A fusion protein preparation

Abstract

The present invention relates to a fusion protein formulation. In particular, the present invention provides a fusion protein composition comprising a fusion protein, a buffer and a stabilizer. The composition of the present invention is capable of improving the stability of the fusion protein.

Description

A fusion protein preparation Technical Field

[0001] The present invention belongs to the field of biopharmaceuticals and relates to a preparation containing a fusion protein. Background Art

[0002] Generally, factors such as adverse temperature, shear force, vibration, freeze-thaw, UV exposure, excessive pH changes, organic solvents, and microbial contamination can easily lead to changes in the chemical and physical properties of protein drugs. Chemical denaturation includes dissociation, oxidation, deamidation, isomerization, and polymerization of protein molecules, which are affected by the amino acids that make up the protein and the conditions of the solvent containing the protein (salt, pH, and temperature). Physical denaturation includes changes in the tertiary structure, covalent / non-covalent aggregation and adhesion of monomers, and ionic forces between the charges attached to the protein and the charges carried by the solute or solvent. These chemical or physical denaturations will cause the protein to lose its original physicochemical properties and physiological activity.

[0003] As a biological drug, protein denaturation can have a huge impact on the drug's efficacy and safety, such as causing the patient's immune response. Therefore, a liquid preparation that can maintain the protein's physical and chemical properties and enable long-term stable storage of the protein is extremely important to the quality of protein drugs.

[0004] To maintain the long-term stability of proteins, proteins are generally stored for a long time in the form of freeze-dried or liquid preparations.

[0005] Many bio-antibody drugs on the market use freeze-drying for long-term storage. However, freeze-drying also presents challenges, such as ice crystal formation and pH changes during the freeze-drying process, which can alter the protein's properties. Furthermore, the drug must be reconstituted before use, which can also affect the protein's properties. This also complicates drug use and increases the risk of drug contamination. Furthermore, industrial production requires complex freeze-drying equipment and operations, increasing drug production costs.

[0006] Liquid preparations are also a way to store antibody drugs. The buffer system in which they are located maintains the pH within a certain range. Various stabilizers, surfactants, osmotic pressure regulators, and even antimicrobial agents are added to the liquid preparations to keep the protein stable in this buffer system. However, due to different protein structures, each protein requires a different buffer system and stabilizer. Inappropriate buffer systems or stabilizers may even reduce the stability of the protein. Therefore, different protein drugs require different liquid preparation components.

[0007] Therefore, there is a need in the art to develop a fusion protein preparation with high stability to improve the stability of the fusion protein.

[0008] Summary of the Invention

[0009] The purpose of the present invention is to provide a fusion protein composition with high stability.

[0010] In a first aspect, the present invention provides a fusion protein composition comprising a fusion protein, a buffer, and a stabilizer; the fusion protein comprises TNFRII or an active fragment thereof, BCMA or an active fragment thereof, and optionally an antibody Fc region, and the content of the fusion protein is 0.5-100 mg / mL;

[0011] The buffer is selected from the group consisting of phosphate buffer, citrate buffer, or a combination thereof, and the concentration of the buffer is 1-100 mM;

[0012] The stabilizer is selected from the group consisting of salts, sugars, amino acids, or a combination thereof;

[0013] The pH of the composition is 5.0-7.0.

[0014] In another preferred embodiment, the fusion protein has the amino acid sequence shown in SEQ ID NO: 1.

[0015] In another preferred embodiment, the composition is a pharmaceutical composition.

[0016] In another preferred embodiment, the pH of the composition is preferably 5.5-7.0, more preferably 5.5-6.5, and even more preferably 5.5-6.0.

[0017] In another preferred embodiment, the content of the fusion protein is preferably 1-100 mg / mL, more preferably 5-100 mg / mL, more preferably 5-80 mg / mL, more preferably 5-50 mg / mL, more preferably 10-50 mg / mL, and most preferably 25-50 mg / mL.

[0018] In another preferred embodiment, the buffer solution is a citric acid buffer solution.

[0019] In another preferred embodiment, the concentration of the buffer is preferably 1-80 mM, more preferably 1-50 mM, more preferably 10-30 mM, and most preferably 10 mM.

[0020] In another preferred embodiment, the medium of the buffer solution is water.

