Antibody formulations

Abstract

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

Description

[0001] This application claims priority to PCT international application (application number: PCT / CN2024 / 104177; filing date: July 8, 2024). Technical Field

[0002] The present application belongs to the field of immunology and specifically relates to a preparation of a bispecific antibody. Background Art

[0003] Hemophilia A is a genetic disease of blood coagulation dysfunction caused by mutations in genes encoding coagulation factors, which lead to functional defects of the coagulation factors. The main symptom of patients is the risk of long-term bleeding, which is life-threatening in severe cases. 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 introducing viruses during the production process, the inconvenient frequent use of medication every 1 to 7 days, and the easy production of inhibitors that affect the efficacy. @ It is a new drug for the treatment of hemophilia A. Its active ingredient is Emicizumab, which has dual-target binding properties. It can simultaneously bind to coagulation factor IX and coagulation factor X, thereby simulating coagulation factor VIII to restore the patient's coagulation function. It also has no risk of virus introduction, a convenient dosing regimen of up to 4 weeks per dose, and an extremely low inhibitor production rate. This drug is a boon for patients, especially those who have developed inhibitors.

[0004] Existing pharmaceutical preparations containing emicizumab as an active ingredient usually produce antibody aggregates, low-molecular degradation fragments, antibody charge variants and insoluble particles. Among them, the antibody charge variants are mainly acidic peak regions with greatly reduced activity, which are more likely to be produced under high temperature conditions that are very likely to be encountered in reality; in addition, the pharmaceutical preparations also contain high concentrations of aspartic acid, which is neurotoxic, all of which affect the stability and safety of the pharmaceutical preparations. Summary of the invention

[0005] The antibody preparation developed in the present application comprises the active ingredient emicizumab, and is used to treat related diseases caused by congenital FVIII secretion deficiency or dysfunction. In order to provide antibody drugs with better activity stability and safety, it is necessary to develop preparations that are conducive to the stable storage of antibodies, so that the function and structure of the antibodies can be maintained for a long time, especially reducing the components of aggregate formation, fragment formation and / or charge variant formation.

[0006] In order to maintain the stability of Emicizumab, the antibody preparation developed in the present application can significantly inhibit the formation of charge variants (acid peaks), dimers / polymers, degradation products and insoluble particles during freeze / thaw cycles, long-term storage and temperature changes. Specifically, Emicizumab maintains stability in the above-mentioned preparation after at least 3 freeze-thaw cycles, can be stably stored at a high temperature of 40°C for at least 2 weeks, can be stably stored at room temperature for at least 6 months, and can be stably stored at 4°C for at least 24 months. Therefore, the antibody preparation of the present application can be used to stably store Emicizumab 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.

[0007] Specifically, the antibody preparation of the present application contains the following components:

[0008] (1) 20 to 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, 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,

[0009] Preferably, the first polypeptide comprises an H chain comprising the heavy chain variable region amino acid sequence shown in SEQ ID NO: 13; the second polypeptide comprises an H chain comprising the heavy chain variable region amino acid sequence shown in SEQ ID NO: 14; and the third polypeptide and the fourth polypeptide comprise a common light chain variable region amino acid sequence shown in SEQ ID NO: 15, more preferably, the first polypeptide comprises an H chain comprising the amino acid sequence of SEQ ID NO: 10; the second polypeptide comprises an 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;

[0010] (2) a buffer having a concentration of 5-40 mM, selected from a histidine-acetate buffer or a histidine-aspartate buffer;

[0011] (3) Stabilizer, the stabilizer is a combination of 20-90 mM arginine and one or more selected from the following substances: mannitol, sucrose, trehalose and sorbitol or amino acids (such as glycine, proline), which is used to adjust the osmotic pressure, stabilize the antibody and / or inhibit antibody aggregation. The stabilizing arginine is sometimes also expressed as arginine-aspartic acid, in which aspartic acid is mainly used to neutralize basic amino acids. It is known in the art that the conventional osmotic pressure of antibody preparations is 200-600 mOsm / kg. The concentration of the mannitol, sucrose, trehalose, sorbitol or amino acids can be 25-200mM, for example, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM, 55mM, 60mM, 65mM, 70mM, 75mM, 80mM, 85mM, 90mM, 95mM, 100mM, 110mM, 120mM, 130mM, 140mM, 150mM, 160mM, 170mM, 180mM, 190mm or 200mM;

[0012] 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

[0013] (4) a surfactant, wherein the surfactant is selected from one or more of polysorbate 20, polysorbate 80, poloxamer 188, and hydroxypropyl β-cyclodextrin. Preferably, the surfactant is selected from poloxamer 188; more preferably, the surfactant is selected from 0.2-2.0 mg / mL poloxamer 188, more preferably 0.5 mg / mL poloxamer 188; or

[0014] The bispecific antibody preparation comprises:

[0015] (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, the second polypeptide and the fourth polypeptide form a pair, 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, the third polypeptide and the fourth polypeptide comprise a common light chain, the light chain comprising LCDR1-3 as shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, respectively;

[0016] (2) Histidine-acetate buffer at a concentration of 5-40 mM;

[0017] (3) a stabilizer, when the buffer is a histidine-acetate buffer, the stabilizer is >90-300 mM arginine; and

[0018] (4) a surfactant, wherein the surfactant is selected from one or more of polysorbate 20, polysorbate 80, poloxamer 188, and hydroxypropyl β-cyclodextrin.

