Stable ophthalmic compositions containing teneganosertib without or substantially without a stabilizer

Abstract

The present invention relates to a stable ophthalmic composition containing tanfanercept without the use of a stabilizer, and methods of making and using the same. According to the present invention, it was found that the use of a stabilizer such as histidine or sucrose resulted in the generation of impurities such as tanfanercept derived acidic / basic variants and affected bioactivity. The ophthalmic pharmaceutical composition according to the present invention can control pH without excluding the use of such a stabilizer to significantly reduce the generation of impurities under refrigerated storage conditions as well as accelerated and stress conditions, thereby making a stable tanfanercept ophthalmic composition.

Description

Technical Field

[0001] This invention relates to a stabilized ophthalmic composition containing tanfanercept without the use of a stabilizer, and a method for preparing and using the composition. Background Technology

[0002] Typically, therapeutic agents for topical ophthalmic administration are formulated into liquid or gel forms and stably maintained in a sterile state prior to administration. These ophthalmic solutions contain buffers, various surfactants, stabilizers, and isotonic agents, contributing to greater user comfort. Solution stability is particularly important for the solution's effectiveness and commercialization. Solution stability can vary depending on the interactions between all compounds present in the formulation, as well as temperature and pH.

[0003] Pharmaceutical compositions containing proteins undergo physicochemical denaturation under suboptimal conditions. In particular, factors such as protein concentration, type of buffer solution, type and concentration of stabilizer, type and concentration of organic cosolvent, salt concentration, pH, temperature, and exposure to air significantly affect protein oxidation, deamidation, isomerization, and polymerization. This denaturation can reduce physiological activity by generating protein aggregates, fragments, and isomers.

[0004] Meanwhile, tumor necrosis factor (TNF-α) is a cytokine produced by various cell types, including monocytes and macrophages. TNF-α is associated with a variety of other human immune diseases, including infections, autoimmune diseases, sepsis, and transplant rejection. Due to the negative consequences of TNF-α overexpression causing human diseases, therapeutic agents have been designed to control or attenuate TNF-α activity. Therefore, antibodies have been developed to bind to and neutralize TNF-α and are marketed in various protein formulations. However, due to their large molecular weight, these anti-TNF-α antibody formulations have limitations in effectively reaching the inflammatory areas caused by the disease. Therefore, the applicant has developed a polypeptide molecule that is suitable for treating localized inflammatory diseases due to its small size and high activity. The TNF-α inhibitor of the present invention (which is a modified human TNF receptor-1 polypeptide, Tenvastatin) is disclosed in Korean Patent Publication No. 2012-0072323 filed by the applicant in this case, and its use for treating dry eye syndrome is disclosed in Korean Patent Publication No. 2013-0143484.

[0005] Related technical documents

[0006] Patent documents

[0007] Korean Patent Publication No. 2012-0072323

[0008] Korean Patent Publication No. 2013-0143484 Summary of the Invention

[0009] Protein components may have a shorter shelf life compared to chemically synthesized drugs, and during this storage period, physicochemical impurities (such as charge variants or aggregates) may develop, thereby reducing biological activity.

[0010] The present invention aims to provide a stable ophthalmic composition containing tempanoseptide without the use of a stabilizer.

[0011] Technical solutions

[0012] The inventors of this invention conducted various studies on the stability of tempanosecept to develop an ophthalmic composition that minimizes the formation of tempanosecept-derived impurities (acidic / basic variants) under refrigerated storage conditions as well as under accelerated and stress conditions. Specifically, the inventors conducted various formulation studies, including buffer solutions, isotonic reagents, pH ranges, and functional excipients. As a result, the inventors discovered that the use of stabilizers (such as histidine and sucrose) allows for the formation of acidic / basic variants even at specific pH levels, thereby affecting the stability of the tempanosecept composition. Therefore, this invention provides a stable tempanosecept ophthalmic composition with a pH range of 5.0 to 6.5, which is substantially free of stabilizers.

[0013] Specifically, the present invention provides a stable ophthalmic composition containing tempanoseptide, comprising tempanoseptide and a buffer system with pH 5.0 to pH 6.5, and substantially free of stabilizers.

[0014] Tenfano Septo is the TNFRI variant disclosed in Korean Publication No. 2013-0143484, and is represented by an amino acid sequence (TNFRI171) consisting of amino acids 41 to 211 of the amino acid sequence of wild-type TNFRI, which contains amino acid modifications of L68V / S92M / H95F / R97P / H98G / K161N.

[0015] Since Tempranopeptide is a polypeptide composed of a total of 171 amino acids, like ordinary protein-containing pharmaceutical components, one of the most important issues in drug development is ensuring the stability of this component and its optimal efficacy before it is given to patients.