[0021] In another preferred embodiment, the salt is selected from the group consisting of sodium chloride, potassium chloride, calcium chloride, magnesium chloride, ammonium chloride, sodium sulfate, potassium sulfate, calcium sulfate, magnesium sulfate, ammonium sulfate, sodium nitrate, potassium nitrate, calcium nitrate, magnesium nitrate, ammonium nitrate, or a combination thereof.

[0022] In another preferred embodiment, the salt is sodium chloride.

[0023] In another preferred embodiment, the concentration of the salt is 0-150 mM, preferably 0-140 mM, more preferably 0-110 mM, more preferably 10-110 mM, more preferably 10-70 mM, and most preferably 70 mM.

[0024] In another preferred embodiment, the sugar is selected from the group consisting of mannose, sucrose, trehalose, sorbitol, or a combination thereof.

[0025] In another preferred embodiment, the sugar is mannitol.

[0026] In another preferred embodiment, the content of the sugar is 0-10 wt %, preferably 0-8 wt %, more preferably 0-5 wt %, more preferably 1-5 wt %, more preferably 2-5 wt %, and most preferably 2 wt %, based on the total weight of the composition.

[0027] In another preferred embodiment, the amino acid is selected from the group consisting of glycine, histidine, lysine, glutamic acid, or a combination thereof.

[0028] In another preferred embodiment, the amino acid is glycine.

[0029] In another preferred embodiment, the concentration of the amino acid is 0-200 mM, preferably 0-100 mM, more preferably 50-100 mM, and most preferably 50 mM.

[0030] In another preferred embodiment, the stabilizer is selected from a combination of salts and sugars.

[0031] In another preferred embodiment, the stabilizer is selected from a combination of sodium chloride and mannitol.

[0032] In another preferred embodiment, the composition may further include a surfactant, and the surfactant includes (but is not limited to): a cationic surfactant, a negative ionic surfactant, a nonionic surfactant, or a combination thereof.

[0033] In another preferred embodiment, the surfactant is selected from the group consisting of polysorbate, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene hydrogenated castor oil, glycerol fatty acid ester, poloxamer, or a combination thereof.

[0034] In another preferred embodiment, the polysorbate is selected from the group consisting of polysorbate 20 (PS-20, Tween-20), polysorbate 40 (PS-40, Tween-40), polysorbate 60 (PS-60, Tween-60), polysorbate 80 (PS-80, Tween-80), or a combination thereof.

[0035] In another preferred embodiment, the surfactant is polysorbate 20 (Tween-20).

[0036] In another preferred embodiment, the content of the surfactant is 0-5wt%, preferably 0-3wt%, more preferably 0-1wt%, more preferably 0-0.5wt%, more preferably 0-0.3wt%, more preferably 0-0.2wt%, more preferably 0-0.1wt%, based on the total weight of the composition.

[0037] In another preferred embodiment, the composition does not contain a surfactant.

[0038] In another preferred embodiment, the composition comprises:

[0039] In another preferred embodiment, the composition comprises:

[0040] In another preferred embodiment, the composition comprises:

[0041] In another preferred embodiment, the composition comprises:

[0042] The second aspect of the present invention provides a method for preparing the composition of the first aspect of the present invention, comprising the steps of:

[0043] After mixing the components of the composition of the first aspect of the present invention, a fusion protein composition is obtained.

[0044] The third aspect of the present invention provides use of the composition of the first aspect of the present invention, wherein the composition is used for preparing a medicament for treating an immune disease.

[0045] In another preferred embodiment, the composition is a liquid preparation.

[0046] In another preferred embodiment, the composition is an oral liquid preparation or an injectable liquid preparation.

[0047] The fourth aspect of the present invention provides a method for preventing and / or treating an immune disease, comprising: administering the composition of the first aspect of the present invention to a subject in need thereof, thereby treating the immune disease.

[0048] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. DETAILED DESCRIPTION

[0049] Through extensive and in-depth research, the present inventors have developed a composition that effectively maintains the stability of a fusion protein, and the fusion protein in the composition can maintain stability under high temperature conditions. Therefore, the composition of the present invention can provide stable fusion protein quality, extend the shelf life of the product, and improve the safety of clinical application. Based on this, the present invention was completed.

[0050] the term

[0051] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0052] As used herein, the terms "comprise," "include," and "contain" are used interchangeably to include not only closed definitions but also semi-closed and open definitions. In other words, the terms include "consisting of," "consisting essentially of."

