Liquid compositions containing antibodies against human interleukin-4 receptor alpha

Abstract

The present invention provides a liquid composition containing an antibody against human interleukin-4 receptor alpha. The liquid composition contains the antibody at a concentration of 50 to 200 mg / ml, and auxiliary agents such as a buffer, a protective agent, and a surfactant. The liquid composition has a pH of 5.4 to 6.4.

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] 2. 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. Thus, blocking antibodies targeting the protein are useful for treating and alleviating such diseases.

[0004] Currently, monoclonal antibody drugs targeting hIL-4R are in clinical trials, such as dupilumab, which has shown good efficacy in Phase II clinical trials for the treatment of atopic dermatitis. However, for antibody drugs, the best mode of administration is subcutaneous injection, and a relatively high dose is required to exert its effect, so it is generally necessary to prepare a highly concentrated antibody formulation. As 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 and inject with a syringe, may lead to large deviations in dosage due to high drug residues in the container or cartridge holding the formulation, may cause pain at the injection site, etc. In addition, highly viscous formulations may bring about serious process problems during production. 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. Or, the high concentration of antibodies in such formulations tends to aggregate and form insoluble particles, which leads to unstable formulations, increased immunogenicity, and more side effects of drug application, etc.

[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 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, as well as 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), 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 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. TIFF0007681315000001.tif106161TIFF0007681315000002.tif215162

[0012] Preferably, the antibody is present at a concentration of 100-200 mg / ml, more preferably 130-165 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 5-50 mmol / L; preferably, the buffer is an amino acid buffer at a concentration of 5-50 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 in a concentration of 5 to 20 mmol / L.

[0016] Preferably, the buffer is 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.

[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 40-220 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 40-220 mmol / L, and / or the protective agent is a chloride salt, and the protective agent is present at a concentration of 40-150 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 0.01-0.03% Tween80, more preferably 0.02% Tween80.

[0023] The pharmaceutical composition provided by the present invention is a colorless to pale yellow, transparent, sterile solution with slight opalescence. As can be detected, the liquid composition provided by the present invention has 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 formulation provided by the present invention is a colorless or light yellow, transparent, sterile solution, which has favorable long-term stability (it can be stored at 2-8°C for 2 years and meets quality standards) and is free of aggregates (≦10.0%). The formulation of the present invention has low viscosity (<30 cP) and is characterized by a pH and osmolality (290-310 mOsmol / kg) suitable for subcutaneous injection. Please refer to Table 14 below for details in this regard.

[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 a use of the liquid composition 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, etc.

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

[0031] The present invention provides a container comprising 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 invention provides a kit comprising a container provided by the invention; and further comprising instructions.

[0033] In yet another aspect, the present invention provides a method of preventing, treating, or ameliorating an inflammatory or allergic disorder, comprising administering to a subject in need thereof a liquid composition of the present invention. 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 disease.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 sequentially.