[0016] To develop formulations that ensure the stability of tempanoseceptide, the inventors first tested the storage stability of tempanoseceptide and observed that tempanoseceptide formed charge variants during storage (Experimental Example 1). As used herein, the term "charge variant" refers to a protein or polypeptide whose charge has changed from its native state due to modification. In some embodiments, the charge variant is more acidic than the original protein or polypeptide, i.e., its pI value is lower than the original protein or polypeptide. In another embodiment, the charge variant is more basic than the original protein or polypeptide, i.e., its pI value is higher than the original polypeptide. Such modifications can be the result of engineered or natural processes (e.g., oxidation, deamination, C-terminal treatment of lysine residues, formation of N-terminal pyroglutamic acid, and non-enzymatic glycosylation). In some embodiments, a protein or polypeptide charge variant is a glycoprotein that has a changed charge relative to the parent glycoprotein due to the addition of a glycan linking the protein, such as sialic acid or a derivative thereof. As used herein, the term "tempanoseceptide charge variant" refers to a material in which the charge of tempanoseceptide has changed from its native state due to modification.

[0017] Generally, since charge variants are known to typically lead to decreased drug activity, it is necessary to control the generation of charge variants to a specific value or lower. Therefore, the inventors of this case have confirmed that components suitable for use as ophthalmic stabilizers can be used to minimize the generation of impurities (such as charge variants), and such compositions may primarily contain sucrose and histidine (Experimental Example 2). However, in further studies determining the appropriate pH of compositions containing tefanoseptide, it was confirmed that the production rate of the charge variant is high even when a stabilizer is used at a specific pH, and methods to minimize the production rate of the charge variant include increasing the production rate of the charge variant and adjusting the pH to 5.0 to 6.5 without using a stabilizer (Experimental Example 3).

[0018] Therefore, the present invention provides an ophthalmic composition containing tempanoseptide, which comprises tempanoseptide and a buffer system with pH 5.0 to pH 6.5, and is substantially free of stabilizers.

[0019] Tempanosecept is preferably included in the composition in an appropriate amount, because the higher the amount, the higher the content of impurities (such as aggregates). In the ophthalmic composition containing tempanosecept of the present invention, the tempanosecept content may be 0.01 to 10% (w / v), for example 0.01 to 8% (w / v), 0.01 to 6% (w / v), 0.01 to 4% (w / v), 0.01 to 2% (w / v), 0.01 to 1% (w / v), 0.02 to 1% (w / v), 0.05 to 0.8% (w / v), 0.1 to 0.7% (w / v), or 0.2 to 0.6% (w / v). For commercial purposes, this composition may contain 0.25% (w / v), 0.5% (w / v), 1% (w / v), 2% (w / v), 3% (w / v), 4% (w / v), 5% (w / v), 6% (w / v), 7% (w / v), 8% (w / v), 9% (w / v), or 10% (w / v) of tempanoceptor. The content of tempanoceptor may vary depending on the type and severity of the patient's disease to be administered.

[0020] The ophthalmic composition containing tempanoseptide according to the present invention comprises a buffer system with a pH of 5.0 to 6.5. A pH of 5.0 to 6.5 is sufficient for this buffer system, and all values ​​within the pH range of 5.0 to 6.5 (e.g., buffer systems with pH 5.0 to pH 6.0, pH 5.5 to pH 6.5, pH 5.5 to pH 6.0, and pH 5.8 to pH 6.3) are included within the scope of the present invention. In one embodiment of the present invention, the ophthalmic composition containing tempanoseptide according to the present invention comprises a buffer system with a pH of 5.0 to pH 6.0. In another embodiment of the present invention, the ophthalmic composition containing tempanoseptide according to the present invention comprises a buffer system with a pH of 5.5 to pH 6.0.

[0021] In ophthalmic compositions containing tempanoseptide according to the present invention, the method of implementing a buffering system is well known to those skilled in the art. A buffering system of pH 5.0 to pH 6.5 may contain one or more buffers selected from the group consisting of: phosphate buffer, histidine buffer, acetate buffer, succinate buffer, citrate buffer, glutamate buffer, and lactate buffer. Regardless of the buffering system used, if the buffering system meets the pH 5.0 to pH 6.5 condition, it has been proven to ensure the stability of tempanoseptide. However, the use of a particular buffering system may be relatively ideal. According to the following examples, citrate buffer has been proven to be relatively advantageous compared to acetate buffer in controlling the generation of aggregates or charge variants (Experimental Example 4). Therefore, in one specific embodiment of the invention, the buffering system may be a buffering system containing citrate buffer, such as a citrate buffer system or a citrate-phosphate buffer system, but the invention is not limited thereto. The buffer contained in the buffering system may consist of a combination of conjugate acid and conjugate base to improve the buffering effect. For example, in one specific embodiment of the invention, the buffer system comprises a citrate buffer, which comprises trisodium citrate (conjugate base) and citric acid (conjugate acid).

[0022] The buffer system of the present invention contains 5 mM to 50 mM buffer solution, for example, 10 mM to 30 mM buffer solution.

