Liquid composition comprising antibody of human interleukin-4 receptor alpha

Abstract

To provide a liquid composition comprising an antibody of human interleukin-4 receptor alpha.SOLUTION: The liquid composition comprises the antibody at a concentration of 50-200 mg / ml, and a buffer, a protective agent, and a surfactant as adjuvants. The liquid composition has a pH of 5.4-6.4.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application claims the benefit of priority from Chinese Patent Application No. 201910187179.9, filed on March 13, 2019, the entire contents of which are incorporated herein by reference.

[0002] Technical Field The present invention relates to the field of biopharmaceutical formulations. In particular, the present invention relates to stable liquid formulations containing antibodies at high concentrations. [Background technology]

[0003] Background of the Invention Human interleukin-4 receptor is known to produce a soluble form of the protein (shIL-4Rα) that inhibits cell proliferation mediated by T cell-mediated upregulation of IL-4 and IL-5. Two forms of the receptor are associated with allergic reactions that manifest as diseases such as allergic rhinitis, sinusitis, asthma, eczema, etc. Therefore, blocking antibodies targeting the protein are useful for treating and alleviating such diseases.

[0004] Monoclonal antibody drugs targeting hIL-4R, such as dupilumab, which has shown good efficacy in Phase II clinical trials for the treatment of atopic dermatitis, are currently undergoing clinical trials. However, the best administration mode for antibody drugs is subcutaneous injection, which requires a relatively high dose to exert its effect. Therefore, it is generally necessary to prepare highly concentrated antibody formulations. As is known in the prior art, the manufacture and application of highly concentrated antibody formulations usually involves many difficulties. For example, such formulations with high viscosity may be difficult to draw up and inject with a syringe, may lead to large deviations in dosage due to high drug residue in the container or cartridge holding the formulation, and may cause pain at the injection site. Furthermore, highly viscous formulations may cause serious process problems during manufacturing. For example, extremely high pressure may be required during the concentration and filtration steps, or even the formulation may not be able to pass through the filtration membrane at all. Alternatively, high concentrations of antibodies in such formulations tend to aggregate and form insoluble particles, resulting in unstable formulations, increased immunogenicity, and more side effects of drug application.

[0005] Therefore, there remains a need in the art to develop novel antibody formulations targeting the human interleukin-4 receptor that can meet the manufacturing and clinical application requirements for high antibody concentration, long-term stability, no aggregation, and low viscosity, among others. Summary of the Invention

[0006] An object of the present invention is to provide a liquid composition and a formulation thereof comprising an antibody against human interleukin-4 receptor alpha, which liquid composition and a formulation thereof can enable the antibody to be present stably at a high concentration and have a low viscosity.

[0007] The technical solutions provided by the present invention are as follows:

[0008] In one aspect, the present invention provides a liquid composition comprising an antibody against human interleukin-4 receptor alpha, the liquid composition comprising the antibody at a concentration of 50 to 200 mg / ml, and excipients such as a buffer, a protective agent, and a surfactant, and having a pH of 5.4 to 6.4.

[0009] In the liquid composition, the antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the light chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO:1, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:2, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:3, and the heavy chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO:5, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:6, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:7.

[0010] Preferably, the antibody comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:4, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:8.

[0011] According to a particular embodiment of the present invention, the antibody has a kappa light chain constant region (CL) and a gamma heavy chain constant region (CH). More preferably, the antibody comprises a light chain constant region comprising the amino acid sequence set forth in SEQ ID NO:9 and a heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO:10. More preferably, the antibody is a monoclonal antibody comprising two light chains and two heavy chains. TIFF2025118750000001.tif106161TIFF2025118750000002.tif215162

[0012] Preferably, the antibody is present at a concentration of 100 to 200 mg / ml, more preferably 130 to 165 mg / ml, and even more preferably 150±5 mg / ml.

[0013] In the liquid composition, the buffer is one or more selected from the group consisting of acetate buffer, phosphate buffer, and amino acid buffer, and the buffer is present at a concentration of 5 to 50 mmol / L; preferably, the buffer is an amino acid buffer at a concentration of 5 to 50 mmol / L.

[0014] Preferably, the acetate buffer is a sodium acetate buffer, the phosphate buffer is a sodium dihydrogen phosphate buffer, or the amino acid buffer is a histidine hydrochloride buffer.

[0015] Preferably, the buffer is present at a concentration of 5 to 20 mmol / L.

[0016] Preferably, the buffer is a histidine hydrochloride buffer at a concentration of 5 to 20 mmol / L, more preferably a histidine hydrochloride buffer at a concentration of 10 mmol / L.

[0017] In the liquid composition, the protective agent is one or more selected from the group consisting of sugars, alcohols, amino acids, and chloride salts, and the protective agent is present at a concentration of 40 to 220 mmol / L.

[0018] Preferably, the protective agent is one or more selected from the group consisting of a sugar, an alcohol, and an amino acid, and the protective agent is present at a concentration of 40 to 220 mmol / L, and / or the protective agent is a chloride salt, and the protective agent is present at a concentration of 40 to 150 mmol / L.

[0019] Preferably, the sugar is trehalose and / or sucrose at a concentration of 40 to 150 mmol / L, more preferably 60 to 150 mmol / L; the alcohol is mannitol at a concentration of 40 to 220 mmol / L, more preferably 110 to 150 mmol / L; the amino acid is one or more selected from the group consisting of proline, arginine hydrochloride, and glycine at a concentration of 40 to 220 mmol / L, more preferably 120 to 220 mmol / L; or the chloride salt is sodium chloride at a concentration of 40 to 150 mmol / L, more preferably 80 to 120 mmol / L.

[0020] More preferably, the protective agent is a combination of trehalose and sodium chloride; even more preferably, the protective agent is a combination of 40-150 mmol / L trehalose and 40-150 mmol / L sodium chloride; even more preferably, the protective agent in the liquid composition is a combination of 60-150 mmol / L trehalose and 80-120 mmol / L sodium chloride, more preferably a combination of 60 mmol / L trehalose and 100 mmol / L sodium chloride.

[0021] In the liquid composition, the surfactant may be a non-ionic polymer, such as one or more selected from the group consisting of Tween 80, Tween 20, poloxamer, and polyethylene glycol, and the surfactant is present at a concentration of 0.01% to 0.2%.

[0022] Preferably, the surfactant is 0.01 to 0.03% Tween80, more preferably 0.02% Tween80.

[0023] The pharmaceutical compositions provided by the present invention are clear, sterile solutions that are colorless to pale yellow with a slight opalescence. As detected, the liquid compositions provided by the present invention have an osmolality of 230-330 mOsmol / kg, a viscosity of <30 cP, and a pH of 6.2±0.2.

[0024] According to a particular embodiment of the invention, the liquid composition is a formulation for injection, preferably for subcutaneous or intravenous injection; preferably, the liquid composition is a formulation for subcutaneous or intravenous injection.

[0025] Preferably, the liquid composition comprises: 130-165 mg / ml, preferably 150±5 mg / ml, of an antibody against human interleukin-4 receptor alpha; 10 mmol / L histidine hydrochloride; 60mmol / L trehalose; 100mmol / L sodium chloride; 0.02% Tween 80; Including, The liquid composition has a pH of 6.2±0.2, preferably 6.2±0.05.

[0026] The formulations provided by the present invention are colorless or pale yellow, transparent, sterile solutions with favorable long-term stability (they can be stored at 2-8°C for 2 years and meet quality standards) and are free of aggregates (≦10.0%). The formulations of the present invention have low viscosity (<30 cP) and are characterized by a pH and osmolality (290-310 mOsmol / kg) suitable for subcutaneous injection. See Table 14 below for details.

[0027] The above concentrations are all based on the total volume or weight of the liquid composition or liquid formulation. In this context, the terms "liquid formulation" and "liquid composition" can be used interchangeably.

[0028] According to a particular embodiment of the invention, the liquid composition is a formulation for subcutaneous injection and further comprises sterile water for injection.