[0053] As used herein, "mM" is a unit of mmol / L, for example, 1 mM = 1 mmol / L.

[0054] In the composition of the present invention, the weight content (wt.%) of each component is based on the weight of the composition.

[0055] Fusion protein

[0056] In the present invention, the composition of the present invention includes a purified fusion protein. The fusion protein includes the following elements fused together: (a) TNFRII or an active fragment thereof; (b) BCMA or an active fragment thereof; and optionally (c) an Fc fragment. Preferably, the fusion protein of the present invention has the structure of TNFRII-BCMA-Fc.

[0057] The amino acid sequence of the fusion protein of the present invention is as follows:

[0058] Composition and preparation method thereof

[0059] The present invention provides a fusion protein composition, which comprises a fusion protein, a buffer and a stabilizer.

[0060] The composition of the present invention is preferably a pharmaceutical composition.

[0061] The composition of the present invention is preferably a liquid preparation, such as an oral liquid preparation or an injectable liquid preparation.

[0062] In a preferred embodiment of the present invention, the content of the fusion protein is 0.5-100 mg / mL, preferably 1-100 mg / mL, more preferably 5-100 mg / mL, more preferably 5-80 mg / mL, more preferably 5-50 mg / mL, more preferably 10-50 mg / mL, and most preferably 25-50 mg / mL.

[0063] In a preferred embodiment of the present invention, the composition may further include a surfactant, and the surfactant includes (but is not limited to): a cationic surfactant, a negative ionic surfactant, a nonionic surfactant, or a combination thereof.

[0064] Typically, the surfactant includes (but is not limited to): polysorbate, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene hydrogenated castor oil, glycerol fatty acid ester, poloxamer, or a combination thereof.

[0065] Typically, the polysorbate includes (but is not limited to): polysorbate 20 (PS-20, Tween-20), polysorbate 40 (PS-40, Tween-40), polysorbate 60 (PS-60, Tween-60), polysorbate 80 (PS-80, Tween-80), or a combination thereof.

[0066] Typically, the surfactant comprises polysorbate 20 (PS-20, Tween-20).

[0067] In a preferred embodiment of the present invention, the content of the surfactant is 0-5wt%, preferably 0-3wt%, more preferably 0-1wt%, more preferably 0-0.5wt%, more preferably 0-0.3wt%, more preferably 0-0.2wt%, more preferably 0-0.1wt%, based on the total weight of the composition.

[0068] In a preferred embodiment of the present invention, the buffer includes (but is not limited to): phosphate buffer, citrate buffer, or a combination thereof.

[0069] In another preferred embodiment, the medium of the buffer solution is water.

[0070] In a preferred embodiment of the present invention, the concentration of the buffer is 1-100 mM, preferably 1-80 mM, more preferably 1-50 mM, more preferably 5-40 mM, more preferably 5-25 mM, more preferably 5-20 mM, and most preferably 10 mM.

[0071] Typically, the buffer comprises a citrate buffer.

[0072] In a preferred embodiment of the present invention, the concentration of the citric acid buffer is 1-100 mM, preferably 1-80 mM, more preferably 1-50 mM, more preferably 10-30 mM, and most preferably 10 mM.

[0073] In a preferred embodiment of the present invention, the stabilizer includes (but is not limited to) an osmotic pressure regulator.

[0074] In a preferred embodiment of the present invention, the stabilizer includes (but is not limited to): amino acids, sugars, salts, or a combination thereof.

[0075] Typically, the amino acids include (but are not limited to): arginine, glycine, histidine, or a combination thereof.

[0076] Typically, the sugars include (but are not limited to): sucrose, mannitol, trehalose, maltose, sorbitol, or a combination thereof.

[0077] In a preferred embodiment of the present invention, the salts include (but are not limited to): sodium chloride, potassium chloride, calcium chloride, magnesium chloride, or a combination thereof.

[0078] In another preferred embodiment, the stabilizer includes mannitol.

[0079] Typically, the stabilizer includes mannitol, sodium chloride, glycine or an amino acid with the same properties, or a combination thereof.