[0023] The ophthalmic composition of this invention substantially does not contain stabilizers. Here, "stabilizer" refers to an additional component included in the formulation to prevent a decrease in the chemical and physical stability or biological activity of tempanosecept, which is used as the active ingredient. For example, to inhibit protein aggregation in this ophthalmic composition, it is well known that sugars (such as sucrose or mannitol) or amino acid stabilizers (such as proline, arginine, glycine, lysine, or methionine) can be used to ensure protein stabilization. This invention substantially does not contain stabilizers because, as has been found (unlikely) in the following specific embodiments, such stabilizers have an adverse effect on the stability of tempanosecept. The buffer or isotonic reagent described in this invention is not included in the stabilizer.

[0024] "Substantially free" or "substantially absent" indicates that the stabilizer is present in amounts of less than 0.1% (w / v), 0.05% (w / v), 0.03% (w / v), 0.02% (w / v), 0.01% (w / v), 0.005% (w / v), or 0.001% (w / v), with the best being completely absent.

[0025] The osmotic pressure of ophthalmic components can be 260 to 320 mOsm / kg.

[0026] In addition to the active ingredient, fenestrate, and the buffer system, the ophthalmic composition according to the invention may further contain an isotonic agent. This isotonic agent is used to adjust the osmotic pressure of the ophthalmic composition according to the invention. In this invention, the isotonic agent is included to make the osmotic pressure of the ophthalmic composition according to the invention 260 to 320 mOsm / kg. This osmotic pressure is measured by measuring the number of dissolved particles per unit volume of water. In a solution, since the number of solute particles decreases proportionally with the number of units of water (solvent), the concentration of a low-osmotic-pressure solution is low. When solutions of different solute concentrations are separated using a semi-permeable membrane (a membrane that only solvent molecules can pass through), osmosis occurs, where solvent molecules move from a low concentration to a high concentration across the membrane to form a concentration equilibrium. The pressure driving this movement is called osmotic pressure and is controlled by the number of solute "particles" in the solution. Solutions containing the same particle concentration and subjected to the same osmotic pressure are called isotonic. Applying low-osmotic or high-osmotic-pressure solutions to the eye may damage the eye; therefore, ophthalmic medications must use isotonic solutions. This invention uses sodium chloride as an isotonic reagent. In ophthalmic compositions according to the invention, the content of the isotonic reagent can be 0.5% to 1% (w / v). In ophthalmic compositions according to the invention, the concentration of the isotonic reagent can be 100 to 150 mM.

[0027] In one specific embodiment of the invention, the ophthalmic composition according to the invention comprises tempanosecta, a buffer system with pH 5.5 to pH 6.0, an isotonic reagent, and water. In another specific embodiment, the ophthalmic composition according to the invention comprises tempanosecta, a buffer system with pH 5.5 to pH 6.0 containing citrate buffer, sodium chloride, and water.

[0028] The ophthalmic composition containing tavanoxeptide according to the present invention is very stable even under accelerated or stressed conditions.

[0029] The ophthalmic composition containing tavanoxeptide according to the present invention may have 20% or less of a charge variant after being stored under accelerated conditions for 6 months.

[0030] The ophthalmic composition containing tavanoxeptide according to the present invention may have 10% or less of a basic variant after being stored under accelerated conditions for 6 months.

[0031] The ophthalmic composition containing tavanoxeptide according to the present invention may have 10% or less of an acidic variant after being stored under accelerated conditions for 6 months.

[0032] The ophthalmic composition containing tavanoropeptide according to the present invention may have 20% or less of the tavanoropeptide charge variant after being stored under long-term storage conditions for 36 months.

[0033] The ophthalmic composition containing tavanoropeptide according to the present invention may have 10% or less of a basic variant of tavanoropeptide after being stored under long-term storage conditions for 36 months.

[0034] The ophthalmic composition containing tempanoseptide according to the present invention may have 10% or less of the tempanoseptide acid variant after being stored under long-term storage conditions for 36 months.

[0035] Because of the improved physicochemical and biological stability of Tenvastatin, the ophthalmic pharmaceutical composition according to the present invention can be administered to patients with TNF-mediated eye diseases (such as dry eye) by conventional methods (such as instillation).

[0036] Effects of the invention

[0037] According to the present invention, the use of stabilizers (such as histidine or sucrose) has been found to lead to the generation of impurities (such as acidic / basic variants derived from tempanosec) and affect bioactivity. Ophthalmic pharmaceutical compositions according to the present invention can control pH, rather than exclude, the use of such stabilizers to significantly reduce the generation of impurities under refrigerated storage conditions as well as under accelerated and stress conditions, thereby preparing stable tempanosec ophthalmic compositions.

[0038] Simple Explanation of the Diagram

[0039] Figure 1 The results of isoelectric focusing (IEF) are shown for Tenvanovate after storage at 37°C for 0 to 4 weeks.

