparation is about 5.0, 5.1 or 5.2.

[0024] Preferably, the amino acid sequence of the TACI-Fc fusion protein is as shown in SEQ ID NO: 1; more preferably, it is Telitacicept.

[0025] SEQ ID NO: 1

[0026] Furthermore, the TACI-Fc fusion protein liquid preparation described in any of the aforementioned items contains about 10 mmol / L of succinic acid, about 150 mmol / L of hydroxypropyl-β-cyclodextrin, and about 80 mg / mL of the TACI-Fc fusion protein, and the pH of the liquid preparation is about 5.1.

[0027] Furthermore, the TACI-Fc fusion protein liquid preparation described in any of the aforementioned items contains succinic acid at a concentration of 10 mmol / L, 150 mmol / L hydroxypropyl-β-cyclodextrin, and 80 mg / mL of the TACI-Fc fusion protein, and the pH of the liquid preparation is approximately 5.1.

[0028] Furthermore, each milliliter of the TACI-Fc fusion protein liquid preparation described above contains about 80 mg of TACI-Fc fusion protein, about 1.18 mg of succinic acid, and about 209.85 mg of hydroxypropyl-β-cyclodextrin, and has a pH of about 5.1.

[0029] Furthermore, each milliliter of the TACI-Fc fusion protein liquid preparation described above contains 80 mg of TACI-Fc fusion protein, 1.18 mg of succinic acid, and 209.85 mg of hydroxypropyl-β-cyclodextrin, and has a pH of 5.1.

[0030] The present invention also relates to the use of any of the above-mentioned TACI-Fc liquid preparations in the preparation of a medicament, wherein the medicament is used to prevent, alleviate and / or treat autoimmune diseases.

[0031] The present invention also relates to any of the above-mentioned TACI-Fc liquid preparations for use in preventing, alleviating and / or treating autoimmune diseases.

[0032] The present invention also relates to a method for preventing, alleviating and / or treating an autoimmune disease, comprising administering any of the above-described TACI-Fc liquid preparations to a subject in need thereof.

[0033] The present invention also relates to a packaged pharmaceutical product comprising any one of the above-mentioned TACI-Fc liquid drugs.

[0034] The present invention also relates to a prefilled syringe comprising any one of the above-mentioned TACI-Fc liquid preparations. The prefilled syringe is further preferably a prefilled automatic syringe. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1: pH main effect diagram fitted by MODDE software;

[0036] Figure 2: Main effect diagram of arginine concentration fitted by MODDE software;

[0037] Figure 3: Main effect diagram of sucrose concentration fitted by MODDE software;

[0038] Figure 4: Main effect diagram of protein concentration fitted by MODDE software;

[0039] Figure 5: Contour plot fitted by MODDE software. From the contour plot, we can conclude that the optimal formulation in the DOE experimental design is when Arg is not added, the sucrose concentration is between 270 mmol / L and 300 mmol / L, the pH is between 4.95 and 5.1, and the protein concentration is 80 mg / mL. DETAILED DESCRIPTION

[0040] Unless otherwise defined, all technical terms used herein have the same meanings as understood by those of ordinary skill in the art. For definitions and terms in this field, professionals can refer to Current Protocols in Molecular Biology (Ausubel).

[0041] The three-letter and one-letter codes for amino acids used in the present invention are as described in J. biol. chem, 243, p3558 (1968).

[0042] The term "TACI" as used herein stands for transmembrane activator and CAML interactor, a member of the tumor necrosis factor receptor superfamily. The term "BLys" as used herein refers to B lymphocyte stimulator, a member of the TNF ligand superfamily that exists 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) as used herein refers to a tumor necrosis factor (TNF) analog that stimulates the proliferation of naive B cells and T cells in the body, promoting B cell accumulation. APRIL specifically binds to TACI and BCMA, preventing APRIL from binding to B cells and inhibiting APRIL-stimulated naive B cell proliferation. Furthermore, APRIL competes with BLys for binding to receptors (BCMA and TACI).

[0043] The term "TACI-Fc fusion protein" involved in the present invention refers to a transmembrane activator, calcium regulator and cyclophilin ligand interactor (TACI)-immunoglobulin fusion protein (i.e., TACI-Fc fusion protein). The TACI-immunoglobulin fusion protein provided by the present invention includes: (i) the TACI extracellular region or a fragment thereof that binds to Blys and / or APRIL; and (ii) a human immunoglobulin constant region fragment.