[0080] In a preferred embodiment of the present invention, the content of the stabilizer is 0.5-50wt%, preferably 0.5-40wt%, more preferably 0.8-30wt%, more preferably 1-20wt%, more preferably 1-15wt%, more preferably 1-10wt%, more preferably 1-8wt%, more preferably 2-7wt%, and most preferably 5wt%, based on the total weight of the composition.

[0081] In a preferred embodiment of the present invention, the pH of the composition is 5.0-7.0, preferably 5.0-6.5, more preferably 5.2-6.4, more preferably 5.3-6.3, and most preferably 5.4-6.0, for example 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0.

[0082] In a preferred embodiment of the present invention, the composition comprises:

[0083] In a preferred embodiment of the present invention, the composition comprises:

[0084] In a preferred embodiment of the present invention, the composition comprises:

[0085] In a preferred embodiment of the present invention, the composition comprises:

[0086] use

[0087] The present invention also provides a use of the composition of the present invention for preparing a medicament for preventing and / or treating immune diseases.

[0088] The present invention also provides a method for preventing and / or treating an immune disease, which comprises administering the composition of the present invention to a subject in need thereof, thereby treating the immune disease.

[0089] In a preferred embodiment of the present invention, the immune disease includes rheumatoid arthritis (RA), ankylosing spondylitis (AS), psoriasis (PS), psoriatic arthritis (PsA), juvenile idiopathic arthritis (JIA), systemic lupus erythematosus (SLE), Behcet's disease (BD), multiple sclerosis (MS), Sjögren's syndrome (SS), Graves' disease, Crohn's disease (CD), ulcerative colitis (UC), primary glomerulonephritis, IgA nephropathy, autoimmune vasculitis, polymyositis (PM), non-infectious uveitis, autoimmune hemolytic anemia (AIHA), autoimmune purpura (ATTP), N-methyl-d-aspartate receptor (NMDAR) encephalitis, myasthenia gravis, hidradenitis suppurativa (HS), myelin-oligodendrocyte glycoprotein spectrum disorder (MOGSD) and neuromyelitis optica spectrum disorder (NMOSD), or a combination thereof.

[0090] The main advantages of the present invention include:

[0091] The composition containing the fusion protein provided by the present invention can reduce the degradation and aggregation of the fusion protein and improve the stability of the fusion protein.

[0092] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0093] Example

[0094] The citrate buffer and phosphate buffer in the following examples are as follows:

[0095] Citric acid buffer: the medium is water, and the solutes are citric acid and sodium citrate.

[0096] Phosphate buffer: The medium is water, and the solutes are sodium dihydrogen phosphate and disodium hydrogen phosphate.

[0097] The sequence of the recombinant fusion protein in the following examples is shown in SEQ ID NO.: 1.

[0098] Example 1: Protein Stability pH Range

[0099] The stability of proteins in the 10 mM citric acid-disodium hydrogen phosphate buffer system with the same composition (citric acid-disodium hydrogen phosphate) but different pH was studied.

[0100] The above different pH values ​​refer to 10 mM citric acid-disodium hydrogen phosphate buffers of pH 3.0, pH 4.0, pH 5.0, pH 6.0, pH 7.0, pH 8.0, and pH 8.5.

[0101] After the protein solution was exchanged to the corresponding buffer system, the stability of the protein was studied at 2-8°C and 40°C.

[0102] SE-HPLC is a size exclusion chromatography method that separates high molecular weight proteins and protein degradation products from the target protein based on molecular weight. Protein stability is determined by the formation of high molecular weight protein aggregation and degradation products.

[0103] Table 1. pH stability screening: SEC test results after 3 days at 40°C

[0104] In this example, it was found that when pH ≤ 5.0, the sample degraded severely under accelerated conditions at 40°C, and the degree of degradation increased as the pH decreased. When pH ≥ 7.0, the sample began to aggregate significantly at 40°C, and the aggregation gradually increased as the pH increased.

[0105] According to the above SEC results, the sample stability is relatively the best at pH 6.0. The preferred pH range should be higher than 5.0 and lower than 7.0, and more preferably 5.5-6.5. This example basically determines the pH range of target protein stability.

[0106] Table 2. pH stability screening: SEC test results after 2 weeks at 4°C

[0107] The results of the long-term storage test at 2-8°C were basically consistent with the high-temperature accelerated results, and the optimal pH range was 5.5-6.5.