[0040] Figure 2 This shows the IEX-HPLC analysis results of Tenvanovate after storage at 37°C for 0 to 4 weeks.

[0041] Figure 3 The results of IEF analysis of the charge variants are shown;

[0042] Figure 4 The results of IEX-HPLC analysis of the charge variants are shown;

[0043] Figure 5 The changes in the main peak in IEX-HPLC are shown for the control group without stabilizers and the stabilizer screening solutions containing methionine, glycine, histidine salt and sucrose as stabilizers, respectively.

[0044] Figure 6 The changes of acidic variants in IEX-HPLC are shown in the control group without stabilizers and the stabilizer screening solutions containing methionine, glycine, histidine salt and sucrose as stabilizers, respectively.

[0045] Figure 7The changes in basic variants in IEX-HPLC are shown in the control group without stabilizers and the stabilizer screening solutions containing methionine, glycine, histidine salt and sucrose as stabilizers, respectively.

[0046] Figure 8 The results show the stability of the Tenvanovate ophthalmic composition prepared under various pH and stabilizer conditions by variant analysis in RP-HPLC after storage at 40°C for 4 weeks.

[0047] Figure 9 The stability of the TenvaSeptember ophthalmic composition prepared under various pH and stabilizer conditions after storage at 40°C for 4 weeks is shown by the change of the main peak in IEX-HPLC.

[0048] Figure 10 The stability of the TenvaSeptember ophthalmic composition prepared under various pH and stabilizer conditions after storage at 40°C for 4 weeks is shown in HEX-HPLC by means of changes in acidic variants.

[0049] Figure 11 The stability of the TenvaSeptember ophthalmic composition prepared under various pH and stabilizer conditions after storage at 40°C for 4 weeks is shown in HEX-HPLC by means of changes in basic variants.

[0050] Figures 12 to 14 The results of SEC-HPLC analysis were shown when the four types of Tenvanovate ophthalmic components were stored at 4°C, 25°C and 40°C, respectively.

[0051] Figures 15 to 17 The results of analysis of the acidic variants using IEX-HPLC are shown when the four types of TenvaSeptember ophthalmic components were stored at 4°C, 25°C, and 40°C, respectively.

[0052] Figures 18 to 20 The results of analysis of basic variants using IEX-HPLC are shown when the four types of Tenvanovose ophthalmic components are stored at 4°C, 25°C and 40°C, respectively.

[0053] Figure 21 The stability of the Tevanosept ophthalmic composition was demonstrated by the results of aggregate analysis using SEC-HPLC.

[0054] Figures 22 to 23 The stability of the Tenvanosecept ophthalmic composition was demonstrated by analyzing the charge variants using IEX-HPLC.

[0055] Implementation

[0056] Detailed description of preferred embodiments of the present invention

[0057] The advantages and features of the present invention, as well as the methods for implementing the present invention, are illustrated by referring to the following preparation examples, illustrative examples, and experimental examples. However, these examples are provided only to aid in understanding the present invention and are not intended to be construed as limiting the present invention.

[0058] Experimental Example 1: Determination of the Tenvanosector Charge Variant

[0059] The generation of charge variants can affect drug activity, stability, and safety. Therefore, the inventors in this case first analyzed the charge variants of tempanoseceptide. More specifically, tempanoseceptide was stored at 37°C for 4 weeks, followed by isoelectric focusing (IEF) and IEX-HPLC analysis.

[0060] Isoelectric focusing method

[0061] 10 μg was loaded into each well of a gel at pH 3.0 to pH 7.0, followed by electrophoresis at 100 V for 1 hour, 200 V for 1 hour, and then 500 V for 30 minutes. The product was fixed with 12% trichloroacetic acid for 30 minutes and then stained with Coomassie blue.

[0062] IEX-HPLC (Ion Exchange-High Performance Liquid Chromatography)

[0063] Ion-exchange high-performance liquid chromatography (IEX-HPLC) uses ion exchange resins to separate proteins based on their affinity for the stationary phase in the column, which is related to the protein's net charge. The experimental method of this invention utilizes a cation exchange column, a temperature controller (set to 25°C), an autosampler (set to 4°C), a UV detector operating at 280 nm, and an HPLC system capable of maintaining a flow rate of 0.7 mL / min.

[0064] Isolation and purification of charge variants

[0065] To separate and analyze the charge variants present in ophthalmic compositions containing tefanoseptide based on their properties, SP-HP columns and liquid chromatography-particle chromatography (FPLC) for protein separation were used to separate and purify the proteins according to their salt concentrations. Based on the charge variant properties, acidic variant sample A, main peak sample B, and basic variant sample C were prepared and subsequently analyzed by IEF and IEX-HPLC, respectively.

[0066] Figure 1 This shows the results of isoelectric focusing (IEF) after the Tenvanovate was stored at 37°C for 0 to 4 weeks.