[0044] The term "TACI extracellular region or a fragment thereof that binds to Blys and / or APRIL" can be specifically referred to the extracellular domain of TACI disclosed in U.S. Patent Nos. 5,969,102, 6,316,222 and 6,500,428 and U.S. patent applications 09 / 569,245 and 09 / 627,206 (the contents of which are incorporated herein by reference), as well as specific fragments of the TACI extracellular domain that can interact with the TACI ligand, or the amino acid fragment of position 13-118 of the TACI extracellular domain disclosed in Chinese Patent Publication No. CN101323643A.

[0045] In the term "human immunoglobulin constant region fragment", the immunoglobulin portion is preferably IgG1, which may include a heavy chain constant region, such as a human heavy chain constant region. Preferred "human immunoglobulin constant region fragments" of the present invention are amino acid fragments containing part of the hinge region domain, CH2 domain, and CH3 domain.

[0046] The term "treatment" as used herein is related to a given disease or condition, including but not limited to: inhibiting 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 disappear; or alleviating the symptoms caused by the disease or condition, such as alleviating, preventing or treating the symptoms of the disease or condition.

[0047] The term "Telitacicept" (or "Telitacicept" (which can be used interchangeably in the present invention) in the present invention is a TACI-Fc fusion protein, whose amino acid sequence is shown in SEQ ID NO: 1, or as shown in https: / / extranet.who.int / soinn / mod / page / view.php?id=137&inn_n=10932. Its preparation can refer to the contents disclosed in patent publication No. CN101323643A or CN113613675A.

[0048] 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 used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.

[0049] Example 1: Formulation Development

[0050] After screening and comparing various excipients, this example focuses on the effects of different concentrations of amino acids (such as arginine hydrochloride), surfactants (such as polysorbate 20 (Tween-20)), protective agents (such as sucrose), and different pH values ​​on the stability of the TACI-Fc protein (amino acid sequence: SEQ ID NO: 1). Histidine was used as a buffer (containing hydrochloric acid to adjust the pH), and the MODDE software CCF model was used to perform a 5-factor 3-level experimental design.

[0051] A TACI-Fc protein (SEQ ID NO: 1) solution was dialyzed and concentrated, mother liquor was added, volume was fixed, and filtration and packaging were performed. The resulting formula information is shown in Table 1. The prepared sample was stored at 25°C for 5 weeks (up to one month). SEC-purity was tested at time points of 3 days (i.e., 3 days, the same below), 7 days, 2 weeks (i.e., 2 weeks, the same below), and 5 weeks. The 5-week research results were analyzed using MODDE software.

[0052] Table 1 Recipe information

[0053] The experimental analysis results are shown in Figures 1, 2, 3, and 4. The results show that both arginine hydrochloride and sucrose concentrations had statistically significant effects on protein purity (P < 0.05). Increasing arginine hydrochloride concentration increased aggregates, resulting in decreased protein purity. Conversely, increasing sucrose concentration reduced aggregates, leading to increased protein purity. Surprisingly, the presence of Tween-20 had no effect on reducing aggregates or degradation, suggesting that surfactants can be omitted from the formulation. pH had no significant effect on monomer content (P > 0.05), indicating that a pH range of 4.8-5.2 is suitable for the tetasip liquid protein formulation. The contour plot in Figure 5 shows that when arginine hydrochloride is omitted, a sucrose concentration between 270 mmol / L and 300 mmol / L and a protein concentration of 80 mg / mL are the most suitable ranges.

[0054] Example 2: Buffer system screening

[0055] This example compared the effectiveness of three buffer systems: histidine-histidine hydrochloride, citric acid, and succinic acid. The optimal buffer system for the TACI-Fc protein formulation was selected. The TACI-Fc protein (SEQ ID NO: 1) concentration was 160 mg / mL, pH 5.0, and the sucrose concentration was set at 290 mmol / L.

[0056] TACI-Fc protein (SEQ ID NO: 1) was dialyzed and concentrated into the formula in Table 2, and then the volume was fixed, filtered and aliquoted. The prepared sample was then stored at 25°C, and SEC-purity was tested at time points of 3 days and 9 days.