[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 the manufacture of medicines. The liquid composition provided by the present invention contains a high concentration of antibodies against human interleukin-4 receptor alpha, which can meet the demands of drug application and improve the therapeutic effect by providing a high dose of antibodies when administered subcutaneously or intravenously. On the one hand, even if a high concentration of antibodies is contained, the liquid composition does not show antibody aggregation, and on the other hand, it has a fairly low viscosity, which allows the composition to be easily delivered through fine needles and needle tubes, thereby minimizing the discomfort of patients. The liquid composition of the present invention also has the advantages of easy production and storage. In addition, 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 ≦60000 / vial, and the number of insoluble particles with a particle size of ≧25 μm was ≦600 / vial). The liquid composition also can 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 of blocking STAT-6 signal transduction.The results showed that the liquid composition provided by the present invention can stably and effectively bind with antigen IL-4Rα and effectively block STAT-6 signal transduction. [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, as well as a buffer, a protective agent, and a surfactant, which 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 a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:1, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:2, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:3; the heavy chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:5, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:6, and a 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; The liquid composition of any one of claims 1001 to 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 said 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 sugars, alcohols, amino acids, and chloride salts; 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 sugars, alcohols, and amino acids; the protective agent is present at a concentration of 40-220 mmol / L, and / or the protective agent is a chloride salt and the protective agent is present at a concentration of 40-150 mmol / L; Preferably, the sugar is trehalose and / or sucrose at a concentration of 40-150 mmol / L, more preferably 60-150 mmol / L; the alcohol is mannitol at a concentration of 40-220 mmol / L, more preferably 110-150 mmol / L; the amino acid is one or more selected from the group consisting of proline, arginine hydrochloride, and glycine at a concentration of 40-220 mmol / L, more preferably 120-220 mmol / L; or the chloride salt is sodium chloride at a concentration of 40-150 mmol / L, more preferably 80-120 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 40-150 mmol / L trehalose and 40-150 mmol / L sodium chloride; even more preferably, the protective substance 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. [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% Tween80 Including, The liquid composition according to 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 the present inventions 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 includes 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 description 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 of formulations tested during pH screening are shown. [Diagram 2] SEC purity results of formulations tested during pH screening are shown. [Diagram 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. [Diagram 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] Each shows the results of the CEX neutral peak change for the formulations tested during pH screening. [Figure 8]Each shows the results of the CEX neutral peak change for the formulations tested during pH screening. [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 protective agent screening. [Figure 16] Each shows the results of the CEX neutral peak change for the formulations tested during protective agent screening. [Figure 17] 1 shows a comparison of the viscosity of formulations tested during protective agent screening. [Figure 18] 4 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 PREFERRED EMBODIMENTS

[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 in no way limit the scope of the present invention, 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 with reference to the pH measurement method set out in General Rule 0631, Volume IV, Pharmacopoeia of the People's Republic of China (2015 edition). (2)Accelerated stability test at 40℃: 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 formulation. (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, where "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 5.0mg / 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 TIFF0007681315000003.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 is performed with reference to ion chromatography as described in General Rule 0514, Volume III, Pharmacopoeia of the People's Republic of China (2015 edition). The column used is BiomAb NP5, PK, 4.6 x 250 mm available from Agilent; the results of chromatographic peak integration are evaluated with reference to the reference, the highest peak is the main peak, the peak integrated earlier than the retention time of the main peak is defined as an acidic peak, and the peak integrated later than the retention time of the main peak is defined as an alkaline peak. (8)DSC detection: DSC thermal analysis, also called differential scanning calorimetry, is a technique for measuring the phase transition temperatures of a sample by recording the rate at which heat is absorbed or released by the sample using a differential scanning calorimeter, with heat flux dH / dt (millijoules / second) as the vertical axis and temperature T or time t as the horizontal axis, and then the stability of the sample can be determined. (9) Viscosity detection: The detection is carried out using a DV2T viscometer available from Brook Field, with reference to the viscosity determination method--the third method (rotational viscosity measurement method) described in General Rule 0633, Volume IV, Pharmacopoeia of the People's Republic of China (2015 edition). (10) Visible foreign object detection: The detection is carried out with reference to the methods for checking for 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 performed with reference 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). EXAMPLES

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

[0045] Table 1. pH and buffer screening solutions TIFF0007681315000004.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, with no visible foreign matter 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 2 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 1-2. After 2 weeks of acceleration, the absolute values ​​of the SEC purity of the test solutions were 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). In addition, the degree of decrease in the SEC purity of the test solutions (compared to that at 0 hours, respectively) 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 of nrCE-SDS are shown in Figures 3-4. After 2 weeks of accelerated storage, the absolute value of nrCE-SDS purity of the test solutions was 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 0 hours, respectively) 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 2 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 of rCE-SDS are shown in Figures 5-6. After 2 weeks of acceleration, the absolute values ​​of 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 the change in the CEX neutral peak are shown in Figures 7-8. After 2 weeks of acceleration, the ratio of the CEX neutral peak of the test solutions was 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 ratio of the CEX neutral peak of the test solutions after 2 weeks of accelerated storage (compared to that at 0 hours) 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 solutions with the His-HCl buffer system and the solutions with the phosphate buffer system, and the viscosities of all these solutions were lower than the solutions with the acetate buffer system. For the solutions with the His-HCl buffer system, the viscosity was in the range of 8.7±0.8 cP, which is lower than the expected 20 cP, indicating a lower viscosity for solutions with higher pH.

