Antibody formulations

Abstract

The invention relates to an antibody preparation, which comprises an antibody serving as an active ingredient, and also comprises a buffer system, a stabilizer and a surfactant.

Description

[0001] This application is a divisional application of the patent application with application number 2025101260911 and application date 2025-01-27, and the patent name is Antibody Preparation.

[0002] This application claims priority to PCT International Application (application number: PCT / CN2024 / 104177; filing date: July 8, 2024). TECHNICAL FIELD

[0003] The present application belongs to the field of immunology. Specifically relates to a preparation of a bispecific antibody. BACKGROUND

[0004] Hemophilia A is a genetic disease of coagulation dysfunction caused by mutations in the gene encoding coagulation factor. The main manifestation of patients is the risk of bleeding all the year round, and it is life-threatening in severe cases. The current treatment drugs are mainly coagulation factor VIII extracted from normal human blood and recombinant human coagulation factor VIII, but these drugs have defects such as the risk of virus introduction in the production process, the inconvenient frequent medication method every 1-7 days / time, and the susceptibility to inhibitors that affect efficacy. Emicizumab @ is a new drug for treating hemophilia A, and its active ingredient is Emicizumab with double target binding characteristics, which can bind to coagulation factor IX and coagulation factor X at the same time, thereby simulating coagulation factor VIII to restore the coagulation function of patients, and has no virus introduction risk, a convenient medication method of up to 4 weeks / time, and a very low inhibitor production rate. This drug brings good news to patients, especially those who have developed inhibitors.

[0005] Among the existing drug preparations containing Emicizumab as the active ingredient, antibody aggregates, low molecular weight degradation fragments, antibody charge variants, and insoluble particles are often produced, among which the antibody charge variants are mainly acidic peak regions with greatly reduced activity, which are more likely to occur under high temperature conditions that may be encountered in reality; In addition, the drug preparation also contains high concentrations of aspartic acid, which is neurotoxic, which affects the stability and safety of the drug preparation. SUMMARY

[0006] The antibody preparation developed by the present application contains the active ingredient Emicizumab and is used for treating diseases related to congenital FVIII secretion deficiency or dysfunction. In order to provide an antibody drug with stable activity and better safety, it is necessary to develop a preparation that is conducive to the stable storage of antibodies, so that the function and structure of the antibodies can be maintained for a long time, especially to reduce the formation of aggregates, fragments, and / or charge variants.

[0007] The antibody formulation of the present application can significantly inhibit the formation of charge variants (acid peak), dimers / multimers, degradants and insoluble particles during freeze / thaw cycles, long-term storage and temperature changes in order to maintain the stability of Amicarumab. Specifically, Amicarumab in the above formulation maintains stability after at least 3 freeze-thaw cycles, can be stably stored at 40°C for at least 2 weeks, at room temperature for at least 6 months, and at 4°C for at least 24 months. Therefore, the antibody formulation of the present application can be used to stably store Amicarumab for a long time and meet more stringent transportation and use conditions, which is of great significance for the treatment of related diseases caused by congenital FVIII secretion deficiency or dysfunction.

[0008] Specifically, the antibody formulation of the present application contains the following components: (1) 20-200 mg / mL of a bispecific antibody or an antigen-binding fragment thereof, wherein a first polypeptide and a third polypeptide form a pair, and a second polypeptide and a fourth polypeptide form a pair, wherein the first polypeptide comprises a heavy chain comprising HCDR1-3 as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, the second polypeptide comprises a heavy chain comprising HCDR1-3 as shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively, and the third polypeptide and the fourth polypeptide comprise a common light chain comprising LCDR1-3 as shown in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively, Preferably, wherein the first polypeptide comprises a H chain comprising a heavy chain variable region amino acid sequence as shown in SEQ ID NO: 13; the second polypeptide comprises a H chain comprising a heavy chain variable region amino acid sequence as shown in SEQ ID NO: 14; and the third polypeptide and the fourth polypeptide comprise a common light chain variable region amino acid sequence as shown in SEQ ID NO: 15, more preferably, wherein the first polypeptide comprises a H chain comprising an amino acid sequence of SEQ ID NO: 10; the second polypeptide comprises a H chain comprising an amino acid sequence of SEQ ID NO: 11; and the third polypeptide and the fourth polypeptide comprise a common L chain of SEQ ID NO: 12; (2) a buffer at a concentration of 5-40 mM, selected from histidine-acetic acid buffer or histidine-aspartic acid buffer; (3) a stabilizer, said stabilizer being 20-90 mM arginine and one or more of the following selected from the group consisting of mannitol, sucrose, trehalose and sorbitol or amino acids (such as glycine, proline) for adjusting the osmotic pressure, stabilizing the antibody and / or inhibiting aggregation of the antibody. Arginine which stabilizes is sometimes also denoted arginine-aspartate, wherein the aspartate is mainly used for neutralizing the basic amino acid. The conventional osmotic pressure of antibody formulations is known in the art to be 200-600 mOsm / kg. The concentration of said mannitol, sucrose, trehalose, sorbitol or amino acids can be 25-200 mM, such as 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM or 200 mM; Preferably, the concentration of arginine or arginine-aspartate is 20-90 mM, more preferably 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, or 90 mM; and (4) a surfactant, said surfactant being one or more of polysorbate 20, polysorbate 80, poloxamer 188, hydroxypropyl beta cyclodextrin, preferably said surfactant is selected from poloxamer 188; further preferably said surfactant is selected from 0.2-2.0 mg / mL poloxamer 188, more preferably 0.5 mg / mL poloxamer 188; or The bispecific antibody formulation comprises: (1) 20-200 mg / mL of bispecific antibody (preferably 20-180 mg / mL, such as 20-150 mg / mL, 120-180 mg / mL, 35-150 mg / mL or 150 mg / mL of bispecific antibody) comprising a first polypeptide, a second polypeptide, a third polypeptide and a fourth polypeptide, wherein the first polypeptide and the third polypeptide form a pair, and the second polypeptide and the fourth polypeptide form a pair, the first polypeptide comprises a heavy chain comprising HCDR1-3 as set forth in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, the second polypeptide comprises a heavy chain comprising HCDR1-3 as set forth in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively, and the third polypeptide and the fourth polypeptide comprise a common light chain comprising LCDR1-3 as set forth in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively; (2) histidine-acetic acid buffer at a concentration of 5-40 mM; (3) a stabilizer, when the buffer is histidine-acetic acid buffer, the stabilizer is >90-300 mM arginine; and (4) a surfactant, the surfactant is selected from one or more of polysorbate 20, polysorbate 80, poloxamer 188, hydroxypropyl β-cyclodextrin.