[0067] From the results of IEF (such as) Figure 1As shown, the bands with low pI values ​​become deeper (or more concentrated) as storage time increases, indicating the production of acidic variants of fenvalerate.

[0068] Figure 2 This describes the IEX-HPLC analysis results of Tomorrow's Vannoseptember after storage at 37°C for 0 to 4 weeks.

[0069] In addition, from the results of IEX-HPLC (such as...) Figure 2 (As shown) This also confirms that the amount of acid variants increases with prolonged storage.

[0070] To study the properties of charge variants, acidic variant samples, main peak samples, and basic variant samples obtained by charge separation using SP-HP columns were analyzed by IEF and IEX-HPLC.

[0071] Figure 3 The results of IEF analysis of the charge variants are shown. Furthermore, Figure 4 Explain the IEX-HPLC analysis results of the charge variants. For example... Figure 3 and 4 As shown, the chromatography results indicate that only the separated acidic variant, sample A, was eluted before the main peak sample. Similarly, the IEF results show that the pI value of sample A is lower than that of the main peak sample. Furthermore, the chromatography results also indicate that the basic variant sample (i.e., sample B) was eluted after the main peak sample. Moreover, the IEF results show that the pI value of sample B is slightly higher than that of the main peak sample.

[0072] Experimental Example 2: Screening of Ophthalmic Stabilizers

[0073] Stabilizers are typically added to protein compositions to stabilize the formulation before it is administered to a patient. In fact, the use of stabilizers significantly reduces impurities (such as aggregates or charge variants) that may develop during storage, thus maintaining a stable formulation. Therefore, selecting an ideal stabilizer that can stabilize the main component of the protein composition to prepare a stable composition is absolutely crucial. To conduct stress tests on the stabilizer (40°C, 4 weeks of storage) and to select the desired type of stabilizer for Tenvanovate, the main component of this invention, the inventors first conducted the following experiments.

[0074] 1) Preparation of Tenvanovate solution samples

[0075] 1) Prepare a 10 mg / mL tenofovir solution in a 20 mM sodium citrate buffer containing 125 mM sodium chloride. 2) Prepare a citrate-phosphate buffer solution with pH 7.0.

[0076] Add 0.37 g of citric anhydride and 2.58 g of disodium hydrogen phosphate to 900 mL of ultrapure water and mix thoroughly. Titrate with 37% hydrochloric acid or 40% sodium hydroxide at pH 7.0, then add ultrapure water to prepare 1 L of final product.

[0077] 3) Preparation of stabilizer screening solution

[0078] Four (4) types of stabilizers (0.149 g methionine, 0.751 g glycine, 1.55 g histidine salt and 6.84 g sucrose) were added to 100 mL of the buffer solution prepared in Project 2) to prepare four (4) types of stabilizer screening components at pH 7.0.

[0079] [Table 1]

[0080]

[0081] 4) Sample preparation and evaluation

[0082] After adding 10 mL of the ophthalmic stabilizer screening solution prepared in Project 3) together with tempanosecide to a 3.5 kDa centrifuge filter, the sample was centrifuged at 4°C and 4000 rpm to replace the tempanosecide buffer in Project 1) with the stabilizer screening buffer from Project 3). The above steps were repeated to produce a stabilizer screening solution (sample) containing 1 mg / mL tempanosecide. The resulting sample was stored at 40°C for 4 weeks, and then analyzed by IEX-HPLC at 0 and 4 weeks to identify any physicochemical impurities in each sample.

[0083] [Table 2]

[0084]

[0085] Table 2 and Figures 5 to 7 The IEX-HPLC analysis results show the results of the control group without any stabilizers and the stabilizer screening groups containing methionine, glycine, histidine salt, and sucrose as stabilizers, respectively.

[0086] Therefore, as shown in Table 2 and Figures 5 to 7 As shown, the presence of sucrose and histamine salts confirms a trend toward reduced production of both basic and acidic variants. Specifically, in the case of histamine salts, the production of acidic variants is significantly reduced compared to the control group or other stabilizers.

[0087] Experimental Example 3: Preparation of ophthalmic components at different pH values ​​and evaluation of their stability

[0088] Since tears have a pH of 7.0 to 7.5, it is ideal to set ophthalmic compositions to similar pH conditions. However, pH can also significantly affect protein stability. Therefore, it is intended to prepare ophthalmic compositions with different pH values ​​and evaluate their stability.

[0089] (1) Preparation of ophthalmic components

[0090] 1) Prepare 20mM citrate-phosphate buffer solutions with pH 5.0 to pH 7.0.