[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, and according to their stabilizing effect, the pH values ​​can be ranked as follows: pH6.4>pH6.2>pH6.0, i.e., proteins can be stabilized under acidic-based conditions of pH6.2±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 added with trehalose, sucrose, mannitol, proline, arginine hydrochloride, glycine, or sodium chloride were prepared, and the solution with added sodium chloride was used as a control. In particular, in an accelerated stability test at 40°C, the following protein solutions containing 133.6 mg / ml CBP-201 were tested to determine suitable protective agents (see Table 2):

[0058] (Table 2) Protective substances TIFF0007681315000005.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 2 weeks of acceleration at 40° C., the following results were obtained from protein solutions with different protective agents:

[0063] 1. The results of SEC purity are shown in Figures 9-10. After 2 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 a protective agent had the lowest SEC purity of 95.3% after 2 weeks, which was acceptable (Figure 9). Furthermore, the degree of decrease in SEC purity of the test solutions (compared to those at 0 hours, respectively) 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 a protective agent had the highest degree of decrease in SEC purity, i.e., 3.04%, but it still had an acceptable SEC purity of 95.4% after 2 weeks of accelerated storage at 40°C (Figure 10).

[0064] 2. The results of nrCE-SDS are shown in Figures 11-12. After 2 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 (compared to those at 0 hours, respectively) 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 2 weeks of acceleration at 40°C, the nrCE-SDS purity, which showed a small degree of decrease compared to those at 0 hours, respectively, and was maintained 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 13-14. After 2 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, the degree of decrease in the rCE-SDS purity of the test solutions compared to those at 0 hours, respectively, 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 2 weeks of acceleration at 40°C, the rCE-SDS purity, which showed a small degree of decrease compared to those at 0 hours, respectively, and was maintained at a relatively high level, was 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 2 weeks of acceleration, the ratio of the CEX neutral peak of the test solutions was 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 ratio of the CEX neutral peak of the test solutions after 2 weeks of accelerated storage (compared to that at 0 hours, respectively) 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 takes the first place in terms of maintaining a stable charge, Suc comes in second place, followed in sequence by Pro, Man, Arg, NaCl, and Gly.

[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 with buffer only (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 formulation. Formulations with the highest concentration were prepared and viscosities were measured to provide a basis for selection of protein concentration in the formulation. In this example, formulations were prepared without NaCl and compared to the NaCl-containing formulations of the above examples.

[0070] The protein was dialyzed with dialysis buffer (10 mmol / L His-HCl, pH 6.0), the dialyzed protein was 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, and then the formulation was filtered. The appearance and viscosity were detected. The results obtained are shown in Table 3 and Figure 18.

[0071] Table 3: Appearance and viscosity detection results of CBP-201 formulation TIFF0007681315000006.tif64170

[0072] The results showed that the viscosity of the protein solution increased with increasing protein concentration. The viscosity of the protein solution with the target concentration of 133.6mg / ml (actual concentration was 137.94mg / ml) was 50.33cP, and only the protein solution containing Suc in the screening for suitable protective agents in Example 2 had a viscosity greater than that value (68.75cP). 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, the protective agents that are preferentially selected for the 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 the CBP-201 formulation. The composition of the protein solution studied is shown in Table 4.

[0074] (Table 4) Combination of ingredients TIFF0007681315000007.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) Visual inspection results TIFF0007681315000008.tif94170

[0077] (Table 6) Protein concentration detection results TIFF0007681315000009.tif90146

[0078] The appearance and protein concentration results 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 1-9, and all of the protein solutions had a whitish appearance, which is associated with the high protein concentration of the solutions and the nature of the proteins themselves. In this regard, based on the appearance and protein concentration results, all of the combinations of ingredients numbered 1-9 were acceptable.