[0009] The pH of each of the above-mentioned bispecific antibody formulations is 5.5-6.5.

[0010] In preferred embodiments, the bispecific antibody formulation is selected from the group consisting of: (1) comprising 150 mg / mL of bispecific antibody, 20 mM of histidine-acetic acid buffer, 150 mM of arginine and 0.5 mg / mL of poloxamer 188, pH 6.0; (2) comprising 150 mg / mL of bispecific antibody, 20 mM of histidine-acetic acid buffer, 80 mM of arginine, 0.5 mg / mL of poloxamer 188 and 140 mM of reagent A, the reagent A being selected from sucrose, trehalose, sorbitol, mannitol, proline or glycine, pH 6.0; (3) comprising 150 mg / mL of bispecific antibody, 20 mM of histidine-aspartic acid buffer, 0.5 mg / mL of poloxamer 188, 80 mM of arginine-aspartic acid, 140 mM of reagent A, the reagent A being selected from sucrose, trehalose, sorbitol, mannitol, proline or glycine, pH 6.0; (4) comprises 150 mg / mL of bispecific antibody, 20 mM histidine-acetate buffer, 80 mM arginine, 0.5 mg / mL poloxamer 188 and 80-200 mM (e.g., 80 mM, 90 mM, 100 mM, 140 mM or 200 mM) of mannitol, pH 6.0; (5) comprises 150 mg / mL of bispecific antibody, 20 mM histidine-acetate buffer, 0.5 mg / mL poloxamer 188 and 90 mM mannitol and 20-90 mM (e.g., 20 mM, 50 mM, 80 mM or 90 mM) of arginine, pH 6.0; (6) comprises 150 mg / mL of bispecific antibody, 80 mM arginine, 90 mM mannitol, 0.5 mg / mL poloxamer 188 and 5-40 mM (e.g., 5 mM, 10 mM, 20 mM or 40 mM) of histidine-acetate buffer, pH 6.0; and (7) comprises 150 mg / mL of bispecific antibody, 20 mM histidine-acetate buffer, 80 mM arginine, 90 mM mannitol, 0.5 mg / mL poloxamer 188, pH 5.5-6.5 (e.g., pH 5.5, pH 5.7, pH 6.0, pH 6.3, or pH 6.5).

[0011] It should be understood that, within the scope of the present application, each of the aforementioned technical features of the present application and each of the technical features specifically described hereinafter (e.g., in the examples) can be combined with each other to form a new or preferred technical solution. Due to the limited space, they are not listed one by one here.

[0012] The terms involved in the present application have the conventional meanings understood by those skilled in the art. When a term has two or more definitions within the art of use and / or acceptance, the definition of the term used herein is used to include all the meanings.

[0013] In the present application, the bispecific antibody is emicizumab. The present application develops an antibody formulation formula by screening the buffer and stabilizer, which can enhance the stability of emicizumab formulation, prevent the generation and / or increase of antibody aggregation, degradation, charge variants (acidic charge isoforms or charge isoform acidic peaks) and insoluble particles. For example, by reducing the content of arginine, the proportion of acidic charge isoforms can be reduced, or by reducing the content of arginine to reduce the content of aspartic acid in the formulation, thereby reducing the neuron toxicity caused by excessive aspartic acid content, or by selecting a specific buffer, histidine-acetic acid buffer, so that the bispecific antibody formulation does not contain aspartic acid. In a preferred embodiment of the present application, the formulation of the present application selects acetic acid as the counterion of the buffer (buffering agent), i.e. selects histidine-acetic acid buffer, which also shows better advantages in inhibiting the increase of aggregates, the generation of small molecule fragments and the formation of acidic peaks.