[0029] In another aspect, the present invention provides use of the liquid composition for manufacturing a medicament for treating an inflammatory or allergic disease; preferably, the inflammatory or allergic disease includes an autoimmune disease, such as allergic dermatitis, asthma, eosinophilic esophagitis, eczema, allergic rhinitis, nasal polyps, rheumatoid arthritis, etc.

[0030] In yet another aspect, the present invention also provides other products related to the liquid compositions.

[0031] The present invention provides a container containing the liquid composition of the present invention. For example, the container can be a 2 mL injection vial made of neutral borosilicate glass tubing, with a liquid composition fill volume of more than 1 mL per vial.

[0032] The present invention provides a kit comprising a container provided by the present invention; and further comprising instructions.

[0033] In yet another aspect, the present invention provides a method for preventing, treating, or ameliorating an inflammatory or allergic disorder, the method comprising administering the liquid composition of the present invention to a subject in need thereof. Preferably, the subject is a mammal, more preferably a human.

[0034] Preferably, the inflammatory or allergic disease includes an autoimmune disease, such as allergic dermatitis, asthma, eosinophilic esophagitis, eczema, allergic rhinitis, nasal polyps, rheumatoid arthritis, and the like.

[0035] The liquid compositions provided by the present invention can be administered to a subject by injection, for example, by subcutaneous or intravenous injection.

[0036] Other medicines can be used in combination with the liquid composition to prevent, treat or improve inflammation or allergic diseases.For example, the method further comprises administering to the subject at least one medicine selected from the group consisting of antiasthmatic drugs, such as albuterol, antihistamines, such as loratadine, immunosuppressants, such as tacrolimus and pimecrolimus, M receptor blockers, such as ipratropium bromide, leukotriene receptor blockers, such as montelukast, phosphodiesterase inhibitors, such as theophylline, nonsteroidal anti-inflammatory drugs, such as 5-aminosalicylic acid, and hormones, such as beclomethasone and budesonide.Preferably, the medicine and the liquid composition of the present invention are administered simultaneously or consecutively.

[0037] The inventors of the present invention have successfully developed a novel liquid composition for antibodies against human interleukin-4 receptor alpha, which provides a basis for pharmaceutical manufacturing. The liquid composition provided by the present invention contains a high concentration of antibodies against human interleukin-4 receptor alpha. When administered subcutaneously or intravenously, it can provide a high dose of antibody, thereby meeting the needs of drug application and improving therapeutic efficacy. Even with a high concentration of antibody, the liquid composition does not exhibit antibody aggregation and has a fairly low viscosity, which allows the composition to be easily delivered through fine needles and needle tubes, thereby minimizing patient discomfort. The liquid composition of the present invention also has the advantages of easy manufacture and storage. Furthermore, the liquid composition has sufficient physical and chemical stability, and the content of insoluble particles is within the range specified in the Chinese Pharmacopoeia (the number of insoluble particles with a particle size of ≥10 μm was ≤60,000 / vial, and the number of insoluble particles with a particle size of ≥25 μm was ≤600 / vial). The liquid composition can also be frozen and thawed repeatedly, is resistant to shaking, has good thermal stability, and meets the requirements for pharmaceutical manufacturing.

[0038] The liquid composition provided by the present invention was evaluated for its binding ability with human interleukin-4 receptor alpha and its biological activity in blocking STAT-6 signaling. The results showed that the liquid composition provided by the present invention can stably and effectively bind to the antigen IL-4Rα and effectively block STAT-6 signaling. [The present invention 1001] A liquid composition comprising an antibody against human interleukin-4 receptor alpha, A liquid composition comprising the antibody at a concentration of 50 to 200 mg / ml, and a buffer, a protective agent, and a surfactant that act as excipients, and having a pH of 5.4 to 6.4. [The present invention 1002] In the liquid composition, the antibody comprises a light chain variable region and a heavy chain variable region; the light chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO:1, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:2, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:3; the heavy chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO:5, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:6, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:7; Preferably, the antibody comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:4 and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:8; Preferably, the antibody is present in a concentration of 100 to 200 mg / ml, more preferably 130 to 165 mg / ml, and even more preferably 150±5 mg / ml. [The present invention 1003] the buffer is one or more selected from the group consisting of an acetate buffer, a phosphate buffer, and an amino acid buffer; 1001 or 1002. The liquid composition of invention 1001 or 1002, wherein said buffering agent is present at a concentration of 5 to 50 mmol / L, preferably said buffering agent is an amino acid buffering agent at a concentration of 5 to 50 mmol / L. [The present invention 1004] the acetate buffer is a sodium acetate buffer, the phosphate buffer is a sodium dihydrogen phosphate buffer, or the amino acid buffer is a histidine hydrochloride buffer; Preferably, the buffer is present at a concentration of 5 to 20 mmol / L; The liquid composition of the present invention 1003, wherein the buffer is preferably a histidine hydrochloride buffer having a concentration of 5 to 20 mmol / L, more preferably a histidine hydrochloride buffer having a concentration of 10 mmol / L. [The present invention 1005] the protective agent is one or more selected from the group consisting of a sugar, an alcohol, an amino acid, and a chloride salt; The liquid composition of any one of claims 1001 to 1004, wherein the protective agent is present at a concentration of 40 to 220 mmol / L. [The present invention 1006] the protective agent is one or more selected from the group consisting of a sugar, an alcohol, and an amino acid; the protective agent is present at a concentration of 40 to 220 mmol / L, and / or the protective agent is a chloride salt and the protective agent is present at a concentration of 40 to 150 mmol / L; Preferably, the sugar is trehalose and / or sucrose at a concentration of 40 to 150 mmol / L, more preferably 60 to 150 mmol / L; the alcohol is mannitol at a concentration of 40 to 220 mmol / L, more preferably 110 to 150 mmol / L; the amino acid is one or more selected from the group consisting of proline, arginine hydrochloride, and glycine at a concentration of 40 to 220 mmol / L, more preferably 120 to 220 mmol / L; or the chloride salt is sodium chloride at a concentration of 40 to 150 mmol / L, more preferably 80 to 120 mmol / L; A liquid composition of the present invention 1005, wherein the protective substance is preferably a combination of trehalose and sodium chloride; more preferably, the protective substance is a combination of 40 to 150 mmol / L trehalose and 40 to 150 mmol / L sodium chloride; even more preferably, the protective substance in the liquid composition is a combination of 60 to 150 mmol / L trehalose and 80 to 120 mmol / L sodium chloride, and more preferably, a combination of 60 mmol / L trehalose and 100 mmol / L sodium chloride. [The present invention 1007] the surfactant is a non-ionic polymer, for example, one or more selected from the group consisting of Tween 80, Tween 20, poloxamer, and polyethylene glycol; the surfactant is present at a concentration of 0.01% to 0.2%, Preferably, the surfactant is 0.01 to 0.03% Tween 80, more preferably 0.02% Tween 80. [The present invention 1008] a formulation for injection, preferably for subcutaneous or intravenous injection, Preferably, 130 to 165 mg / ml, preferably 150±5 mg / ml, of the antibody; 10 mmol / L histidine hydrochloride; 60mmol / L trehalose; 100mmol / L sodium chloride; 0.02% Tween 80 Including, The liquid composition of any one of claims 1001 to 1007, having a pH of 6.2±0.2, preferably 6.2±0.05. [The present invention 1009] Use of any of the liquid compositions according to claims 1001 to 1008 for the manufacture of a medicament for the treatment of an inflammatory or allergic disease, Preferably, the inflammatory or allergic disease comprises an autoimmune disease, such as allergic dermatitis, asthma, eosinophilic esophagitis, eczema, allergic rhinitis, nasal polyps, rheumatoid arthritis, and the like. [The present invention 1010] A container containing the liquid composition of any one of the present inventions 1001 to 1008, or a kit containing said container. [The present invention 1011] A method for preventing, treating, or ameliorating an inflammatory or allergic disease, comprising: Administering any one of the liquid compositions of the present inventions 1001 to 1008 to a subject in need thereof, Preferably, the subject is a mammal, more preferably a human; Preferably, the inflammatory or allergic disease comprises an autoimmune disease, such as allergic dermatitis, asthma, eosinophilic esophagitis, eczema, allergic rhinitis, nasal polyps, rheumatoid arthritis, etc.; Preferably, the liquid composition is administered to the subject by injection, for example, by subcutaneous or intravenous injection. [Brief explanation of the drawings]