[0079] (Table 7) SEC purity TIFF0007681315000010.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 2 weeks of storage at 4° C. Furthermore, the highest decrease in SEC purity was observed for the protein solution having the component combination numbered 4, which was 1.44%, compared to the SEC purity at time 0, but 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 2 weeks of storage at 4° C.

[0081] After 4 weeks of storage at 40° C., the protein solutions with the combinations of components numbered 5 and 8 showed a significant decrease in SEC purity (compared to those at 0 hours, respectively), and therefore had the lowest SEC purity that was not acceptable. The SEC purity of the protein solutions with other different combinations of components after 4 weeks of storage at 40° C. allowed their SEC purity to be ordered and ranked as follows: protein solution with combination numbered 6>protein solution with combination numbered 9>protein solution with combination numbered 1>protein solution with combination numbered 3>protein solution with combination numbered 4>protein solution with combination numbered 2>protein solution with combination numbered 7. With regard to the degree of decrease in SEC purity, the protein solution with the combination of components numbered 6 showed a relatively small degree of decrease, while all the protein solutions with the other combinations of components showed a similar degree of decrease (maintained at about 1.5%), which was still acceptable.

[0082] (Table 8) CE-SDS purity TIFF0007681315000011.tif234166

[0083] The data in Table 8 showed that the lowest nrCE-SDS purity was 99.7% among the protein solutions having nine combinations of components each stored at 4°C for two weeks. In other words, all protein solutions having nine combinations of components maintained the proteins therein stable for two weeks at 4°C. Furthermore, the HC+LC(rCE-SDS) values ​​after two weeks of storage at 4°C were almost unchanged compared with those at 0 hours, i.e., all protein solutions having 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 the component combinations numbered 7 and 8 showed a significant decrease in nrCE-SDS purity (compared to those at time 0, respectively), thereby having unacceptably low nrCE-SDS purity. Furthermore, except for the protein solution having the component combination numbered 1, which had an nrCE-SDS purity of 92.4%, all other protein solutions had nrCE-SDS purity of over 94.0%, showing an acceptable decrease of about 5% (compared to those at time 0, respectively).

[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 greater decrease of about 3% (compared to that at 0 hours, respectively) than the protein solutions with the other combinations of components. The rCE-SDS purity of all protein solutions with combinations of components numbered 1 to 9 after 4 weeks of storage at 40°C was acceptable.

[0086] Table 9. Charge diversity TIFF0007681315000012.tif231158

[0087] The data in Table 9 showed that the protein solutions having the combinations of ingredients numbered 1 to 9 showed different degrees of decrease and increase (respectively compared to that at 0 hours) in the percentage of CEX neutral peak after 2 weeks of storage at 4° C. Among the protein solutions that showed decrease, the combination of ingredients numbered 1 showed the highest decrease of 1%, and the decrease in the percentage of CEX neutral peak was still 89.0%, which was acceptable. In contrast, even the protein solution having the combination of ingredients numbered 5 showed the lowest percentage of CEX neutral peak of 88.5% after 2 weeks of storage at 4° C., and the percentage of CEX neutral peak was increased compared to that at 0 hours, which was still acceptable.

[0088] The proportion of CEX neutral peaks in all nine protein solutions with the combination of components significantly decreased after 4 weeks of storage at 40° C., the proportions decreased were essentially equal, and many of them remained in the range of 70%±2%, and were ranked as follows: protein solution with the combination numbered 2>protein solution with the combination numbered 3=protein solution with the combination numbered 6>protein solution with the combination numbered 1=protein solution with the combination numbered 5>protein solution with the combination numbered 8>protein solution with the combination numbered 4>protein solution with the combination numbered 9. The greatest degree of decrease was observed for the protein solution with the combination of components numbered 9, where the percentage of decrease of the protein solution with the combination of components numbered 9 compared to the proportion of CEX neutral peaks after storage was 39.7%, and the corresponding percentages of the protein solutions with the other combinations of components remained in the range of 30%±5%.