[0014] "Stability", "stable" herein refers to that the antibody (including its antigen binding fragment) in the liquid formulation containing the antibody (including its antigen binding fragment) does not occur or only occurs very little aggregation, degradation or fragmentation under given production, preparation, transportation and / or storage conditions. The "stable" formulation maintains biological activity under given production, preparation, transportation and / or storage conditions. The degree of aggregation, degradation or fragmentation of the formulation can be measured by techniques such as SEC-HPLC, CEX-HPLC, CE-SDS, etc., so as to evaluate the stability of the antibody (including its antigen binding fragment).

[0015] It should be noted that in the present application, if it refers to the formulation containing a buffer or buffer system, it also means that the formulation contains a buffering agent, and the buffering system in the formulation is formed by the buffering agent.

[0016] As used herein, aspects referred to by the expression "comprising" include those referred to by the expressions "consisting essentially of and "consisting of.

[0017] The numerical values described herein can vary within a range, for example, depending on the instrument or equipment, the measurement conditions and the procedures used by the person skilled in the art, and as long as they are within the range that allows the purpose of the present application to be achieved, for example, they can include a deviation of about 10%.

[0018] The emicizumab herein includes four polypeptides, wherein a first polypeptide and a third polypeptide form a pair, and a second polypeptide and a fourth polypeptide form a pair, wherein the first polypeptide comprises a H chain comprising the amino acid sequence of SEQ ID NO: 10; the second polypeptide comprises a H chain comprising the amino acid sequence of SEQ ID NO: 11; and the third polypeptide and the fourth polypeptide comprise a common L chain of SEQ ID NO: 12.

[0019] In one embodiment of the present application, the concentration of the emicizumab in the antibody formulation is about 20-200 mg / mL; as a preferred embodiment, the concentration of the emicizumab in the antibody formulation is 20-180 mg / mL, such as 20-150 mg / mL, 120-180 mg / mL, 35-150 mg / mL or 150 mg / mL.

[0020] In some embodiments of the present application, the effects of different buffer systems, such as phosphate buffer, histidine salt buffer, citrate buffer, succinate, histidine-glutamate, histidine + acetic acid buffer, histidine-hydrochloric acid buffer, histidine-aspartic acid buffer, on the stability of the antibody are evaluated. Among the above-mentioned buffers, the histidine-acetic acid buffer, histidine-glutamate buffer, histidine-hydrochloric acid buffer, or histidine-aspartic acid buffer is preferred, and the histidine-aspartic acid buffer and the histidine-acetic acid buffer are more preferred. In other embodiments, the effects of adding appropriate stabilizers, such as sucrose, trehalose, sorbitol, mannitol, amino acids (such as arginine, proline, glycine), sodium chloride, on the stability of the antibody in the buffer are evaluated. The formulation of arginine, arginine combined with sugars, alcohols, amino acids, etc. is more advantageous for reducing the aggregation and charge heterogeneity of the antibody. In other embodiments, the changes of adding appropriate surfactants, such as polysorbate 20, polysorbate 80 and poloxamer 188, in the formulation containing appropriate buffer and stabilizer under high temperature conditions are evaluated, and poloxamer 188 is more stable and can reduce the generation of insoluble particles in the formulation during storage.

[0021] In a preferred embodiment of the present application, the concentration of aspartic acid is reduced or the aspartic acid is substantially not contained as a counter ion of the buffer (buffering agent) in the formulation of the present application. "Substantially not containing aspartic acid" means that the concentration of aspartic acid is, for example, 5 mM or less, preferably 2 mM or less, and more preferably 1 mM or less. Preferably, the buffer system is a 10-40 mM histidine-acetic acid buffer. @@ The addition of about 175 mM aspartic acid in the formulation mainly as a pH adjuster can cause necrosis of hypothalamic neurons in newborn mice and toxicity causing headache in humans, which has been reported in the literature (Berg, M. J., et al. (2010) Toxicol Sci 114: 45-56). Toxicology, 29(1983), pp. 109-119; NEUROLOGY 1990, 40 1582-1586 Several commercially available products using high doses of aspartic acid have shown headaches as an adverse reaction. In the antibody formulation of this invention, aspartic acid, as an anti-arginine ion, is used as a pH adjuster, and its dosage is essentially the same as that of arginine. For example, the antibody formulation provided by this invention reduces the concentration of aspartic acid by lowering the concentration of arginine; more preferably, an aspartic acid-free buffer solution is selected, thereby reducing the risk of neurotoxicity.

[0022] Therefore, another aspect of the present invention provides a method for reducing the neurotoxicity of a bispecific antibody preparation, comprising reducing the aspartic acid content in the bispecific antibody preparation or making the bispecific antibody aspartic acid-free, while adding 20-200 mM of one or more of the following: mannitol, sucrose, trehalose, sorbitol, and amino acids (such as proline and glycine) to the bispecific antibody preparation, wherein the bispecific antibody is as defined above. Preferably, the bispecific antibody preparation further comprises a buffer solution as defined above, and more preferably, the bispecific antibody preparation further comprises a surfactant, preferably a surfactant as defined above.