[0039] Aspects of the present invention are described in detail below in conjunction with the accompanying drawings: [Figure 1] SEC purity results for formulations tested during pH screening are shown. [Figure 2] SEC purity results for formulations tested during pH screening are shown. [Figure 3] 1 shows the nrCE-SDS purity results of the formulations tested during pH screening. [Figure 4] 1 shows the nrCE-SDS purity results of the formulations tested during pH screening. [Figure 5] 1 shows the rCE-SDS purity results of the formulations tested during pH screening. [Figure 6] 1 shows the rCE-SDS purity results of the formulations tested during pH screening. [Figure 7]The results of the CEX neutral peak change for each formulation tested during pH screening are shown. [Figure 8] The results of the CEX neutral peak change for each formulation tested during pH screening are shown. [Figure 9] Each shows the SEC purity results for the formulations tested during protective agent screening. [Figure 10] Each shows the SEC purity results for the formulations tested during protective agent screening. [Figure 11] Each shows the nrCE-SDS purity results for the formulations tested during protective agent screening. [Figure 12] Each shows the nrCE-SDS purity results for the formulations tested during protective agent screening. [Figure 13] Each shows the rCE-SDS purity results for the formulations tested during protective agent screening. [Figure 14] Each shows the rCE-SDS purity results for the formulations tested during protective agent screening. [Figure 15] Each shows the results of the CEX neutral peak change for the formulations tested during the protective agent screening. [Figure 16] Each shows the results of the CEX neutral peak change for the formulations tested during the protective agent screening. [Figure 17] 1 shows a comparison of the viscosity of formulations tested during protective agent screening. [Figure 18] 1 shows the viscosity results of different concentrations of formulations tested without a protective agent. [Figure 19] 1 shows the results of detecting the biological activity of CBP-201. DETAILED DESCRIPTION OF THE INVENTION

[0040] Detailed Description of the Preferred Embodiments The present invention will be described in more detail in combination with the following specific embodiments. It will be recognized by those skilled in the art that the provided embodiments do not limit the scope of the present invention in any way, but are used only to illustrate the present invention.

[0041] All experimental methods in the following examples are conventional unless otherwise specified. All raw materials and reagents used in the following examples are commercially available products unless otherwise specified.

[0042] The antibody referred to in the following examples as "CBP-201" is a monoclonal antibody comprising a light chain variable region set forth in SEQ ID NO:4 and a heavy chain variable region set forth in SEQ ID NO:8; a light chain constant region set forth in SEQ ID NO:9 and a heavy chain constant region set forth in SEQ ID NO:10; or a light chain set forth in SEQ ID NO:11 and a heavy chain set forth in SEQ ID NO:12. In this context, the terms "CBP-201" and "protein" can be used interchangeably. Furthermore, the terms "CBP-201 formulation" and "CBP-201 liquid composition" can also be used interchangeably.

[0043] The general methods used in the examples below include: (1) Determination of pH value: The pH value is determined by referring to the pH measurement method described in General Rule 0631, Volume IV, Pharmacopoeia of the People's Republic of China (2015 edition). (2) Accelerated stability test at 40°C: Stability studies are conducted by storing test samples of the prepared formulations under high temperature and humidity (40°C ± 2°C / 75 ± 5% RH) for 2 or 4 weeks to examine the stability of the formulations. (3) Protein concentration detection: Protein concentration is detected by UV spectrophotometer using the extinction coefficient (ε). According to the Beer-Lambert law, the absorbance value of the sample is calculated according to the formula: A = ε C L / N. In the formula, "C" represents the concentration of protein in the sample, mg / ml; "L" represents the light path, which is 1 cm; "A" represents the absorbance value; "ε" represents the extinction coefficient; and "N" represents the dilution ratio of the sample. In this regard, the protein concentration in the sample is calculated according to the formula: C = A / ε × N. The theoretical extinction coefficient of a protein (antibody) can be calculated according to the following formula: mass extinction coefficient ε = (5500nw + 1490ny + 125nc) M -1 ·cm -1 In the formula, "nw" represents the number of Trp in the amino acid sequence of the protein; "ny" represents the number of Tyr in the amino acid sequence of the protein; "nc" represents the number of Cys in the amino acid sequence of the protein; "M" represents the molecular weight of the protein, and "cm" represents the optical distance. According to the sequence of the CBP-201 antibody, the mass extinction coefficient ε is calculated to be 1.46. The absorbance value A of the sample at 280 nm is measured by UV spectrophotometer, and the antibody concentration is calculated accordingly. (4) Purity detection by SEC: Size exclusion chromatography (SEC-HPLC) is used using the following chromatographic conditions: Column: TSKgel G3000SW XL 7.8*300mm column; Column temperature: room temperature; Detector: DAD detector; Detection wavelength: 280nm; Flow rate: 0.7mL / min; Sample dilution: Dilute to 5.0 mg / ml with ultrapure water; Injection volume: 10μl; Mobile phase: 25 mM phosphoric acid (pH 6.8 ± 0.1) and 0.3 M sodium chloride; Elution mode: Gradient elution TIFF2025118750000003.tif25128 Detection: The peak area normalization method is used to calculate the peak area percentages of the main peak and the peaks of the HMW and LMW components. (5) Purity detection by nrCE-SDS: The purity of CBP-201 is quantitatively determined based on molecular weight under non-reducing conditions. Detection: According to the area normalization method, the purity of the main peak is calculated as the percentage of the corrected peak area of the IgG main peak relative to the sum of all corrected peak areas. (6) Purity detection by rCE-SDS: The purity of CBP-201 is quantitatively determined based on its molecular weight under reducing conditions. Detection: According to the area normalization method, the purity (i.e., CAP) of each of LC, NGHC, and HC is calculated as the percentage of the corrected peak area of each of LC, NGHC, and HC to the sum of all corrected peak areas. The purity of the sample is the sum of the purity of LC and HC. (7) Charge diversity detection (CEX neutral peak): Detection was performed by ion chromatography as described in General Rule 0514, Volume III, Pharmacopoeia of the People's Republic of China (2015 edition). The column used was a BiomAb NP5, PK, 4.6 x 250 mm, available from Agilent. Chromatographic peak integration results were evaluated with reference to the reference. The highest peak was the main peak, and peaks integrated earlier than the main peak's retention time were defined as acidic peaks, and peaks integrated later than the main peak's retention time were defined as alkaline peaks. (8) DSC detection: DSC thermal analysis, also known as differential scanning calorimetry, is a technique for measuring the phase transition temperatures of a sample by recording the endothermic or exothermic rate of the sample using a differential scanning calorimeter, with the heat flux dH / dt (millijoules / second) as the vertical axis and the temperature T or time t as the horizontal axis, and then determining the stability of the sample. (9) Viscosity detection: The detection is carried out using a DV2T viscometer available from Brookfield, with reference to the viscosity determination method - Method 3 (rotational viscosity measurement method) as set out in General Rule 0633, Volume IV, Pharmacopoeia of the People's Republic of China (2015 edition). (10) Visible foreign body detection: The detection is carried out with reference to the method for detecting visible foreign bodies - Method 1 (lamp test) set out in General Rule 0904, Volume IV, Pharmacopoeia of the People's Republic of China (2015 edition). (11) Particle size detection: The detection is carried out according to the insoluble particle inspection method--method 1 (light blocking) described in General Rule 0903, Volume IV, Pharmacopoeia of the People's Republic of China (2015 edition). [Example]

[0044] Example 1 pH and buffers In this example, to study the pH range and buffering agent for CBP-201 formulations, six pH values were used: 5.4, 5.6, 5.8, 6.0, 6.2, and 6.4, and buffering agents selected from sodium acetate, histidine hydrochloride, and sodium dihydrogen phosphate. Sodium chloride was also added to each sample. Specifically, in an accelerated test at 40°C, the following protein solutions containing 133.6 mg / ml CBP-201 were tested to determine the appropriate pH range and buffering agent (see Table 1).