[0089] (Table 10) DSC TIFF0007681315000013.tif80128

[0090] The data in Table 10 showed that, except for the protein solution having a combination of components 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) TIFF0007681315000014.tif80146

[0092] The data in Table 11 show the different degree of viscosity reduction of all protein solutions with combinations of ingredients numbered 1 to 9, and the viscosity of the solutions was ranked as follows: protein solution with combination numbered 1<protein solution with combination numbered 2<protein solution with combination numbered 8<protein solution with combination numbered 6<protein solution with combination numbered 5<protein solution with combination numbered 7<protein solution with combination numbered 4<numbered 9<protein solution with combination numbered 3. Furthermore, a comparison between protein solutions with combinations of ingredients numbered 3, 4, and 5 showed that Tween 80 can reduce the viscosity of the protein solution, but the degree of reduction is not linearly proportional to the concentration of Tween 80. For example, the protein solution containing 0.2% Tween 80 showed a viscosity about 0.94 cP (6.2%) lower than the 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 terms of 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 on reducing the viscosity of the protein solution than Tre.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 the viscosity of formulations Viscosity test of formulations (1): A protein solution was prepared 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, without trehalose, and then concentrated with an ultracentrifugal filter to three protein concentrations of 133.6 mg / ml, 151.4 mg / ml, and >151.4 mg / ml, respectively. The viscosity of the concentrated protein solutions was tested and then compared to that of the protein solutions containing Tre. The results are shown in Table 12.

[0094] (Table 12) Viscosity detection results TIFF0007681315000015.tif32164

[0095] The data in Table 12 show that the viscosity increases with increasing protein concentration, and care must be taken beforehand to determine the degree of overconcentration when the protein solution is overconcentrated.Comparing the protein solution with a concentration of 137.23 mg / ml, which had a viscosity of 19.71 cP (24.5°C), with the protein solution having the combination of components numbered 3 in Example 4 (which further contained Tre compared to the protein solution in this example, and had a viscosity of 25.64 cP (25.1°C)), it was shown that the viscosity of the protein solution with the 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 (1×) by tangential flow. SEC purity and viscosity were detected and the results are shown in Table 13.

[0097] (Table 13) Viscosity detection results TIFF0007681315000016.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 results obtained from the protein solution with the combination of components numbered 3 in Example 4 (which had a viscosity of 25.64 cP (25.1 ° C)), which indicates 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, the cell culture debris was removed by a filtration clarification process. A combination of MD0HC and MA1HC pod filters from Millipore Corporation was used in 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 completed, the inlet liquid was changed to cell culture and the intake pressure was set to 0-20 psi. After the supply was finished, the pod filter was washed with PBS and all the discharged liquid 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 theoretical plate number ≥ 2000 N / m. The column was equilibrated with 4–6 column volumes of equilibration buffer for affinity chromatography (0.025 mol / L Tris, 0.10 mol / L NaCl, pH 7.40 ± 0.20) and loaded onto it 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 rinsing solution 1 (0.025 mol / L Tris, 1.00 mol / L NaCl, pH 7.40 ± 0.20) and rinsing solution 2 (0.05 mol / L NaAc, pH 5.50 ± 0.10), respectively. After rinsing was completed, the proteins were eluted with elution solution for affinity chromatography (0.10 mol / L NaAc-HAc, pH 3.60 ± 0.10). Protein collection was started when the UV absorption value rose to 0.1000–0.1500 AU / 5 mm and stopped when the UV absorption value fell to 0.2000–0.3000 AU / 5 mm.