[0091] Citric anhydride and disodium hydrogen phosphate were added to 400 mL of ultrapure water to achieve the desired pH (0.62 g citric anhydride and 0.97 g disodium hydrogen phosphate in pH 5.0 / 5.5 buffer; 0.43 g citric anhydride and 1.11 g disodium hydrogen phosphate in pH 6.0 / 6.5 buffer; 0.19 g citric anhydride and 1.29 g disodium hydrogen phosphate in pH 7.0 buffer). Furthermore, after titrating to pH 5.0 to pH 7.0 using 37% hydrochloric acid or 40% sodium hydroxide to meet different conditions, ultrapure water was added to prepare 500 mL of the final product.

[0092] 2) Preparation of ophthalmic formulations with added stabilizers (pH 5.0 to pH 7.0)

[0093] 6.85 g of sucrose and 1.55 g of histidine were added to each of the five (5) pH 5.0 to pH 7.0 buffer solutions prepared in Project 1) at 100 mL to prepare five (5) types of 20 mM citrate-phosphate buffer solutions at pH 5.0 to pH 7.0 (each containing 200 mM sucrose) and five (5) types of 20 mM citrate-phosphate buffer solutions at pH 5.0 to pH 7.0 (each containing 100 mM histidine). As a stabilizer-free experimental group, the five (5) pH 5.0 to pH 7.0 20 mM citrate-phosphate buffer solutions prepared in Project 1) above were used directly as the stabilizer-free experimental group. Therefore, a total of 15 types of buffer solutions with / without stabilizers at pH 5.0 to pH 7.0 were prepared.

[0094] 3) Sample preparation and evaluation

[0095] After adding 4 mL of the buffer solutions prepared in Project 2) and Tempanosec, the samples were centrifuged using a 3.5 kDa centrifuge filter. The existing Tempanosec buffer was then replaced with a buffer containing stabilizers at pH 5.0 to 7.0. This process was repeated to obtain 15 different types of samples with varying stabilizers and pH values. The samples were stored at 40°C for 4 weeks and analyzed at weeks 0 and 4 to identify any physicochemical impurities.

[0096] [Table 3] Preparation of ophthalmic compositions with added stabilizers (pH 5.0 to pH 7.0)

[0097] pH 5.0 0mM 100mM 200mM pH 5.5 0mM 100mM 200mM pH 6.0 0mM 100mM 200mM pH 6.5 0mM 100mM 200mM pH 7.0 0mM 100mM 200mM

[0098] (2) Evaluate the stability of Tenvanovate to pH and stabilizers.

[0099] 1) Reverse phase chromatography analysis

[0100] RP-HPLC (Reverse-Phase Chromatography) is a method for assessing protein purity based on protein polarity. The experimental method of this invention utilizes a reverse-phase chromatography column, a temperature controller (set to 60°C), an autosampler (set to 4°C), a UV detector operating at 214 nm, and a high-performance liquid chromatography (HPLC) system capable of maintaining a flow rate of 1.0 mL / min.

[0101] The 15 types of samples were stored under stress conditions (at 40°C) for 4 weeks, and then analyzed at 0 and 4 weeks to analyze the changes in the identified variants in each sample. Therefore, as shown in Table 4 and... Figure 8 As shown, the results confirm that the experimental group containing sucrose or histidine as a stabilizer at pH 7.0 exhibited a lower variant generation rate than the experimental group without a stabilizer. However, in the case of the experimental group containing histidine as a stabilizer, it was shown that the variant generation rate was higher than that of the control group at pH 5.0 to pH 6.5. Furthermore, in the experimental group containing sucrose as a stabilizer, it was shown that the variant generation rate was higher than that of the control group at pH 5.0 to pH 6.0.

[0102] [Table 4]

[0103]

[0104] 2) Ion-exchange high-performance liquid chromatography (IEX-HPLC)

[0105] Due to the net charge of proteins, IEX-HPLC uses ion exchangers to separate proteins based on their affinity for the stationary phase of the column. The experimental method of this invention is performed using a cation exchange column, a temperature controller (set to 25°C), an autosampler (set to 4°C), a UV detector operating at 280 nm, and a high-performance liquid chromatography (HPLC) system capable of maintaining a flow rate of 0.7 mL / min.

[0106] The 15 types of samples were stored under stress (at 40°C) for 4 weeks, and then analyzed at 0 and 4 weeks. The changes in the acidic / basic variants identified in each sample were then compared.

[0107] Therefore, as shown in Table 5 and Figures 9 to 11 As shown, the stabilizer-free group (pH 5.0 to pH 6.5) exhibited better results than the groups containing sucrose or histidine in terms of producing acidic variants. Specifically, the stabilizer-free group (pH 5.0 to pH 6.0) showed less than 10% variation in producing acidic variants. For basic variants, the stabilizer-free group (pH 5.0 to pH 6.0) showed better results than the groups containing sucrose or histidine. At pH 6.5 to pH 7.0, the sucrose-containing group showed the least variation in producing basic variants, while the histidine-containing group showed no significant difference from the stabilizer-free group.