[0045] Table 1. pH and buffer screening solutions TIFF2025118750000004.tif48143

[0046] Protein solutions were tested for appearance, protein concentration, SEC purity, CE-SDS purity, charge diversity, DSC, and viscosity by accelerated stability testing at 40°C.

[0047] Visual inspection results: All protein solutions were whitish in appearance, and no visible foreign matter was observed.

[0048] Protein concentration measurement results: The protein concentrations of the protein solutions were all within the range of 133.6±5% mg / ml, and no obvious increase or decrease in concentration was observed.

[0049] Furthermore, after two weeks of acceleration at 40°C, the following results were obtained from protein solutions at different pH values:

[0050] 1. The results of SEC purity are shown in Figures 1 and 2. After two weeks of acceleration, the absolute SEC purity of the test solutions was ranked as follows: pH 6.2 solution > pH 6.4 solution > pH 6.0 solution > pH 5.8 solution > pH 5.6 solution > pH 5.4 solution (Figure 1). Furthermore, the degree of decrease in SEC purity of the test solutions (compared to that at time 0) was ranked as follows: pH 5.4 solution > pH 5.6 solution > pH 5.8 solution > pH 6.0 solution > pH 6.2 solution > pH 6.4 solution (Figure 2).

[0051] 2. The results for nrCE-SDS are shown in Figures 3 and 4. After two weeks of accelerated storage, the absolute values of the nrCE-SDS purity of the test solutions were ranked as follows: pH 6.4 solution > pH 6.2 solution > pH 5.8 solution > pH 6.0 solution > pH 5.6 solution > pH 5.4 solution (Figure 3). Furthermore, the degree of decrease in nrCE-SDS purity of the test solutions (compared to those at time 0) was ranked as follows: pH 5.4 solution > pH 5.6 solution > pH 6.0 solution > pH 5.8 solution > pH 6.2 solution > pH 6.4 solution (Figure 4). Furthermore, the nrCE-SDS purity of the pH 6.0 solution was 97.5% after two weeks of accelerated storage, which was a fairly high purity. The nrCE-SDS purity of the pH 6.2 and pH 6.4 solutions was higher than that of the pH 6.0 solution.

[0052] 3. The results for rCE-SDS are shown in Figures 5 and 6. After two weeks of acceleration, the absolute values of the rCE-SDS purity of the test solutions were ranked as follows: pH 6.4 solution > pH 6.2 solution > pH 6.0 solution > pH 5.8 solution > pH 5.6 solution = pH 5.4 solution (Figure 5). Furthermore, the degree of decrease in rCE-SDS purity of the test solutions was ranked as follows: pH 5.4 solution = pH 5.6 solution > pH 5.8 solution > pH 6.0 solution > pH 6.2 solution > pH 6.4 solution (Figure 6).

[0053] 4. The results of changes in the CEX neutral peak are shown in Figures 7 and 8. After two weeks of accelerated storage, the CEX neutral peak ratios of the test solutions were ranked as follows: pH 6.4 solution > pH 6.2 solution > pH 5.6 solution > pH 6.0 solution > pH 5.8 solution > pH 5.4 solution (Figure 7). Furthermore, the degree of change (decrease) in the CEX neutral peak ratios of the test solutions after two weeks of accelerated storage (compared to those at time 0) was ranked as follows: pH 5.8 solution > pH 5.6 solution > pH 5.4 solution > pH 6.0 solution > pH 6.2 solution > pH 6.4 solution (Figure 8).

[0054] 5. DSC results: The protein was stable in all six solutions with different pH values and buffers shown in Table 1, with no significant differences between them.

[0055] 6. Viscosity results: A comparison of the viscosities of the test protein solutions with different buffers showed that there was a small difference in viscosity between the His-HCl and phosphate buffer systems, and that the viscosities of all these solutions were lower than those of the acetate buffer system. For the His-HCl buffer system, the viscosity was in the range of 8.7 ± 0.8 cP, which was lower than the expected 20 cP, indicating that solutions with higher pH had lower viscosities.

[0056] Based on the results obtained from the above SEC, nrCE-SDS, rCE-SDS, CEX, DSC, and viscosity detection, it can be concluded that the higher the pH, the better the stabilizing effect on proteins. According to their stabilizing effect, the pH values can be ranked as follows: pH 6.4 > pH 6.2 > pH 6.0. That is, proteins can be stabilized under acidic conditions of pH 6.2 ± 0.2.

[0057] Example 2 protective agent In this example, suitable protective agents for CBP-201 formulations were investigated. Protein solutions with a pH of 6.0 and supplemented with trehalose, sucrose, mannitol, proline, arginine hydrochloride, glycine, or sodium chloride were prepared, with the sodium chloride solution used as a control. Specifically, in an accelerated stability study at 40°C, the following protein solutions containing 133.6 mg / ml of CBP-201 were tested to determine suitable protective agents (see Table 2):

[0058] (Table 2) Protective substances TIFF2025118750000005.tif64128

[0059] Protein solutions were tested for appearance, protein concentration, SEC purity, CE-SDS purity, charge diversity, viscosity, and DSC by accelerated stability testing at 40°C.

[0060] Visual inspection results: All protein solutions were whitish in appearance.

[0061] Protein concentration measurement results: The protein concentrations of all protein solutions were within the range of 133.6±5%mg / ml.

[0062] Furthermore, after two weeks of acceleration at 40° C., the following results were obtained from protein solutions with different protective agents:

[0063] 1. SEC purity results are shown in Figures 9-10. After two weeks of acceleration, the absolute SEC purity of the test solutions was ranked as follows: solution with Pro > solution with Gly > solution with Tre > solution with Suc > solution with Man > solution with Arg > solution with NaCl. The protein solution with NaCl as the protective agent had the lowest SEC purity of 95.3% after two weeks, which was acceptable (Figure 9). Furthermore, the degree of decrease in SEC purity of the test solutions (compared to that at time 0) was ranked as follows: solution with Arg > solution with NaCl > solution with Suc > solution with Tre > solution with Gly > solution with Pro > solution with Man. Even the protein solution with Arg as the protective agent had the greatest decrease in SEC purity, i.e., 3.04%, but it still had an acceptable SEC purity of 95.4% after two weeks of accelerated storage at 40°C (Figure 10).

[0064] 2. The results of nrCE-SDS are shown in Figures 11-12. After two weeks of acceleration, the nrCE-SDS purity of the test solutions was ranked as follows: solution with Tre > solution with NaCl > solution with Suc > solution with Gly > solution with Man > solution with Pro > solution with Arg (Figure 11). Furthermore, the degree of decrease in nrCE-SDS purity of the test solutions (each compared to that at time 0) was ranked as follows: solution with Arg > solution with Pro > solution with Man > solution with Gly > solution with Suc > solution with NaCl > solution with Tre (Figure 12). It can be seen that after two weeks of acceleration at 40°C, the nrCE-SDS purity, which showed a small decrease compared to that at time 0 and remained at a relatively high level, was ranked as follows: solution with Tre > solution with NaCl > solution with Suc > solution with Gly > solution with Man > solution with Pro > solution with Arg.