[0102] Viral inactivation at low pH An acid 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, and the resulting protein solution was placed at 20-25°C for 2-3 hours for virus inactivation at low pH. Then, a basic titrant (2.00 mol / L Tris) was used to adjust the pH of the protein solution to 5.40-5.60, and the conductivity of the protein solution was detected. By using a sterile filter, the protein solution was filtered into a disposable liquid container, and then sampled for protein concentration, pH, conductivity, bacterial endotoxin, SEC-HPLC, CEX-HPLC, CE-SDS, and microbial limit tests.

[0103] Cation exchange chromatography For cation exchange chromatography, Capto S ImpAct resin, column height of 18–22 cm, symmetry factor of 0.8–1.8, and theoretical plate number ≥ 2000 N / m were used. The column was equilibrated with 4–6 column volumes of equilibration buffer for cation exchange chromatography (0.05 mol / L NaAc, 0.05 mol / L NaCl, pH 5.50 ± 0.05), followed by loading the protein solution onto it. After loading, the column was further equilibrated with 2–4 column volumes of equilibration buffer for cation exchange chromatography. Then, the column was rinsed with 5–7 column volumes of rinsing solution for cation exchange chromatography (0.05 mol / L NaAc, 0.08 mol / L NaCl, pH 5.50 ± 0.05). Once rinsing was completed, the protein was eluted with elution solution for cation exchange chromatography (0.05 mol / L NaAc, 0.22 mol / L NaCl, pH 5.50 ± 0.05). Protein collection was started 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, column height of 18-22 cm, symmetry factor of 0.8-1.8, and theoretical plate number ≥ 2000 N / m were used. 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 2 The protein solution was then filtered into a disposable liquid container by using a filtration filter.

[0105] Nanofiltration The nanofiltration system was assembled and the prefiltration membrane was rinsed with equilibration buffer for anion exchange chromatography. Once the membrane was rinsed, the sterile filter was connected to the nanofiltration system and nanofiltration of proteins was performed at a pressure of 28-32 psi within 4 h.

[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. Then, if it passed the test, 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 as above was stirred to mix thoroughly, and then loaded onto the membrane and preconcentrated to 40-60 mg / ml. After preconcentration, dialysis was performed with 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 liquid obtained from the rinsing was collected. The volume of dialysis buffer used to rinse the membrane each time was about 1.5 times the system hold-up volume, and the total volume used to rinse the membrane was about 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 detected.

[0107] Preparation of stock solutions (dilution and excipient addition) The protein solution was diluted to 150±5 mg / ml with Tween80 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), and the final concentration of Tween80 in it was 0.02%. Then, the diluted protein solution was filtered into a disposable liquid container, and the obtained filtered protein solution was used as the stock solution. The prepared stock solution was divided into PETG bottles and stored at -80±10 °C.

[0108] Preparation of the formulation The stock solution was taken out, thawed at room temperature, mixed well, and then filtered. Rubber stoppers and aluminum covers were filled, sterilized, and transferred to the corresponding sites for filling into vials that were cleaned to remove pyrogens. Then, all stoppered filled vials were transferred to the capping room for capping. After that, visual inspection was performed on each vial to exclude those with defects such as inaccurate loading amounts, disintegration, visible particles, foreign matters, damaged caps, and empty vials. Then, the vials were labeled, packed in boxes, and then a carton label was pasted in the center of the upper surface of the box.

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

[0110] (Table 14) Acceptance criteria and detection results TIFF0007681315000017.tif242166TIFF0007681315000018.tif64166