[0108] [Table 5]

[0109]

[0110] Analysis of the above measurements showed that the experimental groups containing sucrose or histidine at pH 7.0 tended to produce fewer variants than the group without stabilizers. Conversely, when the pH was lowered to pH 5.5 or pH 6.0, a significant increase in variant production was observed in the experimental groups containing sucrose or histidine compared to the group without stabilizers.

[0111] Based on the experimental results, contrary to expectations, it was determined that the experimental group containing tempanoseceptide ophthalmic composition at pH 5.5 to pH 6.0 and without any stabilizers (i.e., the stabilizer-free group) showed the least variant production.

[0112] Typically, ophthalmic formulations are set at pH 7.0, which is similar to the body's pH. However, the stability of the active ingredient in ophthalmic formulations is a key factor in demonstrating medical efficacy, leading to the conclusion that the ophthalmic formulations of the present invention containing tefanoseptide are preferably set at pH 5.5 to pH 6.0, while being free of sucrose or histidine.

[0113] Experimental Example 4: Evaluating the stability of the buffer system

[0114] To compare the basic composition of the buffer solution and the differences between the two functional excipients with and without the addition, and to fix the concentration of tempanoseceptide, pH, sodium chloride concentration, osmotic pressure, etc., four (4) experimental groups were prepared and screened at 4℃, 25℃, and 40℃ respectively to select the final formulation. Considering the above experimental results, the pH of the ophthalmic composition was fixed at pH 5.5. In addition, sodium acetate (20mM) and sodium citrate (20mM) were used as common buffer solutions that are generally applicable to the above range to evaluate tempanoseceptide, relative to the buffer system.

[0115] As shown in Table 6 below, four types (4) of ophthalmic compositions containing tempanosecide were prepared and used to conduct stability tests on tempanosecide.

[0116] [Table 6]

[0117]

[0118] Stability testing of ophthalmic components containing tenofotropic was performed using SEC-HPLC to analyze aggregates and charge variants. The stability test was conducted for a total of 2 months at 4°C, 25°C, and 40°C. Aggregate analysis was performed by SEC-HPLC at 0 weeks, 2 weeks, 1 month, and 2 months, and charge variant analysis was performed by IEX-HPLC.

[0119] (1) Analysis of aggregates by SEC-HPLC

[0120] Size exclusion high-performance liquid chromatography (SEC-HPLC) typically separates proteins based on size differences by introducing a stationary phase sample into a column filled with a porous gel. The experimental method of this invention utilizes a size exclusion column, a temperature controller (set to 25°C), an autosampler (set to 4°C), a UV detector operating at 214 nm, and an HPLC system capable of maintaining a flow rate of 0.5 mL / min.

[0121] Tables 7 to 9 and Figures 12 to 14 The results of SEC-HPLC analysis of the four (4) types of ophthalmic formulations containing tefanoseptide stored at 4°C, 25°C, and 40°C are presented. The SEC-HPLC analysis results show that the higher the temperature, the higher the concentration of aggregates. Specifically, in formulations containing buffer, aggregates are rapidly formed. Among the three (3) types of formulations prepared with citrate buffer, FFS3 showed a high aggregate content after storage at 4°C for 2 months. Furthermore, FFS2, without any excipients, showed the most stable results under all conditions including 25°C, 40°C, 2 weeks, 1 month, and 2 months.

[0122] [Table 7] SEC Aggregate Analysis [4℃]

[0123]

[0124] [Table 8] SEC Aggregate Analysis [25℃]

[0125]

[0126] [Table 9] SEC Aggregate Analysis [40℃]

[0127]

[0128] (2) Analysis of acid variants by IEX-HPLC

[0129] Tables 10 to 12 and Figures 15 to 17 The results of acid variant analysis by IEX-HPLC are presented for the above four (4) types of ophthalmic compositions containing tavanoxeptide when stored at 4°C, 25°C and 40°C.

[0130] [Table 10] IEX-acidic variants [4℃]

[0131]

[0132] [Table 11] IEX-acidic variants [25℃]

[0133]

[0134] [Table 12] IEX-acidic variant [40℃]

[0135]

[0136] (3) Basic variant analysis by IEX-HPLC

[0137] Tables 13 to 15 and Figures 18 to 20 The results of basic variant analysis by IEX-HPLC are presented for the above four (4) types of ophthalmic compositions containing tavanoxeptide when stored at 4°C, 25°C and 40°C.

[0138] [Table 13] IEX-basic variant [4℃]

[0139]

[0140] [Table 14] IEX-basic variant [25℃]

[0141]

[0142] [Table 15] IEX-basic variant [40℃]

[0143]

[0144] IEX-HPLC analysis showed that all formulations exhibited an increasing trend in both acidic and basic variants under high-temperature storage conditions of 40°C. Specifically, the acetate buffer (FFS1) formulation showed a pattern of significant increase in basic variants at all temperatures.