[0065] 3. The results of rCE-SDS are shown in Figures 13 and 14. After two weeks of acceleration, the absolute values of the rCE-SDS purity of the test solutions were ranked as follows: solution with Suc > solution with Man = solution with Tre > solution with Pro > solution with NaCl > solution with Gly > solution with Arg (Figure 13). Furthermore, compared to the solution at time 0, the degree of decrease in rCE-SDS purity of the test solutions was ranked as follows: solution with Arg > solution with Gly > solution with NaCl > solution with Pro > solution with Tre = solution with Man > solution with Suc (Figure 14). It can be seen that after two weeks of acceleration at 40°C, the rCE-SDS purity showed a small decrease compared to the solution at time 0 and remained at a relatively high level, ranked as follows: solution with Suc > solution with Man = solution with Tre > solution with Pro > solution with NaCl > solution with Gly > solution with Arg.

[0066] 4. The results of the change in the CEX neutral peak are shown in Figures 15-16. After two weeks of accelerated storage, the CEX neutral peak ratios of the test solutions were ranked as follows: solution with Tre > solution with Pro > solution with Suc > solution with Man > solution with Arg > solution with NaCl > solution with Gly (Figure 15). Furthermore, the degree of change (decrease) in the CEX neutral peak ratios of the test solutions after two weeks of accelerated storage (compared to those at time 0) was ranked as follows: solution with Gly > solution with Man > solution with Arg > solution with NaCl > solution with Pro > solution with Suc (increase) > Tre (increase) (Figure 16). It can be concluded from the results that Tre ranks first in maintaining a stable charge, Suc comes in second, followed by Pro, Man, Arg, NaCl, and Gly, in that order.

[0067] 5. Viscosity results are shown in Figure 17. After adding different protective agents, the viscosity of the protein solutions was ranked as follows: solution with Suc>solution with Pro>solution with Tre>solution with Man>solution with Gly>solution with NaCl>solution with Arg.

[0068] 6. DSC results: For the solutions containing the seven protective agents respectively, the Tm1 and Tm2 values were all acceptable, the proteins therein were structurally stable, and there was no significant difference between them.

[0069] Example 3 Viscosity evaluation for protein solutions with buffer only This example was conducted to explore the change in viscosity with concentration of CBP-201 formulations containing only buffer (10 mmol / L His-HCl, pH 6.0) (no added protective agents) to evaluate the effect of protective agents in reducing the viscosity of the formulations. To provide a basis for selecting the protein concentration in the formulations, formulations with the highest concentrations were prepared and viscosities were measured. In this example, formulations were prepared without NaCl and compared to the NaCl-containing formulations in the previous examples.

[0070] The proteins were dialyzed against dialysis buffer (10 mmol / L His-HCl, pH 6.0), and the dialyzed proteins were concentrated to concentrations of 71.23 mg / ml, 89.04 mg / ml, 106.85 mg / ml, 133.56 mg / ml, and >151.37 mg / ml (detected protein concentrations), respectively. The formulations were then filtered. Appearance and viscosity were detected. The results are shown in Table 3 and Figure 18.

[0071] Table 3: Appearance and viscosity of CBP-201 formulation TIFF2025118750000006.tif64170

[0072] The results showed that the viscosity of the protein solution increased with increasing protein concentration. The viscosity of the protein solution with a target concentration of 133.6 mg / ml (the actual concentration was 137.94 mg / ml) was 50.33 cP, and only the protein solution containing Suc in the screening for suitable protective agents in Example 2 had a viscosity greater than that value (68.75 cP). In other words, among the seven protective agent alternatives, only Suc increased the viscosity of the protein solution, while the other protective agents reduced the viscosity of the protein solution to various levels. Therefore, protective agents that are preferentially selected for their effect of reducing the viscosity of the formulation can be ranked as follows: Arg, NaCl, Gly, Man, Tre, and Pro.

[0073] Example 4 Combination of ingredients in the formulation Based on the results obtained from the screening test, a study was conducted on the combination of ingredients in CBP-201 formulations. The compositions of the protein solutions studied are shown in Table 4.

[0074] (Table 4) Combinations of ingredients TIFF2025118750000007.tif88166

[0075] The protein solution contained 133.6 mg / ml of CBP-201 and was examined by accelerated stability testing at 4°C and 40°C. The accelerated stability testing at 4°C and 40°C detected the solution for visible foreign matter, specific size (MFI / FLOWCAM), protein concentration, SEC purity, DSC, osmolality, viscosity, CE-SDS, and CEX, and the results obtained are shown in Tables 5-11.

[0076] (Table 5) Results of visual inspection TIFF2025118750000008.tif94170

[0077] (Table 6) Protein concentration detection results TIFF2025118750000009.tif90146

[0078] The results of appearance and protein concentration showed that after storage at 4° C. for 2 weeks and at 40° C. for 4 weeks, no significant decrease in protein concentration was observed for the protein solutions having combinations of ingredients numbered 1 to 9, and all of the protein solutions had a whitish appearance, which was associated with the high protein concentration of the solutions and the properties of the proteins themselves. In this regard, based on the results of appearance and protein concentration, all of the combinations of ingredients numbered 1 to 9 were acceptable.

[0079] (Table 7) SEC purity TIFF2025118750000010.tif106168

[0080] The data in Table 7 showed that the protein solution having the component combination numbered 2 had the lowest, but acceptable, SEC purity of 96.8% after two weeks of storage at 4° C. Furthermore, compared to the SEC purity at time 0, the greatest decrease in SEC purity was observed for the protein solution having the component combination numbered 4, at 1.44%, although the decrease in SEC purity (97.1%) was still acceptable. In this regard, all solutions having component combinations numbered 1 through 9 had acceptable SEC purity after two weeks of storage at 4° C.

[0081] After 4 weeks of storage at 40°C, the protein solutions having the component combinations numbered 5 and 8 showed a significant decrease in SEC purity (compared to that at time 0), thereby having the lowest, unacceptable SEC purity. The SEC purity of the protein solutions having other different component combinations after 4 weeks of storage at 40°C was ranked as follows: protein solution having the component combination numbered 6 > protein solution having the component combination numbered 9 > protein solution having the component combination numbered 1 > protein solution having the component combination numbered 3 > protein solution having the component combination numbered 4 > protein solution having the component combination numbered 2 > protein solution having the component combination numbered 7. Regarding the degree of decrease in SEC purity, the protein solution having the component combination numbered 6 showed a relatively small decrease, while all the protein solutions having the other component combinations showed a similar decrease (maintained at about 1.5%), which was still acceptable.

[0082] (Table 8) CE-SDS purity TIFF2025118750000011.tif234166

[0083] The data in Table 8 show that the lowest nrCE-SDS purity was 99.7% among the protein solutions containing nine combinations of components stored at 4°C for two weeks. In other words, all protein solutions containing nine combinations of components maintained the protein therein stably for two weeks at 4°C. Furthermore, the HC+LC(rCE-SDS) values after two weeks of storage at 4°C remained almost unchanged compared to those at time 0. In other words, all protein solutions containing combinations of components numbered 1 to 9 were relatively stable at 4°C.

[0084] After 4 weeks of storage at 40°C, the protein solutions having component combinations numbered 7 and 8 showed a significant decrease in nrCE-SDS purity (compared to that at time 0), thereby resulting in unacceptably low nrCE-SDS purity. Furthermore, except for the protein solution having component combination numbered 1, which had an nrCE-SDS purity of 92.4%, all other protein solutions had nrCE-SDS purity greater than 94.0%, resulting in an acceptable decrease of approximately 5% (compared to that at time 0).

[0085] On the other hand, after 4 weeks of storage at 40°C, the rCE-SDS purity of protein solutions with different combinations of components was ranked as follows: protein solution with combination numbered 9 > protein solution with combination numbered 3 > protein solution with combination numbered 2 = protein solution with combination numbered 4 > protein solution with combination numbered 1 > protein solution with combination numbered 6 = protein solution with combination numbered 5 > protein solution with combination numbered 7 = protein solution with combination numbered 8. It can be seen that the protein solutions with combinations of components numbered 7 and 8 showed the lowest purity, i.e., 96.6%, each with a decrease of about 3% (compared to that at time 0), which was greater than that of the protein solutions with other combinations of components. After 4 weeks of storage at 40°C, the rCE-SDS purity of all protein solutions with combinations of components numbered 1 to 9 was acceptable.