[0023] Another aspect of the present invention provides a method for preparing the bispecific antibody formulation, comprising adding a buffer, a surfactant, and a stabilizer as defined above to a solution containing the bispecific antibody.

[0024] The pH of the solution of the formulation of the present invention is preferably 5.5 to 6.5, more preferably 6.

[0025] If desired, the formulations of the present invention may additionally contain suitable cryoprotectants, suspending agents, solubilizers, isotonic agents, preservatives, adsorption inhibitors, diluents, excipients, pH adjusters, analgesics, sulfur-containing reducing agents, antioxidants, etc. These are conventional choices in the art. Detailed Implementation

[0026] The embodiments of the present invention will be described in detail below with reference to examples. Those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the examples, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Where the manufacturers of the reagents or instruments used are not specified, they are conventional products that can be purchased from the market.

[0027] Example 1 Screening of pH / buffer system for emecizumab formulation I 1. Experimental objective: To study antibody stability within the pH range of 5.0-7.0 and to select a pH value that reduces the formation of soluble aggregates.

[0028] 2. Materials and Methods: Liquid compositions containing emecizumab were prepared, comprising 150 mg / mL emecizumab, 150 mM NaCl, and 20 mM histidine / histidine hydrochloride buffer (His / His-HCl), 20 mM citrate / sodium citrate buffer (Cit / Cit-Na), and 20 mM disodium hydrogen phosphate / sodium dihydrogen phosphate buffer (PB) at different pH values. The trends of aggregate (SEC-HPLC) and small molecule fragment (rCE-SDS) changes were investigated after 28 days of storage at 40 °C for various formulations.

[0029] 3. Detection method for emecizumab multimers The polymers in the sample were detected by size exclusion chromatography (SEC-HPLC) using a G3000 SWXL (Tosoh) column. The mobile phase was citrate buffer (50 mmol / L, pH 6.0) containing 100 mmol / L sodium chloride, and the flow rate was 0.5 mL / min.

[0030] Among the detected peaks, the peak with the largest area and height is identified as a monomer. Peaks detected earlier than monomers are collectively referred to as polymers or high molecular weight species (HMWS). The percentage of peak area for polymers and monomers is recorded.

[0031] 4. Detection method for non-reducing purity of CE-SDS Sample purity was determined by capillary electrophoresis (CE-SDS) using an uncoated capillary column (total length 30.2 cm, effective length 20 cm, inner diameter 50 μm, outer diameter 375 μm). The filling solution was SDS-MW Gel Buffer (pH 8, 0.2% SDS), and the buffer solution was SDS-MW Sample Buffer (0.05 mol / L Tris-HCl, 1% SDS, pH 7.5). Separation and detection were performed using capillary electrophoresis (capillary temperature 25℃, sample chamber temperature 8℃, detection wavelength 214 nm).

[0032] Record the retention time of each peak and the area ratio of the correction peak for the sample, and report the percentage of the main peak area and the percentage of the other peak areas.

[0033] 5. Experimental Results After being stored at 40°C for 4 weeks, the increases in aggregate content and small fragments in both the citrate / sodium citrate buffer (pH 6.0) and the histidine / histidine hydrochloride buffer (pH 6.0) were relatively small, demonstrating good physical and chemical stability. The higher the pH in the same buffer, the more pronounced the increase in small fragments.

[0034]

[0035] Example 2 Amiselimab Formulation Prescription pH / Buffer System Screening II 1. Purpose of the experiment: Based on the results of Amiselimab Formulation Prescription pH / Buffer System Screening I, the effects of different buffer salts on the stability of Amiselimab were evaluated.

[0036] 2. Materials and methods: The pH range of the buffer system for stability testing was 5.8-6.2, including 20mM citric acid-sodium citrate buffer, 20mM succinic acid-sodium succinate buffer (SA / SA-Na), 20mM histidine-acetic acid buffer (His / Ace), 20mM histidine-glutamic acid buffer (His / Glu), 20mM histidine-hydrochloric acid buffer (His / HCl), and 20mM histidine-aspartic acid buffer (His / Asp). In the buffer salt system, 150mg / mL of Amiselimab antibody, 150mM of arginine and 0.5mg / mL of poloxamer 188 were contained.

[0037] The trends of aggregate and small molecule fragment changes of various formulations were studied under the condition of high temperature 40℃ for 28 days.

[0038] 3. Detection method of CE-SDS reduced purity The purity of the sample was detected by capillary electrophoresis (CE-SDS) using a non-coated capillary column (total length 30.2 cm, effective length 20 cm, inner diameter 50 microns, outer diameter 375 microns), with SDS-MW Gel Buffer (pH 8, 0.2% SDS) as the filling fluid and SDS-MW Sample buffer (0.05 mol / L Tris-HCl, 1% SDS, pH 7.5) as the buffer. Capillary electrophoresis was used for separation and detection (capillary temperature 25℃, sample chamber temperature 8℃, detection wavelength 214nm).