[0057] Table 2 Buffer system formula information

[0058] Table 3 Purity (SEC-purity) (%) Results

[0059] Table 4 Visible foreign matter results

[0060] Table 5 Viscosity (mPa.s) Results

[0061] Table 6 Protein concentration (mg / ml) results

[0062] Table 7 pH changes

[0063] The experimental results are detailed in Tables 3 to 7. The results show that after being placed at 25°C for 9 days, the main peak purity of the histidine-histidine hydrochloride buffer system and the citric acid buffer system decreased by 1.2% and 2.6%, respectively, and the succinic acid buffer system decreased by 1.8%. After being placed at 25°C for 9 days, all three buffer systems had an increase in aggregates, with the increase in aggregates of 4.1%, 4.8%, and 3.7% in the histidine-histidine hydrochloride buffer system, the citric acid buffer system, and the succinic acid buffer system, respectively. Visible foreign matter and viscosity measurements indicate that the citric acid buffer system is less stable than the other two groups, exhibiting a whitish appearance and high viscosity, making it unsuitable. Furthermore, the citric acid buffer system made TACI-Fc protein more difficult to dialyze than the other two groups. After dialysis at 4°C, the protein became a milky suspension, but returned to a clear whitish liquid after a period of time at room temperature. Furthermore, the viscosity of the TACI-Fc protein after dialysis in the citric acid buffer system was significantly higher than that in the other two groups, making the protein solution more viscous. In summary, the histidine-histidine hydrochloride and succinate buffer systems were superior to the citric acid buffer system. Furthermore, the study found that at pH 5.0, the succinate buffer system had a superior buffering capacity to the histidine-histidine hydrochloride buffer system.

[0064] Example 3: Protective agent screening experiment

[0065] In Example 1, it was found that protein stability increased with increasing sucrose concentration. The possible reason is that the hydroxyl groups in sucrose act as proton donors, which inhibit the deprotonation of thiols to a certain extent, thereby reducing the formation rate of disulfide bond aggregates. However, high concentrations of sucrose may lead to a significant increase in the osmotic pressure of the liquid preparation, thereby affecting the isotonicity of the preparation required for subcutaneous injection and may cause higher levels of endotoxins. After evaluating and comparing a variety of different protective agents, this example focuses on the effect of hydroxypropyl-β-cyclodextrin as a protective agent on the stability of the preparation.

[0066] The TACI-Fc protein (SEQ ID NO: 1) was dialyzed and concentrated into the formula in Table 8, then fixed to volume, filtered and aliquoted. The prepared samples were then stored at 25°C and 40°C, respectively. SEC-purity, capillary electrophoresis (nrCE-SDS) changes at 40°C, and protein cell activity and binding activity at 25°C were tested at 4d and 7d at 40°C and 2 weeks, 3 weeks, and 4 weeks at 25°C.

[0067] Table 8 Protective agent formula information

[0068] Table 9 40℃ Purity (SEC-purity) (%) Results

[0069] Table 10 25℃ Purity (SEC-purity) (%) Results

[0070] Table 11 40℃ nrCE-SDS results

[0071] Table 12 Cell activity results at 25°C

[0072] Table 13 Binding activity results at 25°C

[0073] The experimental results are shown in Tables 9 to 13. The results show that the SEC results at 40°C and 25°C (see Tables 9 to 10) show that the higher the concentration of hydroxypropyl-β-cyclodextrin, the better the stability of the TACI-Fc protein, the higher the sucrose concentration, the better the protein stability, and the protective effect of hydroxypropyl-β-cyclodextrin on the TACI-Fc protein is better than that of sucrose on the protein; the nrCE-SDS results (see Table 11) show that the higher the concentration of hydroxypropyl-β-cyclodextrin, the better the protein stability, the higher the sucrose concentration, the better the protein stability, and the protective effect of hydroxypropyl-β-cyclodextrin on the protein is better than that of sucrose on the protein, which is consistent with the results in Tables 9 and 10; the results of cell activity at 25°C (see Table 12) show that there is no downward trend in the cell activity of the two; and the binding activity at 25°C (see Table 13) shows that there is no downward trend in the binding activity of the two. From the above results, it can be seen that the presence of sucrose or hydroxypropyl-β-cyclodextrin reduces the formation of protein aggregates and degradation, and the protective effect of hydroxypropyl-β-cyclodextrin on TACI-Fc protein is better than sucrose.

[0074] Example 4: pH confirmation experiment

[0075] This example focuses on investigating the effect of different pH values ​​on the stability of TACI-Fc protein based on a hydroxypropyl-β-cyclodextrin concentration of 140 mmol / L and a succinic acid buffer system of 10 mmol / L.

[0076] TACI-Fc protein (SEQ ID NO: 1) was dialyzed into 10 mmol / L succinic acid. When the protein reached 40 mg / mL, a quantitative amount was taken. The mother liquor was added and the sample was concentrated to the target concentration. The final formulation information is shown in Table 14. The prepared sample was then stored at 40°C and tested for SEC purity at 2 and 7 days.