[0086] Table 9. Charge diversity TIFF2025118750000012.tif231158

[0087] The data in Table 9 showed that the protein solutions having the component combinations numbered 1 to 9 showed different degrees of decrease and increase (each compared to that at time 0) in the CEX neutral peak ratio after two weeks of storage at 4°C. Among the protein solutions that showed a decrease, the component combination numbered 1 showed the largest decrease of 1%, and the decrease in the CEX neutral peak ratio was still 89.0%, which was acceptable. In contrast, even the protein solution having the component combination numbered 5 showed the lowest CEX neutral peak ratio of 88.5% after two weeks of storage at 4°C, and the CEX neutral peak ratio increased compared to that at time 0, which was still acceptable.

[0088] The CEX neutral peak ratios in all nine protein solutions with component combinations significantly decreased after 4 weeks of storage at 40°C, with the ratios of decrease essentially equal, with many of these maintained within a range of 70% ± 2% and ranked as follows: protein solution with component combination number 2 > protein solution with component combination number 3 = protein solution with component combination number 6 > protein solution with component combination number 1 = protein solution with component combination number 5 > protein solution with component combination number 8 > protein solution with component combination number 4 > protein solution with component combination number 9. The greatest decrease was observed for the protein solution with component combination number 9, where the percentage decrease in the CEX neutral peak ratio after storage for the protein solution with component combination number 9 was 39.7%, and the corresponding percentages for the protein solutions with the other component combinations were maintained within a range of 30% ± 5%.

[0089] (Table 10) DSC TIFF2025118750000013.tif80128

[0090] The data in Table 10 showed that, except for the protein solution having the component combination numbered 7, which only yielded one Tm value (not significantly different compared to the other eight protein solutions), the Tm1 and Tm2 values of the other protein solutions were all acceptable and there was no significant difference between them.

[0091] Table 11: Viscosity and osmolality (0 hours) TIFF2025118750000014.tif80146

[0092] The data in Table 11 show the different degrees of viscosity reduction for all protein solutions having component combinations numbered 1 to 9, and the solution viscosities were ranked as follows: protein solution having a combination numbered 1 < protein solution having a combination numbered 2 < protein solution having a combination numbered 8 < protein solution having a combination numbered 6 < protein solution having a combination numbered 5 < protein solution having a combination numbered 7 < protein solution having a combination numbered 4 < numbered 9 < protein solution having a combination numbered 3. Furthermore, a comparison between protein solutions having component combinations numbered 3, 4, and 5 showed that Tween 80 can reduce the viscosity of a protein solution, but the degree of reduction is not linearly proportional to the Tween 80 concentration. For example, a protein solution containing 0.2% Tween 80 exhibited a viscosity approximately 0.94 cP (6.2%) lower than a protein solution containing 0.02% Tween 80. A comparison between the protein solutions having the component combinations numbered 4, 7, and 9 showed that the protein solutions having the component combinations numbered 4 and 7 had similar viscosities, and the protein solution having the component combination numbered 9 exhibited a viscosity 2.27 cP (13.0%) higher than the protein solution having the component combination numbered 7 and 2.20 cP (12.6%) higher than the protein solution having the component combination numbered 4, indicating that pH 6.0 and pH 6.2 performed better than pH 6.4 in reducing the viscosity of the protein solution, and pH 6.2 was a reasonable choice. A comparison between the protein solutions having the component combinations numbered 1, 2, and 4 showed that the protein solution having the component combination numbered 4 exhibited a viscosity 29.7% higher than the protein solution having the component combination numbered 2 and 48.8% higher than the protein solution having the component combination numbered 1, indicating that NaCl had a better effect than Tre in reducing the viscosity of the protein solution.Furthermore, proteins in protein solutions that contained only NaCl as a protective agent were difficult to stabilize, thereby requiring a combination of NaCl and Tre in an appropriate ratio.

[0093] Example 5 Effect of trehalose on formulation viscosity Viscosity test of formulation (1): A protein solution containing CBP-201 antibody at a concentration of 102.8 mg / ml, 10 mmol / L His-HCl buffer (pH 6.2), and 40 mmol / L NaCl, but without trehalose, was prepared and then concentrated using an ultracentrifugal filter to three protein concentrations: 133.6 mg / ml, 151.4 mg / ml, and >151.4 mg / ml, respectively. The viscosity of the concentrated protein solutions was tested and compared with that of the protein solution containing Trehalose. The results are shown in Table 12.

[0094] (Table 12) Viscosity detection results TIFF2025118750000015.tif32164

[0095] The data in Table 12 show that the viscosity increases with increasing protein concentration, and that if the protein solution is over-concentrated, care must be taken to determine the degree of overconcentration. A comparison of the 137.23 mg / ml protein solution, which had a viscosity of 19.71 cP (24.5°C), with the protein solution having the combination of ingredients numbered 3 in Example 4 (which further contained Tre compared to the protein solution of this example and had a viscosity of 25.64 cP (25.1°C)), showed that the viscosity of the protein solution having the above composition can be increased.

[0096] Viscosity test of formulation (2): A protein solution containing the CBP-201 antibody was prepared and then dialyzed and concentrated in dialysis buffer (10 mmol / L His-HCl, pH 6.2, 40 mmol / L NaCl) supplemented with 150 mmol / L trehalose (1x) by tangential flow. SEC purity and viscosity were determined, and the results are shown in Table 13.

[0097] (Table 13) Viscosity detection results TIFF2025118750000016.tif35149

[0098] The SEC purity results showed that the SEC purity did not decrease significantly during dialysis and concentration, indicating that the processes and methods for dialysis and concentration did not affect the SEC purity of the protein.

[0099] The viscosity detection results showed that the viscosity of the protein solution with a protein concentration of 133.37 mg / ml was 23.18 cP (25.0°C), which was basically consistent with the result obtained from the protein solution having the component combination numbered 3 in Example 4 (which had a viscosity of 25.64 cP (25.1°C)), demonstrating that the technical solution of the present invention is reproducible. A viscosity of 45.22 cP (24.9°C) was exhibited by the protein solution with a protein concentration of 155.15 mg / ml, which was 49.0% higher than the viscosity (23.07 cP (24.9°C)) exhibited by the protein solution with a protein concentration of 156.85 mg / ml in viscosity test (1). Obviously, the addition of Tre increases the viscosity of the protein solution, which should be added appropriately under the premise of ensuring the stability of the protein.

[0100] Example 6 Generation of CBP-201 antibody formulations Clarification by filtration First, a filtration clarification process was performed to remove debris from the cell culture. A combination of Millipore Corporation MD0HC and MA1HC pod filters was used for the filtration clarification process. The filters were installed and secured in the pod holder, then rinsed with water for injection and PBS, respectively. After rinsing was complete, the inlet fluid was replaced with the cell culture fluid, and the suction pressure was set to 0-20 psi. After the supply was completed, the pod filter was washed with PBS, and all expelled fluid was collected.

[0101] Affinity chromatography The chromatography column was packed with MabSelect SuRe LX resin to obtain a column height of 18–22 cm, ensuring a symmetry factor of 0.8–1.0 and a theoretical plate count of ≥ 2000 N / m. The column was equilibrated with 4–6 column volumes of equilibration buffer (0.025 mol / L Tris, 0.10 mol / L NaCl, pH 7.4 ± 0.20) for affinity chromatography and loaded with the liquid collected during clarification by filtration. After loading, the column was further equilibrated with 3–4 column volumes of equilibration buffer for affinity chromatography. The column was then rinsed with 2–3 column volumes of rinse solution 1 (0.025 mol / L Tris, 1.00 mol / L NaCl, pH 7.4 ± 0.20) and rinse solution 2 (0.05 mol / L NaAc, pH 5.5 ± 0.10), respectively. After rinsing, the proteins were eluted with affinity chromatography elution buffer (0.10 mol / L NaAc-HAc, pH 3.60 ± 0.10). Protein collection began when the UV absorbance rose to 0.1000–0.1500 AU / 5 mm and stopped when the UV absorbance fell to 0.2000–0.3000 AU / 5 mm.