[0039] The retention time and corrected peak area ratio of each peak of the sample were recorded, and the purity (sum of LC and HC peak area percentage) and other peak peak area percentage were reported.

[0040] 4. Experimental results After 28 days at 40℃, 20mM histidine-hydrochloride, 20mM histidine-glutamate, 20mM histidine-acetate and 20mM histidine-aspartate had good protective effect on inhibiting the increase of aggregates and the production of small molecule fragments, among which the protective effect of histidine-acetate was outstanding.

[0041]

[0042] Screening of stabilizers for Example 3 1. Purpose of the experiment: To screen stabilizers that can stabilize the protein.

[0043] 2. Materials and Methods: Prepare a mixed solution containing 150 mg / mL of Emapalumab, pH 6.0, containing 20 mM Histidine-Aspartate buffer or 20 mM Histidine-Acetic acid buffer, 0.5 mg / mL Poloxamer 188 and different stabilizers, including 150 mM sodium chloride, 150 mM arginine (or arginine-aspartate) or 80 mM arginine (or arginine-aspartate), and a combination of Reagent A, which is 140 mM sucrose, 140 mM trehalose, 140 mM sorbitol, 140 mM mannitol and 140 mM proline or 140 mM glycine. Study the trend of aggregate (SEC-HPLC) and charge variant (acidic peak charge isoform) changes of various formulations under the condition of high temperature 40°C for 28 days.

[0044] 3. Method for measuring and calculating the charge variants of Emapalumab The charge variants in the sample were detected by ion exchange chromatography (IEX-HPLC) using a strong cation exchange column (MabPac SCX-10, 5 μm, 4 x 150 mm, Thermo Scientific) with 20 mmol / L methanesulfonic acid (pH 6.0) and 20 mM methanesulfonic acid + 500 mmol / L sodium chloride (pH 6.0) as the mobile phase. Separation was achieved by gradient elution using liquid chromatography (detection wavelength: 280 nm, flow rate: 0.5 mL / min, column temperature: 40°C).

[0045] Among the detected peaks, the peak with the largest area and height was determined as the main peak, and the peaks detected earlier than the main peak were collectively referred to as acidic peaks (Acidic Peaks), and the peaks detected later than the main peak were collectively referred to as basic peaks (Basic Peaks). Record the results of the acidic peaks, the main peaks and the basic peaks.

[0046] 4. Experimental results The results obtained are shown in the following table. The antibody containing 150 mM arginine, or containing 80 mM arginine (or arginine-aspartate) in combination with sugars, alcohols or amino acids showed better advantages in reducing protein aggregation and reducing the formation of acidic peaks, especially the combination of arginine and mannitol showed lower aggregates and acidic peaks.

[0047]

[0048] Example 4 Concentration of mannitol on the inhibitory effect of aggregates and acidic peaks 1. Experimental Purpose: Based on the results of stabilizer screening, arginine and mannitol combination was selected as stabilizer, and the effect of mannitol concentration on the aggregation of Emapalumab and acidic peak was investigated.

[0049] 2. Materials and Methods: Liquid compositions containing 150 mg / mL of Emapalumab, 20 mM histidine-acetate buffer, 80 mM arginine and 80-200 mM mannitol and 0.5 mg / mL poloxamer 188, pH 6.0 were prepared. The change trend of aggregation (SEC-HPLC) and acidic peak of charge isoforms of various formulations was studied under the condition of high temperature 40℃ for 28 days.

[0050] 3. Experimental Results Different mannitol concentrations effectively inhibited the change of aggregation and acidic peak.

[0051]

[0052] Example 5 Arginine concentration on the inhibition effect of aggregation and acidic peak 1. Experimental Purpose: Based on the results of stabilizer screening, arginine and mannitol combination was selected as stabilizer, and the effect of arginine concentration on the aggregation of Emapalumab and acidic peak was investigated.

[0053] 2. Materials and Methods: Liquid compositions containing 150 mg / mL of Emapalumab, 20 mM histidine-acetate buffer, 90 mM mannitol, 20-90 mM arginine and 0.5 mg / mL poloxamer 188 were prepared. The change trend of aggregation (SEC-HPLC) and acidic peak of charge isoforms of various formulations was studied under the condition of high temperature 40℃ for 28 days.

[0054] 3. Experimental Results Formulations containing 20-90 mM arginine in combination with mannitol showed comparable acidic peak inhibition and better aggregation inhibition effect than formulations containing single 150 mM arginine.

[0055]

[0056] Example 6 Histidine concentration on the inhibition effect of aggregation and acidic peak 1. Experimental Purpose: Based on the results of stabilizer screening, arginine and mannitol combination was selected as stabilizer, and the effect of histidine concentration on the aggregation of Emapalumab and acidic peak was investigated.