[0108] Example 1 By conducting stability tests on the target protein in a citric acid-sodium dihydrogen phosphate buffer system under different pH conditions, it was finally determined that the preferred pH range of the target protein under high temperature acceleration and 2-8°C long-term storage conditions was pH 5.5-6.5.

[0109] Example 2: Screening of buffer systems

[0110] After determining that the preferred stability pH range of the target protein is 5.5-6.5 through Example 1, a buffer screening test is required to find the buffer system most suitable for the target protein.

[0111] Common buffer systems for antibody drug formulations in the pH range of 5.5-6.5 include histidine buffer, phosphate buffer, and citrate buffer. However, histidine buffer is susceptible to degradation at high temperatures and exhibits significant pH variations with temperature, making it unsuitable for rapid formulation development trials. Therefore, this buffer system was not considered for this purpose. In this example, the stability of the screened protein was compared in phosphate and citrate buffer systems to determine the buffer most suitable for the target protein.

[0112] The factors affecting protein stability are mainly the concentration and pH of the buffer system. The buffer system concentration is set at 10-30 mM, and the pH range is 5.5-6.5.

[0113] Table 3. SEC results of phosphate buffer samples treated at 50°C for 1 day

[0114] Table 4. SEC results of citrate buffer samples treated at 50°C for 1 day

[0115] As shown in Tables 3 and 4, the samples at 50°C showed a significant increase in aggregation and degradation. Aggregate content decreased with increasing buffer concentration, while degradation increased. In the phosphoric acid system, both aggregation and degradation increased with increasing pH. In the citric acid system, aggregation increased with increasing pH, while degradation decreased.

[0116] Comparing the citric acid and phosphate buffer systems, under the same conditions, the citric acid system exhibited relatively less aggregate growth. Taking both degradation and aggregate content into account, the monomer content of the samples decreased with increasing pH, with a relatively high monomer content at pH below 6.0. When the pH was above 6.0, sample stability was relatively poor, while monomer content tended to increase with increasing citric acid buffer concentration.

[0117] Example 2 determined that citric acid buffer was the preferred buffer for the sample by a comparative experiment of citric acid buffer and phosphate buffer. The concentration and pH range of the buffer solution were confirmed by a test to investigate the influence of citric acid buffer concentration and pH on sample stability. From a comprehensive analysis of polymer, monomer, and degradation, it was finally concluded that the sample had better stability in a citric acid buffer system of pH 5.5-6.5. A more preferred pH range was pH 5.5-6.0, and a more preferred buffer concentration was a high-concentration citric acid buffer, with 30mM citric acid buffer being optimal. However, the final product of the sample was an intramuscular injection drug, and the citric acid content needed to be reduced to reduce the irritation of the injection. The test showed that high-concentration citric acid buffer had a better sample stability than low-concentration citric acid, which may be due to the high concentration of salt ions in the high-concentration citric acid, which had a certain stabilizing effect on the sample. The salt ion concentration could be increased by adding sodium chloride in the subsequent formulation excipient screening, so the citric acid buffer concentration could be reduced to 10mM to reduce the irritation of the injection.

[0118] Finally, the optimal buffer system for the sample was determined to be 10 mM citric acid system, pH 5.5-6.0.

[0119] Example 3: Effect of salt concentration on protein stability

[0120] In order to further improve the stability of the formulation composition on the protein and ensure the osmotic pressure of the formulation, it is necessary to examine the effect of adding excipients on the protein stability when the buffer solution is determined.

[0121] Since the effect of buffer concentration on sample stability may be caused by salt concentration in Example 2, the effect of ion concentration on sample stability was first investigated, and sodium chloride was selected as the salt excipient for investigation. At the same time, in order to control the osmotic pressure, the salt concentration was set to 150mM for the experiment.

[0122] Table 5. SEC results of the effect of salt concentration on sample stability

[0123] As can be seen from Table 5, increasing salt concentration has a significant effect on sample stability. As the salt concentration increases, the aggregates in samples at pH 5.5-6.0 are significantly reduced. Under pH 5.5 conditions, degradation also decreases to a certain extent as the salt concentration increases, while under pH 6.0 conditions, increasing salt concentration has no significant effect on degradation. Comparing pH 5.5 and pH 6.0, it is found that the aggregate content of pH 5.5 samples is significantly lower than that of pH 6.0, and pH 5.5 also has an advantage in monomer content. However, from the perspective of degradation, the sample degrades more at pH 5.5 than at pH 6.0. It can be seen that providing a certain concentration of salt in the preparation significantly improves the stability of the sample.