[0077] Table 14 pH confirmation formula information

[0078] Table 15 40℃ Purity (SEC-purity) (%) Results

[0079] The experimental results are shown in Table 15. The results show that as the pH increases, the polymer tends to increase and the degradation tends to decrease, but the increase in the polymer is greater than the decrease in the degradation, which makes the monomer tend to decrease. In order to balance the polymer and degradation degree, the pH is determined to be 5.0-5.2.

[0080] Example 5: Hydroxypropyl-β-cyclodextrin concentration confirmation experiment

[0081] This example focuses on the effect of 50 mmol / L, 100 mmol / L and 140 mmol / L hydroxypropyl-β-cyclodextrin on protein stability at pH 5.1, 10 mmol / L succinic acid and 80 mmol / L TACI-Fc (SEQ ID NO: 1).

[0082] TACI-Fc (SEQ ID NO: 1) was dialyzed and concentrated into the formula in Table 16, and then the volume was fixed, filtered and aliquoted. The prepared sample was then stored at 40°C. SEC-purity, nrCE-SDS results, and osmotic pressure were tested at time points of 3d and 7d.

[0083] Table 16 Recipe information

[0084] Table 17 40℃ Purity (SEC-purity) (%) Results

[0085] Table 18 nrCE-SDS results

[0086] Table 19 Osmotic pressure (mOsm / kg) results

[0087] The experimental results are shown in Tables 17 to 19, where the results in Tables 17 and 18 show that as the concentration of hydroxypropyl-β-cyclodextrin decreases, the purity decreases successively; the results in Table 19 show that the isotonic effect can only be initially achieved when the concentration of hydroxypropyl-β-cyclodextrin is 140 mmol / L. The concentration of hydroxypropyl-β-cyclodextrin will be increased subsequently to obtain a more reasonable solution osmotic pressure. The concentration of hydroxypropyl-β-cyclodextrin will be further set at 150 mmol / L and confirmed by an osmotic pressure experiment.

[0088] Example 6: Osmotic pressure confirmation

[0089] The experiment was carried out in two parallel groups A and B. The specific formula information is shown in Table 20.

[0090] Table 20 Recipe information

[0091] Table 21 Osmotic pressure (mOsm / kg) and pH results

[0092] The experimental results are shown in Table 21. When the concentration of TACI-Fc (SEQ ID NO: 1) was 80 mg / mL, the osmotic pressures of both groups A and B were approximately 330. When the concentration of TACI-Fc (SEQ ID NO: 1) was 120 mg / mL, the osmotic pressures of both groups A and B were approximately 400. The osmotic pressures measured before and after filtration were similar, indicating that filtration had no effect on the osmotic pressures. The pH values ​​were both within the range of 5.1 ± 0.1. Based on these findings, the formulation composition was tentatively determined to be: 10 mmol / L succinic acid, 150 mmol / L hydroxypropyl-β-cyclodextrin, 80 mg / mL protein, and sodium hydroxide for adjustment to pH 5.1 ± 0.1.

[0093] Example 7: Long-term stability test

[0094] This example focuses on the long-term and accelerated stability of the finalized prescription solution packaged into prefilled syringes.

[0095] TACI-Fc (SEQ ID NO: 1) was dialyzed into the following formulations. After dialysis, the samples were concentrated, fixed to volume, filtered, and packaged. Specific formulation information is shown in Table 22. The long-term stability of the samples under different temperature conditions was investigated according to the temperature and time parameters in Table 23.

[0096] Table 22 Recipe information

[0097] Table 23 Long-term stability test time points

[0098] According to SEC-purity data, after 24 months of storage at -20-4°C, the aggregate content of TACI-Fc (SEQ ID NO: 1) increased from 1.3% to 2.88%, and the purity decreased from 98.6% to 94.7%, both within the quality standard range. No increase in particulate matter (visible foreign matter and insoluble particles) was observed, and all results were within the acceptable standard range. Other product quality attributes, including peptide mapping, appearance, osmotic pressure, pH, CE-SDS (r / nr), RP-HPLC, activity, protein content, bacterial endotoxin content, and container closure (CCI), showed little change, all within the acceptable standard range. After rigorous long-term stability evaluation, the test results showed that the formulation of the present invention, when filled in a prefilled automatic syringe (PFS) and stored at -20-4°C for a long period of time, showed good stability and met all requirements of the stability quality standard. Even at 25°C storage conditions, after 6 months of storage, SEC-purity testing showed that although the aggregates increased slightly, all test results were within the quality standard range.