[0057] 2. Materials and Methods: Liquid compositions containing 150 mg / mL of emicizumab, 5-40 mM histidine-acetic acid buffer, 90 mM mannitol, 80 mM arginine, and 0.5 mg / mL of poloxamer 188 were prepared. The change trends of aggregates (SEC-HPLC) and acidic peak of charge isoforms were investigated for various formulations under the condition of high temperature 40°C for 28 days.

[0058] 3. Experimental Results The buffer containing 5 mM or more of histidine-acetic acid showed a better effect of inhibiting aggregates.

[0059]

[0060] Example 7 Effect of pH on Inhibition of Aggregates and Acidic Peak 1. Purpose of Experiment: Effect of pH on emicizumab aggregates and acidic peak during high temperature storage.

[0061] 2. Materials and Methods: Liquid compositions containing 150 mg / mL of emicizumab antibody, 20 mM histidine-acetic acid buffer, 90 mM mannitol, 80 mM arginine, and 0.5 mg / mL of poloxamer 188 were prepared at pH 5.0, 5.5, 5.7, 6.0, 6.3, and 6.5, respectively. The change trends of aggregates (SEC-HPLC) and acidic peak of charge isoforms were investigated for various formulations under the condition of high temperature 40°C for 28 days.

[0062] 3. Experimental Results The samples showed a better effect of inhibiting aggregates and acidic peak at pH 5.5-6.5.

[0063]

[0064] Example 8 Screening of Surfactant Type and Concentration 1. Purpose of Experiment: Screening of surfactant type and concentration capable of stabilizing the antibody.

[0065] 2. Materials and Methods: Liquid compositions containing 150 mg / mL of emicizumab, 20 mM histidine-acetic acid buffer, 80 mM arginine, 90 mM mannitol, and different surfactants were prepared, and the surfactants included 0.5 mg / mL of polysorbate 20, 0.5 mg / mL of polysorbate 80, 0.5 mg / mL of poloxamer 188, and hydroxypropyl beta cyclodextrin. The change trends of aggregates, surfactant content, and visible particles were investigated for various formulations under the condition of high temperature 40°C for 28 days.

[0066] 3. Method for measuring and calculating the content of polysorbate 20, polysorbate 80 and poloxamer 188 in an ameisabum antibody sample The content of surfactants in the sample was detected by high performance liquid chromatography with evaporative light scattering detector (ELSD-HPLC) using Waters OASIS MAX column (30 μm, 2.1 x 20 mm) with 2% formic acid in water and 2% formic acid in isopropanol as mobile phase at a flow rate of 1 mL / min. The content of poloxamer 188, polysorbate 20 and polysorbate 80 in the sample was quantitatively detected by external standard method.

[0067] 4. Experimental results After 28 days at high temperature 40°C, no obvious increase in aggregates was observed, but the content of polysorbate 20 and polysorbate 80 decreased, indicating that polysorbate 20 and polysorbate 80 were unstable and degraded in the sample, while poloxamer 188 showed better stability. No obvious visible particles were generated in the samples containing surfactants.

[0068]

[0069] N / A: not detected.

[0070] Example 9 Effect of antibody concentration on the formulation 1. Purpose of the experiment: to explore the effect of different antibody concentrations on the stability of the formulation.

[0071] 2. Materials and methods: mixed solutions containing 20-200 mg / mL ameisabum were prepared, which contained 20 mM histidine-acetic acid buffer, 0.5 mg / mL poloxamer 188, 80 mM arginine and 90 mM mannitol combination. The trend of aggregate (SEC-HPLC) and acidic peak change of charge isoforms of various formulations was studied at high temperature 40°C for 28 days.

[0072] The method for measuring and calculating the charge variants of ameisabum is described in Example 4.

[0073] 3. Experimental results The results showed that when the antibody concentration reached 200 mg / mL, the formulation containing 20 mM histidine-acetic acid buffer, 80 mM arginine and 90 mM mannitol combination could well inhibit the formation of aggregates and acidic peaks.

[0074]

[0075] All documents referred to in this disclosure are incorporated herein by reference as if each were individually incorporated by reference. In addition, it is to be understood that various alterations and modifications can be made to the present application upon reading and understanding the above lecture of the present application. Such equivalent forms are intended to fall within the scope of the appended claims.