[0124] Example 3 investigated the salt concentration and determined that increasing the salt concentration significantly improved sample stability, which also provided factual support for the use of a lower buffer concentration in Example 2.

[0125] Finally, the optimal concentration of sodium chloride in the prescription was determined to be 0-150 mM.

[0126] Example 4: Effects of excipients such as sugars and amino acids on sample stability

[0127] In addition to sodium chloride as an isotonicity regulator, sucrose and mannitol are also commonly used as isotonicity regulators and protein stabilizers. To further improve the stability of the formulation on protein, it is necessary to investigate the effects of sucrose, mannitol, glycine, and their combination on protein stability.

[0128] Taking into account that the difficulty of inhibiting protein degradation in the formulation is often higher than that of inhibiting aggregation, the goal will focus on solving degradation problems. According to the results in Example 2, the sample is relatively stable under the conditions of pH 5.5-6.0, but the degradation background content is high, which may make it impossible to distinguish the effect of excipients on degradation during excipient screening. It can be seen that under pH 6.5, the degradation content is lower than pH 5.5 and 6.0, so it is easier to observe the effect of excipients on degradation using pH 6.5 as a pH investigation point. Therefore, while selecting pH 5.5 as a pH investigation point, pH 6.5 is selected as another investigation point to investigate both aggregates and degradation.

[0129] Table 6. SEC results of the effects of carbohydrate excipients on sample stability

[0130] As shown in Table 6, the sample stability at pH 5.5 is better than that at pH 6.5, and the conclusion is consistent with that of Example 2. Under pH 5.5, the degradation of the samples was basically the same regardless of whether or not excipients were added, and whether different excipients were added, which was in line with the expectations during the experimental design. In terms of aggregation, the addition of excipients had a certain inhibitory effect on the formation of aggregates, with the aggregation content being the lowest when the additive was 150mM NaCl, and the aggregation inhibition effect being the weakest when the additive was 5% mannitol. Under pH 6.5, it can be found that there are certain differences in degradation when different excipients are added, with the best inhibitory effect on sample degradation when the additive is 5% mannitol. Considering that inhibition of degradation is the current main goal, the preferred excipient to be added should be mannitol.

[0131] Considering that glycine is also a commonly used osmotic pressure regulator in pharmaceutical formulations, its effect on protein stability was investigated. Furthermore, based on the results in Table 6, the combined use of salt and mannitol was considered for preliminary investigation. Since the samples exhibited a certain tendency to aggregate at high temperatures, the effect of surfactant on sample stability was investigated.

[0132] Table 7. SEC results of the effect of glycine on sample stability

[0133] Table 7 above shows that the addition of Tween 20 has a certain inhibitory effect on sample aggregation. Meanwhile, the addition of glycine has little effect on sample aggregation, but does have a certain effect on inhibiting degradation. Among all experimental groups, the combination of mannitol and salt demonstrated the best results, demonstrating excellent performance in inhibiting both aggregation and degradation. This is a promising candidate for subsequent target formulations.

[0134] Example 4 Through experiments on different excipients and their combinations, mannitol and sodium chloride were determined to be the preferred excipient combination for the target protein.

[0135] Example 5: Determination of Mannitol and Sodium Chloride Concentration Combinations and pH

[0136] Through Example 1, it was finally determined that the preferred pH of the target protein was within the range of 5.5-6.5, so the pH in this range was further investigated to confirm the appropriate pH point. At the same time, according to the results of Example 4, mannitol and sodium chloride were confirmed to be used in combination as the preferred excipient combination for the target protein. According to the results of Examples 3 and 4, considering that mannitol has a certain inhibitory effect on degradation, while sodium chloride has a certain inhibitory effect on degradation, the comprehensive effect of the combination of the two on the overall stability still needs to be further confirmed by the ratio between the two to ensure the optimal result.

[0137] Table 8. SEC results for mannitol, salt concentration, and pH confirmation tests

[0138] Example 6: Investigation and confirmation of protein concentration

[0139] Through Example 5, it was confirmed that the optimal conditions were 10 mM citric acid buffer / 70 mM NaCl / 2% mannitol / pH 5.8, and on this basis, the sample concentration conditions were further investigated.