[0102] Viral inactivation at low pH An acidic titrant (1.00 mol / L acetic acid) was prepared and used to adjust the pH of the protein solution eluted by affinity chromatography to 3.50-3.70. The resulting protein solution was placed at 20-25°C for 2-3 hours for low-pH viral inactivation. The pH of the protein solution was then adjusted to 5.40-5.60 using a basic titrant (2.00 mol / L Tris), and the conductivity of the protein solution was measured. The protein solution was filtered into a disposable liquid container using a sterile filter and then sampled for protein concentration, pH, conductivity, bacterial endotoxin, SEC-HPLC, CEX-HPLC, CE-SDS, and microbial limit testing.

[0103] Cation exchange chromatography For cation exchange chromatography, Capto S ImpAct resin was used, with a column height of 18–22 cm, a symmetry factor of 0.8–1.8, and a theoretical plate count of ≥2000 N / m. The column was equilibrated with 4–6 column volumes of equilibration buffer (0.05 mol / L NaAc, 0.05 mol / L NaCl, pH 5.50 ± 0.05) for cation exchange chromatography, followed by loading of the protein solution. After loading, the column was further equilibrated with 2–4 column volumes of equilibration buffer for cation exchange chromatography. The column was then rinsed with 5–7 column volumes of rinse solution (0.05 mol / L NaAc, 0.08 mol / L NaCl, pH 5.50 ± 0.05). After rinsing, the protein was eluted with cation exchange elution solution (0.05 mol / L NaAc, 0.22 mol / L NaCl, pH 5.50 ± 0.05). Protein collection began when the UV absorbance value rose to 0.2000-0.3000 AU / 5 mm and stopped when the UV absorbance value fell to 1.0000-1.5000 AU / 5 mm.

[0104] Anion exchange chromatography For anion exchange chromatography, POROS 50 HQ resin was used, with a column height of 18–22 cm, a symmetry factor of 0.8–1.8, and a theoretical plate number of ≥2000 N / m. The pH of the protein solution was adjusted to 7.35–7.45 before loading onto the column, and then the conductivity of the protein solution was detected. A sterile filter (loading capacity ≤3000 g / m) was used. 2 The protein solution was then filtered into a disposable liquid container by using a filter.

[0105] Nanofiltration The nanofiltration system was assembled, and the prefiltration membrane was rinsed with the equilibration buffer for anion exchange chromatography. After membrane rinsing was complete, the sterile filter was connected to the nanofiltration system, and protein nanofiltration was performed at a pressure of 28-32 psi for up to 4 hours.

[0106] Concentration by dialysis A Sartocon cassette 30KD was used. First, the cassette was assembled into an ultrafiltration system and tested for integrity and water flux. If the test passed, the membrane in the cassette was rinsed with dialysis buffer (0.060 mol / L trehalose, 0.010 mol / L histidine-HCl, 0.10 mol / L NaCl, pH 6.20 ± 0.05). After rinsing, the protein solution filtered through the nanomembrane obtained above was stirred to mix thoroughly and then loaded onto the membrane and preconcentrated to 40-60 mg / ml. After preconcentration, the protein solution was dialyzed against a volume of dialysis buffer 10-12 times the volume of the preconcentrated protein solution. The protein solution was then overconcentrated to obtain a protein concentration of 170-190 mg / ml. The membrane was rinsed twice with dialysis buffer, and the resulting liquid from the rinse was collected. The volume of dialysis buffer used to rinse the membrane each time was approximately 1.5 times the system hold-up volume, and the total volume used to rinse the membrane was approximately 2-4 times the system hold-up volume. Finally, the protein concentration of the solution was adjusted to 140-180 mg / ml, and the final protein concentration was determined.

[0107] Preparation of stock solutions (dilution and excipient addition) The protein solution was diluted to 150±5 mg / ml with Tween 80 stock solution (2% Tween (w / v), 0.060 mol / L trehalose, 0.010 mol / L histidine-HCl, 0.10 mol / L NaCl) and dialysis buffer (0.060 mol / L trehalose, 0.010 mol / L histidine-HCl, 0.10 mol / L NaCl, pH 6.20±0.05), in which the final Tween 80 concentration was 0.02%. The diluted protein solution was then filtered into a disposable liquid container, and the resulting filtered protein solution was designated as the stock solution. The prepared stock solution was divided into PETG bottles and stored at -80±10°C.

[0108] Preparation of formulations The stock solution was removed, thawed at room temperature, thoroughly mixed, and then filtered. The vials, which were filled with rubber stoppers and aluminum covers, sterilized, and cleaned to remove pyrogens, were transferred to the corresponding site for filling. The filled vials, all of which were stoppered, were then transferred to the capping room for capping. Each vial was then visually inspected to eliminate any defects, such as incorrect loading amounts, collapsed, visible particles, foreign objects, broken caps, and empty vials. The vials were then labeled and packed into boxes, and a carton label was then attached to the center of the top of the box.

[0109] The acceptance criteria and detection results are shown in Table 14.

[0110] Table 14: Acceptance criteria and detection results TIFF2025118750000017.tif242166TIFF2025118750000018.tif64166

[0111] Example 7 Detection of binding activity of CBP-201 antibody preparations Indirect ELISA was used to detect the binding ability of the formulation to the antigen sIL-4Rα.

[0112] The antigen (sIL-4Rα, expressed according to NM_000418.4) was coated and absorbed onto a solid-phase plate for ELISA. The plate was then washed, blocked, and washed again before adding a sample to be tested for binding to the antigen. The sample to be tested was a dilution of a formulation prepared according to Example 6 of the present application. The dilution was obtained by diluting the formulation with 1% BSA to an antibody concentration of 1000 ng / mL, followed by gradient dilution from an initial concentration of 1000 ng / mL to a concentration of 0 ng / mL. After incubation, unbound antibody was removed by washing, and an enzyme-labeled secondary antibody was added for further incubation. After removing unbound enzyme-labeled secondary antibody by washing, an enzyme reaction substrate was added for color development. Finally, the reaction was stopped by adding a stop solution, and the absorbance values were read at 450 nm and 655 nm using a microplate reader. According to the absorbance values, the half-effective concentration EC 50 was calculated by curve fitting.

[0113] The results of detecting the binding activity of the CBP-201 preparation are shown in Table 15.

[0114] Table 15: Binding activity detection results TIFF2025118750000019.tif74156

[0115] Example 8 Detection of biological activity of CBP-201 antibody preparation HEK Blue (商標) IL4 / IL13 cells (available from Invivogen) were used to detect the activity of CBP-201 formulations on blocking STAT-6 signaling.

[0116] HEK Blue (商標)IL4 / IL13 cells were plated in a 384-well cell culture plate, and the test sample was added thereto. The test sample was a dilution of the formulation prepared according to Example 6 of the present application. The dilution was obtained by diluting the formulation with DMEM containing 10% FBS to an antibody concentration of 5000 ng / mL, followed by gradient dilution from an initial concentration of 5000 ng / mL to a concentration of 0 ng / mL. IL4 (available from Invivogen) was then added to the plate to obtain a final concentration of 0.5 ng / mL. After 22 hours of incubation in a cell incubator, the supernatant was taken and Quanti-blue was added thereto for color development, and then the absorbance value was read at 650 nm on a microplate reader. According to the absorbance value, the half-inhibitory concentration IC 50 was calculated by curve fitting.

[0117] The results of detecting the biological activity of the CBP-201 formulation are shown in Table 16 and FIG.

[0118] Table 16: Results of biological activity detection TIFF2025118750000020.tif74157

[0119] HEK Blue (商標) The activity of the prepared CBP-201 preparations was detected using IL4 / IL13 cells, and the average IC50 values of the two batches of CBP-201 preparations were 4.193 ng / ml and 4.513 ng / ml, respectively.