[0076] SEQUENCE LISTING SEQ ID NO: 1, amino acid sequence of bispecific antibody heavy chain CDR1 YYDIQ SEQ ID NO: 2, amino acid sequence of bispecific antibody heavy chain CDR2 SISPSGQSTYYRREVKG SEQ ID NO: 3, amino acid sequence of bispecific antibody heavy chain CDR3 RTGREYGGGWYFDY SEQ ID NO: 4, amino acid sequence of bispecific antibody heavy chain CDR1 DNNMD SEQ ID NO: 5, amino acid sequence of bispecific antibody heavy chain CDR2 DINTRSGGSIYNEEFQD SEQ ID NO: 6, amino acid sequence of bispecific antibody heavy chain CDR3 RKSYGYYLDE SEQ ID NO: 7, amino acid sequence of bispecific antibody light chain CDR1 KASRNIERQLA SEQ ID NO: 8, amino acid sequence of bispecific antibody light chain CDR2 QASRKES SEQ ID NO: 9, amino acid sequence of bispecific antibody light chain CDR3 QQYSDPPLT SEQ ID NO: 10, amino acid sequence of bispecific antibody heavy chain QVQLVESGGGLVQPGGSLRLSCAASGFTFSYYDIQWVRQAPGKGLEWVSSISPSGQSTYYRREVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRTGREYGGGWYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNRYTQKSLSLSP SEQ ID NO: 11, amino acid sequence of the heavy chain of the bispecific antibody QVQLVQSGSELKKPGASVKVSCKASGYTFTDNNMDWVRQAPGQGLEWMGDINTRSGGSIYNEEFQDRVIMTVDKSTDTAYMELSSLRSEDTATYHCARRKSYGYYLDEWGEGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQESLSLSP SEQ ID NO: 12, amino acid sequence of the light chain of the bispecific antibody DIQMTQSPSSLSASVGDRVTITCKASRNIERQLAWYQQKPGQAPELLIYQASRKESGVPDRFSGSRYGTDFTLTISSLQPEDIATYYCQQYSDPPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 13, amino acid sequence of a heavy chain variable region of a bispecific antibody QVQLVESGGGLVQPGGSLRLSCAASGFTFSYYDIQWVRQAPGKGLEWVSSISPSGQSTYYRREVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRTGREYGGGWYFDYWGQGTLVTVSS SEQ ID NO: 14, amino acid sequence of a heavy chain variable region of a bispecific antibody QVQLVQSGSELKKPGASVKVSCKASGYTFTDNNMDWVRQAPGQGLEWMGDINTRSGGSIYNEEFQDRVIMTVDKSTDTAYMELSSLRSEDTATYHCARRKSYGYYLDEWGEGTLVTVSS SEQ ID NO: 15, amino acid sequence of a light chain variable region of a bispecific antibody DIQMTQSPSSLSASVGDRVTITCKASRNIERQLAWYQQKPGQAPELLIYQASRKESGVPDRFSGSRYGTDFTLTISSLQPEDIATYYCQQYSDPPLTFGGGTKVEIK

Claims

1. A bispecific antibody formulation comprising: (1) 20-200 mg / mL of a bispecific antibody (preferably 20-180 mg / mL, such as 20-150 mg / mL, 120-180 mg / mL, 35-150 mg / mL or 150 mg / mL of a bispecific antibody) comprising a first polypeptide, a second polypeptide, a third polypeptide and a fourth polypeptide, wherein the first polypeptide and the third polypeptide form a pair, and the second polypeptide and the fourth polypeptide form a pair, the first polypeptide comprising a heavy chain comprising HCDR1-3 as set forth in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, the second polypeptide comprising a heavy chain comprising HCDR1-3 as set forth in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively, the third polypeptide and the fourth polypeptide comprising a common light chain comprising LCDR1-3 as set forth in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively; (2) a buffer at a concentration of 5-40 mM selected from the group consisting of histidine-acetic acid buffer or histidine-aspartic acid buffer; (3) a stabilizer, the stabilizer being 20-90 mM arginine and one or more selected from the group consisting of mannitol, sucrose, trehalose, sorbitol or amino acids (glycine, proline); and (4) a surfactant, the surfactant being one or more selected from the group consisting of polysorbate 20, polysorbate 80, poloxamer 188, hydroxypropyl β-cyclodextrin; the bispecific antibody formulation having a pH of 5.5 to 6.5; or, (1) 20-200 mg / mL of a bispecific antibody (preferably 20-180 mg / mL, such as 20-150 mg / mL, 120-180 mg / mL, 35-150 mg / mL or 150 mg / mL of a bispecific antibody) comprising a first polypeptide, a second polypeptide, a third polypeptide and a fourth polypeptide, wherein the first polypeptide and the third polypeptide form a pair, and the second polypeptide and the fourth polypeptide form a pair, the first polypeptide comprising a heavy chain comprising HCDR1-3 as set forth in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, the second polypeptide comprising a heavy chain comprising HCDR1-3 as set forth in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively, the third polypeptide and the fourth polypeptide comprising a common light chain comprising LCDR1-3 as set forth in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively; (2) a histidine-acetic acid buffer at a concentration of 5-40 mM; (3) a stabilizer, the stabilizer being 90 mM-300 mM arginine; and (4) a surfactant, the surfactant being one or more selected from the group consisting of polysorbate 20, polysorbate 80, poloxamer 188, hydroxypropyl β-cyclodextrin. ​ (4) a surfactant selected from one or more of polysorbate 20, polysorbate 80, poloxamer 188, hydroxypropyl beta cyclodextrin; the pH of the bispecific antibody formulation is 5.5 to 6.

5.