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

[0112] The antigen (sIL-4Rα, expressed according to NM_000418.4) was coated and absorbed on a solid-phase plate for ELISA, then the plate was washed, blocked, and washed again before adding the sample to be tested for binding to the antigen. The sample to be tested was a dilution of the formulation prepared according to Example 6 of the present application, which 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 the incubation, the unbound antibody was removed by washing, and an enzyme-labeled secondary antibody was added for further incubation. After the unbound enzyme-labeled secondary antibody was removed by washing, an enzyme reaction substrate was added for color development. Finally, the reaction was stopped by adding a stop solution, and the absorbance value was read at 450 nm and 655 nm on a microplate reader. According to the absorbance value, 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 TIFF0007681315000019.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 384-well cell culture plates and the samples to be tested were added therein. The samples to be tested were dilutions of the formulations prepared according to Example 6 of the present application, which were obtained by diluting the formulations with DMEM containing 10% FBS to an antibody concentration of 5000ng / mL, followed by gradient dilution from an initial concentration of 5000ng / mL to a concentration of 0ng / mL. Then, IL4 (available from Invivogen) was added into the plate to obtain a final concentration of 0.5ng / mL. After incubation in a cell incubator for 22 hours, the supernatant was taken and Quanti-blue was added therein for color development, and then the absorbance value was read at 650nm on a microplate reader. According to the absorbance value, the half inhibitory concentration IC 50 was calculated by curve fitting.

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

[0118] Table 16: Biological activity detection results TIFF0007681315000020.tif74157

[0119] HEK Blue (商標) IL4 / IL13 cells were used to detect the activity of the prepared CBP-201 formulations. The average IC50 values ​​of the two batches of CBP-201 formulations were 4.193ng / ml and 4.513ng / 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 modifications and variations according to the present invention, which are within the scope of protection of the claims of the present invention without departing from the spirit of the claims of the present invention.

Claims

1. A liquid composition comprising an antibody against human interleukin-4 receptor alpha, comprising the antibody at a concentration of 100-165 mg / ml, and buffers, protective agents, and surfactants, which act as excipients, and having a pH of 6.2±0.2; the antibody comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:4, a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:8, 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; the buffer is a histidine hydrochloride buffer at a concentration of 5 to 10 mmol / L; The protective agent is a combination of 60 to 150 mmol / L trehalose and 80 to 120 mmol / L sodium chloride; and The surfactant is 0.01% to 0.2% (w / v) polysorbate 80. liquid composition.

2. 2. The liquid composition of claim 1, wherein the antibody is present in a concentration of 130 to 165 mg / ml.

3. The liquid composition of claim 1 , wherein the antibody is present in the liquid composition at a concentration of 150±5 mg / ml.

4. 2. The liquid composition of claim 1, wherein the buffer is a histidine hydrochloride buffer at a concentration of 10 mmol / L.

5. 2. The liquid composition of claim 1, wherein the protective agent is a combination of 60 mmol / L trehalose and 100 mmol / L sodium chloride.

6. 2. The liquid composition of claim 1, wherein the surfactant is 0.01 to 0.03% (w / v) polysorbate 80.

7. 2. The liquid composition of claim 1, wherein the surfactant is 0.02% (w / v) polysorbate 80.

8. 130-165 mg / ml of the antibody; 10 mmol / L histidine hydrochloride; 60 mmol / L trehalose; 100 mmol / L sodium chloride; and 0.02% (w / v) Polysorbate 80 2. The liquid composition of claim 1, comprising:

9. 9. The liquid composition of claim 8, wherein the antibody is present in a concentration of 150±5 mg / ml.

10. 10. The liquid composition of claim 9, having a pH of 6.2±0.

05.

11. The liquid composition according to any one of claims 1 to 7, which is a formulation for injection.

12. The liquid composition according to any one of claims 1 to 7, which is a formulation for subcutaneous injection.

13. A liquid composition according to any one of claims 1 to 7, which is a formulation for intravenous injection.

14. Use of a liquid composition according to any one of claims 1 to 13 for the manufacture of a medicament for the treatment of an inflammatory or allergic disease.

15. 15. The use of claim 14, wherein the inflammatory or allergic disease comprises an autoimmune disease.

16. 15. The use according to claim 14, wherein the inflammatory or allergic disease is allergic dermatitis, asthma, eosinophilic esophagitis, eczema, allergic rhinitis, nasal polyps, or rheumatoid arthritis.

17. A container comprising the liquid composition according to any one of claims 1 to 13.

18. 20. A kit comprising the container of claim 17.

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