[0099] The above describes the spirit of the present invention in detail through the preferred embodiments of the present invention. It is understood by those skilled in the art that any modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention fall within the scope of protection of the present invention.

Claims

1. A TACI-Fc fusion protein liquid preparation, characterized in that: The liquid preparation includes a TACI-Fc fusion protein, a protective agent, a buffer and a pH adjuster, wherein: the TACI-Fc fusion protein includes: (i) a TACI extracellular domain or a fragment thereof that binds to Blys and / or APRIL, and (ii) a human immunoglobulin constant region fragment; the protective agent is hydroxypropyl-β-cyclodextrin; the buffer is succinic acid; and the pH range of the liquid preparation is 5.0-5.

2.

2. The TACI-Fc fusion protein liquid preparation according to claim 1, characterized in that: The concentration of the TACI-Fc fusion protein ranges from 40 mg / ml to 240 mg / ml.

3. The TACI-Fc fusion protein liquid preparation according to claim 2, characterized in that: The concentration of the TACI-Fc fusion protein is about 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.

4. The TACI-Fc fusion protein liquid preparation according to claim 2 or 3, characterized in that: The concentration range of the protective agent is 100mmol / L-200mmol / L.

5. The TACI-Fc fusion protein liquid preparation according to claim 4, characterized in that: The concentration of the protective agent is about 100mmol / L, 110mmol / L, 120mmol / L, 130mmol / L, 140mmol / L, 150mmol / L, 160mmol / L, 170mmol / L, 180mmol / L, 190mmol / L, or 200mmol / L.

6. The TACI-Fc fusion protein liquid preparation according to claim 4 or 5, characterized in that: The concentration of the buffer is 5mmol / L-15mmol / L.

7. The TACI-Fc fusion protein liquid preparation according to claim 6, characterized in that: The concentration of the buffer is about 5mmol / L, 6mmol / L, 7mmol / L, 8mmol / L, 9mmol / L, 10mmol / L, 11mmol / L, 12mmol / L, 13mmol / L, 14mmol / L, or 15mmol / L.

8. The TACI-Fc fusion protein liquid preparation according to claim 6 or 7, characterized in that: The pH regulator is an alkaline inorganic salt.

9. The TACI-Fc fusion protein liquid preparation according to claim 8, characterized in that: The pH regulator is sodium hydroxide.

10. The TACI-Fc fusion protein liquid preparation according to claim 8 or 9, characterized in that: The pH of the liquid formulation is about 5.0, 5.1 or 5.

2.

11. The TACI-Fc fusion protein liquid preparation according to claim 10, characterized in that: The amino acid sequence of the TACI-Fc fusion protein is shown in SEQ ID NO:

1.

12. The TACI-Fc fusion protein liquid preparation according to claim 11, characterized in that: The TACI-Fc fusion protein is tadalafil.

13. The TACI-Fc fusion protein liquid preparation according to claim 11 or 12, characterized in that: The preparation contains about 10 mmol / L of succinic acid, about 150 mmol / L of hydroxypropyl-β-cyclodextrin, and about 80 mg / mL of the TACI-Fc fusion protein. The pH of the liquid preparation is about 5.

1.

14. The TACI-Fc fusion protein liquid preparation according to claim 11 or 12, characterized in that: Each milliliter of the liquid preparation contains about 80 mg of TACI-Fc fusion protein, about 1.18 mg of succinic acid, and about 209.85 mg of hydroxypropyl-β-cyclodextrin, and has a pH of about 5.

1.

15. Use of the TACI-Fc fusion protein liquid preparation according to any one of claims 1 to 14 in the preparation of a medicament for preventing, alleviating and / or treating autoimmune diseases.

16. The TACI-Fc fusion protein liquid preparation according to any one of claims 1 to 14, which is used for preventing, alleviating and / or treating autoimmune diseases.

17. A method for preventing, alleviating and / or treating an autoimmune disease, comprising administering the liquid preparation according to any one of claims 1 to 14 to a subject in need thereof.

18. A packaged pharmaceutical product comprising the TACI-Fc fusion protein liquid medicine according to any one of claims 1 to 14.

19. A prefilled syringe comprising the TACI-Fc fusion protein liquid preparation according to any one of claims 1 to 14.

20. The syringe of claim 19, which is a prefilled autoinjector.

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