[0120] The above description of the embodiments of the present invention is not intended to limit the present invention, and those skilled in the art can make various changes and modifications according to the present invention, which fall within the scope of protection of the claims of the present invention without departing from the spirit of the claims of the present invention.

[0121] Sequence information Sequence Listing <110> CONNECT BIOPHARMA HONGKONG LIMITED <120> LIQUID COMPOSITION COMPRISING ANTIBODY OF HUMAN INTERLEUKIN-4 RECEPTOR ALPHA <150> CN 201910187179.9 <151> 2019-03-13 <160> 12 <170> PatentIn version 3.3 <210> 1 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Light chain variable region, CDR1 <400> 1 Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala 1 5 10 <210> 2 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Light chain variable region, CDR2 <400> 2 Gly Ala Ser Ser Arg Ala Thr 1 5 <210> 3 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Light chain variable region, CDR3 <400> 3 Gln Gln Tyr Asp His Ser Ala Gly Trp Thr 1 5 10 <210> 4 <211> 109 <212> PRT <213> Artificial sequence <220> <223> Light chain variable region <400> 4 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asp His Ser Ala 85 90 95 Gly Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 5 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Heavy chain variable region, CDR1 <400> 5 Arg Asn Ala Met Phe 1 5 <210> 6 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Heavy chain variable region, CDR2 <400> 6 Gly Ile Gly Thr Gly Gly Ala Thr Ser Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 Arg <210> 7 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Heavy chain variable region, CDR3 <400> 7 Gly Arg Tyr Tyr Phe Asp Tyr 1 5 <210> 8 <211> 115 <212> PRT <213> Artificial sequence <220> <223> Heavy chain variable region <400> 8 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Asn 20 25 30 Ala Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Gly Thr Gly Gly Ala Thr Ser Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Arg Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 9 <211> 107 <212> PRT <213> Artificial sequence <220> <223> Light chain constant region <400> 9 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 10 <211> 326 <212> PRT <213> Artificial sequence <220> <223> Heavy chain constant region <400> 10 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly 325 <210> 11 <211> 216 <212> PRT <213> Artificial sequence <220> <223> Light chain <400> 11 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asp His Ser Ala 85 90 95 Gly Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val 100 105 110 Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys 115 120 125 Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 130 135 140 Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 145 150 155 160 Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 165 170 175 Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys 180 185 190 Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr 195 200 205 Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 12 <211> 441 <212> PRT <213> Artificial sequence <220> <223> Heavy chain <400> 12 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Asn 20 25 30 Ala Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Gly Thr Gly Gly Ala Thr Ser Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Arg Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120 125 Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 180 185 190 Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys 195 200 205 Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys 210 215 220 Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 225 230 235 240 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 245 250 255 Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp 260 265 270 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 275 280 285 Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 290 295 300 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 305 310 315 320 Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 325 330 335 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu 340 345 350 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 355 360 365 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 370 375 380 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 385 390 395 400 Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn 405 410 415 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 420 425 430 Gln Lys Ser Leu Ser Leu Ser Leu Gly 435 440

Claims

1. A liquid composition comprising an antibody against human interleukin-4 receptor alpha, A liquid composition comprising the antibody at a concentration of 50 to 200 mg / ml, and a buffer, a protective agent, and a surfactant, which act as excipients, and having a pH of 5.4 to 6.

4.

2. In the liquid composition, the antibody comprises a light chain variable region and a heavy chain variable region; the light chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO:1, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:2, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:3; the heavy chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO:5, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:6, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:7; Preferably, the antibody comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:4 and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:8; 2. The liquid composition of claim 1, wherein the antibody is preferably present at a concentration of 100 to 200 mg / ml, more preferably 130 to 165 mg / ml, and even more preferably 150±5 mg / ml.

3. the buffer is one or more selected from the group consisting of an acetate buffer, a phosphate buffer, and an amino acid buffer; 3. The liquid composition of claim 1, wherein the buffering agent is present at a concentration of 5 to 50 mmol / L, preferably the buffering agent is an amino acid buffering agent at a concentration of 5 to 50 mmol / L.

4. the acetate buffer is a sodium acetate buffer, the phosphate buffer is a sodium dihydrogen phosphate buffer, or the amino acid buffer is a histidine hydrochloride buffer; Preferably, the buffer is present at a concentration of 5 to 20 mmol / L; 4. The liquid composition according to claim 3, wherein the buffer is preferably a histidine hydrochloride buffer at a concentration of 5 to 20 mmol / L, more preferably a histidine hydrochloride buffer at a concentration of 10 mmol / L.

5. the protective agent is one or more selected from the group consisting of a sugar, an alcohol, an amino acid, and a chloride salt; 5. The liquid composition of any one of claims 1 to 4, wherein the protective agent is present in a concentration of 40 to 220 mmol / L.

6. the protective agent is one or more selected from the group consisting of a sugar, an alcohol, and an amino acid; the protective agent is present at a concentration of 40 to 220 mmol / L, and / or the protective agent is a chloride salt and the protective agent is present at a concentration of 40 to 150 mmol / L; Preferably, the sugar is trehalose and / or sucrose at a concentration of 40 to 150 mmol / L, more preferably 60 to 150 mmol / L; the alcohol is mannitol at a concentration of 40 to 220 mmol / L, more preferably 110 to 150 mmol / L; the amino acid is one or more selected from the group consisting of proline, arginine hydrochloride, and glycine at a concentration of 40 to 220 mmol / L, more preferably 120 to 220 mmol / L; or the chloride salt is sodium chloride at a concentration of 40 to 150 mmol / L, more preferably 80 to 120 mmol / L; The liquid composition according to claim 5, wherein the protective agent is preferably a combination of trehalose and sodium chloride; more preferably, the protective agent is a combination of 40 to 150 mmol / L trehalose and 40 to 150 mmol / L sodium chloride; even more preferably, the protective agent in the liquid composition is a combination of 60 to 150 mmol / L trehalose and 80 to 120 mmol / L sodium chloride, and more preferably, a combination of 60 mmol / L trehalose and 100 mmol / L sodium chloride.

7. the surfactant is a non-ionic polymer, for example, one or more selected from the group consisting of Tween 80, Tween 20, poloxamer, and polyethylene glycol; the surfactant is present at a concentration of 0.01% to 0.2%; The liquid composition according to any one of claims 1 to 6, wherein the surfactant is preferably 0.01 to 0.03% Tween 80, more preferably 0.02% Tween 80.

8. a formulation for injection, preferably for subcutaneous or intravenous injection, Preferably, 130 to 165 mg / ml, preferably 150±5 mg / ml, of the antibody; 10 mmol / L histidine hydrochloride; 60mmol / L trehalose; 100mmol / L sodium chloride; 0.02% Tween 80 Including, 8. The liquid composition according to any one of claims 1 to 7, having a pH of 6.2±0.2, preferably 6.2±0.

05.

9. Use of the liquid composition according to any one of claims 1 to 8 for the manufacture of a medicament for the treatment of an inflammatory or allergic disease, comprising: Preferably, the inflammatory or allergic disease comprises an autoimmune disease, such as allergic dermatitis, asthma, eosinophilic esophagitis, eczema, allergic rhinitis, nasal polyps, rheumatoid arthritis, and the like.

10. A container containing the liquid composition according to any one of claims 1 to 8, or a kit containing said container.

11. A method for preventing, treating, or ameliorating an inflammatory or allergic disease, comprising: administering to a subject in need thereof a liquid composition according to any one of claims 1 to 8, Preferably, the subject is a mammal, more preferably a human; Preferably, the inflammatory or allergic disease comprises an autoimmune disease, such as allergic dermatitis, asthma, eosinophilic esophagitis, eczema, allergic rhinitis, nasal polyps, rheumatoid arthritis, etc.; Preferably, the liquid composition is administered to the subject by injection, for example, by subcutaneous or intravenous injection.

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