2. The bispecific antibody formulation of claim 1, wherein the first polypeptide comprises a H chain comprising the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 13; the second polypeptide comprises a H chain comprising the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 14; and the third and fourth polypeptides comprise the common light chain variable region amino acid sequence set forth in SEQ ID NO: 15, preferably wherein the first polypeptide comprises a H chain comprising the amino acid sequence set forth in SEQ ID NO: 10; the second polypeptide comprises a H chain comprising the amino acid sequence set forth in SEQ ID NO: 11; and the third and fourth polypeptides comprise the common L chain set forth in SEQ ID NO: 12, preferably the pH of the bispecific antibody formulation is 5.5 to 6.5, even more preferably 6.

3. The bispecific antibody formulation of any one of claims 1-2, wherein the concentration of arginine or arginine-aspartate is 20-90 mM, more preferably 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 Mm, 85 mM, or 90 mM.

4. The bispecific antibody formulation of any one of claims 1-3, wherein the concentration of mannitol, sucrose, trehalose, sorbitol, glycine, or proline is 25-200 mM.

5. The bispecific antibody formulation of any one of claims 1-4, wherein the surfactant is selected from poloxamer 188; further preferably, the surfactant is selected from 0.2-1.0 mg / mL poloxamer 188, more preferably 0.5 mg / mL poloxamer 188.

6. The bispecific antibody formulation of any one of claims 1-4, wherein the formulation is essentially free of aspartate when the buffer is a histidine-acetate buffer, by "essentially free of aspartate" is meant that the concentration of aspartate is, for example, 5 mM or less, preferably 2 mM or less, more preferably 1 mM or less, or is free of aspartate, preferably, the bispecific antibody formulation is selected from the following: (1) comprising 150 mg / mL of bispecific antibody, 20 mM of histidine-acetate buffer, 150 mM of arginine, and 0.5 mg / mL of poloxamer 188, pH 6.0; (2) comprising 150 mg / mL of bispecific antibody, 20 mM of histidine-acetate buffer, 80 mM of arginine, 0.5 mg / mL of poloxamer 188, and 140 mM of Reagent A selected from sucrose, trehalose, sorbitol, mannitol, proline, or glycine, pH 6.0; (3) comprising 150 mg / mL of bispecific antibody, 20 mM histidine-aspartate buffer, 0.5 mg / mL poloxamer 188, 80 mM arginine-aspartate, 140 mM of Reagent A selected from the group consisting of sucrose, trehalose, sorbitol, mannitol, proline or glycine, pH 6.0; (4) comprising 150 mg / mL of bispecific antibody, 20 mM histidine-acetate buffer, 80 mM arginine, 0.5 mg / mL poloxamer 188 and 80-200 mM (e.g. 80 mM, 90 mM, 100 mM, 140 mM or 200 mM) of mannitol, pH 6.0; (5) comprising 150 mg / mL of bispecific antibody, 20 mM histidine-acetate buffer, 0.5 mg / mL poloxamer 188 and 90 mM mannitol and 20-90 mM (e.g. 20 mM, 50 mM, 80 mM or 90 mM) of arginine, pH 6.0; (6) comprising 150 mg / mL of bispecific antibody, 80 mM arginine, 90 mM mannitol, 0.5 mg / mL poloxamer 188 and 5-40 mM (e.g. 5 mM, 10 mM, 20 mM or 40 mM) of histidine-acetate buffer, pH 6.0; and (7) comprising 150 mg / mL of bispecific antibody, 20 mM histidine-acetate buffer, 80 mM arginine, 90 mM mannitol, 0.5 mg / mL poloxamer 188, pH 5.5-6.5 (e.g. pH 5.5, pH 5.7, pH 6.0, pH 6.3, or pH 6.5).

7. A method of reducing the neuronal toxicity of a bispecific antibody formulation, comprising adding 5-90 mM arginine or arginine-aspartate to the bispecific antibody formulation to reduce the content of aspartate in the bispecific antibody formulation or to render the bispecific antibody aspartate free, and adding 20-200 mM of one or more of the following reagents to the bispecific antibody formulation: mannitol, sucrose, trehalose, sorbitol, glycine and proline and amino acids (glycine, proline), wherein the bispecific antibody is as defined in any one of claims 1-2.

8. The method of claim 7, wherein the bispecific antibody formulation further comprises a buffer as defined in claim 1 or 3, preferably the bispecific antibody formulation further comprises a surfactant, preferably a surfactant as defined in claim 1 or 6.

9. A method of inhibiting the formation of aggregates and charge variants in a formulation comprising a bispecific antibody, comprising adding arginine (or arginine-aspartic acid) to the solution together with one or more substances selected from the group consisting of sugars, alcohols or amino acids, preferably a combination of arginine (or arginine-aspartic acid) and mannitol in the formulation, wherein the concentration of arginine (or arginine-aspartic acid) ranges from 20 to 90 mM and the concentration of mannitol ranges from 80 to 200 mM, wherein the bispecific antibody is as defined in any one of claims 1 to 2.

10. A method of preparing a formulation of a bispecific antibody according to any one of claims 1 to 6, comprising adding to a solution comprising the bispecific antibody a buffer as defined in claim 1 or 2, a surfactant as defined in claim 1 or 5, a stabilizer as defined in claim 1, 3 or 4.

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