[0140] Table 9. SEC results of high concentration samples at 50°C stability

[0141] Table 10. SEC results of high concentration samples at 4°C stability

[0142] As can be seen from Table 9 above, as the concentration increases to 50 mg / ml, there is no obvious change in degradation at 50°C, but there is a significant increase in aggregates; as can be seen from Table 10, under 4°C conditions, the aggregates of the 50 mg / ml sample have an increasing trend, but the trend is not obvious, and the degradation content is not significantly different from that of the 25 mg / ml sample.

[0143] Example 6 shows that the sample still has good stability under high concentration conditions of 50 mg / ml.

[0144] The above data indicate that, in the combination of mannitol and sodium chloride, within the range of 10mM-70mM sodium chloride, aggregation decreases to a certain extent as the sodium chloride concentration increases, making a higher sodium chloride concentration and a lower mannitol concentration a preferred combination. Further investigation of the pH range revealed that pH 5.8 resulted in the lowest aggregation, and the degradation of the target protein was not significantly affected by the different pH conditions. Therefore, the preferred pH is 5.8.

[0145] Therefore, the formulation of this sample is a combination of citric acid buffer, sugar alcohols, salt ions and surfactant excipients, with a pH of 5.5-6.0. The concentration of citric acid buffer is 10-30mM, sugar alcohols include but are not limited to mannitol, salt ions include but are not limited to sodium chloride and some amino acids, and surfactants include but are not limited to polysorbate 20. The maximum protein concentration can reach 50mg / ml.

[0146] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A fusion protein composition, characterized in that: The composition comprises a fusion protein, a buffer, and a stabilizer; the fusion protein comprises TNFRII or an active fragment thereof, BCMA or an active fragment thereof, and optionally an antibody Fc region.

2. The composition according to claim 1, characterized in that The fusion protein has an amino acid sequence as shown in SEQ ID NO:

1.

3. The composition according to claim 1, characterized in that The pH of the composition is preferably 5.5-7.0, more preferably 5.5-6.5, and even more preferably 5.5-6.

0.

4. The composition according to claim 1, characterized in that The buffer is selected from the group consisting of phosphate buffer, citrate buffer, or a combination thereof; The stabilizer is selected from the following group: salts, sugars, amino acids, or a combination thereof.

5. The composition according to claim 1, characterized in that The content of the fusion protein is 0.5-100 mg / mL, preferably 1-100 mg / mL, more preferably 5-100 mg / mL, more preferably 5-80 mg / mL, more preferably 5-50 mg / mL, more preferably 10-50 mg / mL, and most preferably 25-50 mg / mL.

6. The composition according to claim 1, characterized in that The concentration of the buffer is 1-100 mM, preferably 1-80 mM, more preferably 1-50 mM, more preferably 10-30 mM, and most preferably 10 mM; The concentration of the salt is 0-150mM, preferably 0-140mM, more preferably 0-110mM, more preferably 10-110mM, more preferably 10-70mM, and most preferably 70mM; The content of the sugar is 0-10wt%, preferably 0-8wt%, more preferably 0-5wt%, more preferably 1-5wt%, more preferably 2-5wt%, most preferably 2wt%, based on the total weight of the composition; and / or The concentration of the amino acid is 0-200 mM, preferably 0-100 mM, more preferably 50-100 mM, and most preferably 50 mM.

7. The composition according to claim 1, characterized in that The buffer is a citric acid buffer; the salt is sodium chloride; the sugar is mannitol, and the amino acid is glycine.

8. The composition according to claim 1, characterized in that The composition comprises:

9. The composition according to claim 1, characterized in that The composition comprises:

10. The composition according to claim 1, characterized in that The composition comprises:

11. The composition according to claim 1, characterized in that The composition comprises:

12. A method for preparing the composition of claim 1, the method comprising the steps of: After mixing the components of the composition as claimed in claim 1, a fusion protein composition is obtained.

13. A use of the composition according to any one of claims 1 to 11, characterized in that: The composition is used for preparing medicine, and the medicine is used for treating immune diseases.

14. The use of the composition according to claim 13, characterized in that The composition is a liquid preparation.

15. A method for preventing and / or treating an immune disease, comprising: The composition of any one of claims 1 to 11 is administered to a subject in need thereof to treat an immune disease.