[0145] Therefore, acetate buffer (FFS1) demonstrated worse stability than citrate buffers (FFS2 to 4) in terms of aggregate formation and basic variants. Furthermore, based on the basic composition of citrate buffers, the group with added functional excipients proved to be the most stable when comparing the groups with and without functional excipients. Therefore, considering the overall simplification of the product manufacturing process, contamination during manufacturing, and homogeneity, the formulation using citrate buffer (FFS2) was adopted as the final formulation. However, considering the physiological pH of tears is neutral, a pH of 6.0, which may offer slightly higher patient compliance, was chosen as the pH of the final product, in addition to the acceptable level of stability.

[0146] Preparation Example 1: Production of ophthalmic components containing tefanoseptide

[0147] To prepare a buffer solution, add 5.35 g of trisodium citrate dihydrate, 0.35 g of citric anhydride, and 7.3 g of sodium chloride to 900 ml of ultrapure water and dissolve them completely. After confirming that the pH of the buffer solution is 6.0 ± 0.1, adjust the volume of the buffer solution to 1 L using a graduated cylinder, and then filter it through a 0.22 gm cap filter system.

[0148] Then, tempanoceptor was added to the prepared buffer solution to produce an eye drop composition containing 0.25% tempanoceptor.

[0149] [Table 161]

[0150] Tianfano Sept 2.5 Trisodium citrate dihydrate 5.35 Citric anhydride 0.35 Sodium chloride 7.31 Sodium hydroxide As needed hydrochloric acid As needed Ultrapure water QS

[0151] Experimental Example 5: Evaluation of the stability of ophthalmic compositions containing tefanoseptide

[0152] The stability of the ophthalmic composition containing tefanoseptide according to Preparation Example 1 was evaluated.

[0153] Long-term storage conditions were set at 5°C (unadjusted humidity), while accelerated conditions were set at 25°C / 60% RH. The components in Preparation Example 1 were stored under these conditions and then subjected to aggregate analysis by SEC-HPLC and charge variant analysis by IEX-HPLC.

[0154] [Table 17]

[0155]

[0156] Table 17 and Figure 21 The results of aggregate analysis by SEC-HPLC are shown in Table 17. Figure 21 As shown, when the composition was stored at 5°C for 3 years under long-term storage conditions, the formation of aggregates was within 5%, thus confirming that the composition of Preparation Example 1 was stable. Furthermore, when the composition was stored at 25°C / 60% RH under accelerated conditions for 6 months, it was shown that the formation of aggregates was within 5%.

[0157] [Table 18]

[0158]

[0159] Table 18 and Figures 22 to 23 The results of charge variant analysis via IEX-HPLC are shown in Table 18. Figures 22 to 23 As shown, when the composition was stored at 5°C for 3 years, the formation of charge variants during storage was within 5%, confirming the stability of the composition of Preparation Example 1. Furthermore, the formation of basic variants was also within 10%, demonstrating stability during storage.

[0160] Furthermore, when the composition was stored under accelerated conditions of 25°C / 60% RH for 6 months, the formation of acidic variants was within 10%. Additionally, the formation of basic variants was within 10%, demonstrating stability during storage.

Claims

1. A stable ophthalmic composition containing tanfanercept, comprising: Tenvastatin at 0.1% (w / v) to 1% (w / v) and 5 to 50 mM citrate buffer systems at pH 5.0 to pH 6.

0. It does not contain stabilizers. The stabilizer is a carbohydrate stabilizer or an amino acid stabilizer. The sugar stabilizer is sucrose or mannitol, and the amino acid stabilizer is proline, arginine, glycine, lysine, or methionine.

2. The composition of claim 1, wherein the pH of the buffer system is from pH 5.5 to pH 6.

0.

3. The composition of claim 1, wherein the citrate buffer system comprises trisodium citrate and citric acid as a buffer solution.

4. The composition of claim 1, further comprising an isotonic reagent.

5. The composition of claim 4, wherein the isotonic reagent is sodium chloride.

6. The composition of claim 1, wherein the ophthalmic composition has 20% or less of the Tenvanovose charge variant after being stored under accelerated conditions for 6 months.

7. The composition of claim 1, wherein the ophthalmic composition has 10% or less of the tavanoxept basic variant after being stored under accelerated conditions for 6 months.

8. The composition of claim 1, wherein the ophthalmic composition has 10% or less of the tavanoxetine acid variant after being stored under accelerated conditions for 6 months.

9. The composition of claim 1, wherein the ophthalmic composition has 20% or less of the Tenvanovose charge variant after being stored under long-term storage conditions for 36 months.

10. The composition of claim 1, wherein the ophthalmic composition has 10% or less of the tavanoxept basic variant after being stored under long-term storage conditions for 36 months.

11. The composition of claim 1, wherein the ophthalmic composition has 10% or less of the tavanoxetine acid variant after being stored under long-term storage conditions for 36 months.

12. The composition of claim 1, wherein the osmolality of the ophthalmic composition is 260 to 320 mOsm / kg.

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