[0019] The pH value of each of the above-mentioned bispecific antibody preparations is 5.5-6.5.

[0020] In a preferred embodiment, the bispecific antibody formulation is selected from the group consisting of:

[0021] (1) containing 150 mg / mL of the bispecific antibody, 20 mM histidine-acetate buffer, 150 mM arginine and 0.5 mg / mL poloxamer 188, pH 6.0;

[0022] (2) comprising 150 mg / mL of a bispecific antibody, 20 mM histidine-acetate buffer, 80 mM arginine, 0.5 mg / mL poloxamer 188, and 140 mM reagent A, wherein the reagent A is selected from sucrose, trehalose, sorbitol, mannitol, proline, or glycine, pH 6.0;

[0023] (3) comprising 150 mg / mL of a bispecific antibody, 20 mM histidine-aspartate buffer, 0.5 mg / mL poloxamer 188, 80 mM arginine-aspartate, 140 mM reagent A, wherein the reagent A is selected from sucrose, trehalose, sorbitol, mannitol, proline or glycine, pH 6.0;

[0024] (4) comprising 150 mg / mL of the 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) mannitol, pH 6.0;

[0025] (5) comprising 150 mg / mL of the bispecific antibody, 20 mM histidine-acetate buffer, 0.5 mg / mL poloxamer 188, 90 mM mannitol, and 20-90 mM (e.g., 20 mM, 50 mM, 80 mM or 90 mM) arginine, pH 6.0;

[0026] (6) comprising 150 mg / mL of the 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) histidine-acetate buffer, pH 6.0; and

[0027] (7) comprising 150 mg / mL of the 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).

[0028] It should be understood that within the scope of this application, the above-mentioned technical features of this application and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

[0029] The terms involved in this application have the conventional meanings understood by those skilled in the art. When a term has two or more definitions as used and / or accepted in the art, the definition of the term used herein is intended to include all meanings.

[0030] In the present application, the bispecific antibody is Emicizumab. The present application develops an antibody formulation formula by screening buffer and stabilizer, which can enhance the stability of Emicizumab preparations, prevent antibody aggregation, degradation, charge variants (acidic charge isomers or charge isomer acidic peaks) and insoluble particles. Generation and / or increase. For example, by reducing the content of arginine, the ratio of acidic charge isomers can be reduced, or the content of aspartic acid in the preparation can be reduced by reducing the content of arginine, thereby reducing the neuronal toxicity caused by excessive aspartic acid content, or by selecting a specific buffer histidine-acetate buffer, aspartic acid can be not included in the bispecific antibody preparation. In a preferred embodiment of the present application, acetic acid is selected as the counter ion of buffer (buffer) in the preparation of the present application, i.e., histidine-acetate buffer is selected, which also shows better advantages in suppressing the increase of aggregates, the generation of small molecule fragments and reducing the formation of acidic peaks.

[0031] "Stability" and "stable" herein refer to a liquid formulation containing an antibody (including an antigen-binding fragment thereof) in which the antibody (including an antigen-binding fragment thereof) does not or only rarely aggregates, degrades or fragments under given production, preparation, transportation and / or storage conditions. A "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., thereby evaluating the stability of the antibody (including an antigen-binding fragment thereof).

[0032] It should be noted that, in the present application, when it is mentioned that a preparation contains a buffer or a buffer system, it also means that the preparation contains a buffering agent, and a buffer system is formed in the preparation by the buffering agent.

[0033] As used herein, aspects referred to by the expression "comprising" include those referred to by the expression "consisting essentially of," as well as those referred to by the expression "consisting of. ...

[0034] The numerical values ​​described herein may vary within a certain range, for example, depending on instruments or equipment, measurement conditions and procedures used by those skilled in the art, and as long as they are within the range allowing the purpose of the present application to be achieved, for example, they may contain a deviation of about 10%.

[0035] The emicizumab herein comprises four polypeptides, wherein the first polypeptide and the third polypeptide form a pair, and the second polypeptide and the fourth polypeptide form a pair, wherein the first polypeptide comprises an H chain comprising the amino acid sequence of SEQ ID NO: 10; the second polypeptide comprises an 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.

[0036] In one embodiment of the present application, the concentration of Emicizumab in the antibody preparation is approximately 20-200 mg / mL; as a preferred embodiment, the concentration of Emicizumab in the antibody preparation is 20-180 mg / mL, for example 20-150 mg / mL, 120-180 mg / mL, 35-150 mg / mL or 150 mg / mL.

[0037] In some embodiments of the present application, different buffer systems such as phosphate buffer, histidine buffer, citrate buffer, succinate, histidine-glutamate, histidine + acetate buffer, histidine-hydrochloride buffer, histidine-aspartate buffer are evaluated for the effect on antibody stability. Among the above buffers, histidine-acetate buffer, histidine-glutamate buffer, histidine-hydrochloride buffer or histidine-aspartate buffer are preferred, and histidine-aspartate buffer and histidine-acetate buffer are more preferred. In other embodiments, the effect of adding appropriate stabilizers such as sucrose, trehalose, sorbitol, mannitol, amino acids (such as arginine, proline, glycine), and sodium chloride on antibody stability in the buffer is evaluated. Arginine, arginine and sugar, alcohol, amino acid and other combinations of formula have more advantages in reducing antibody aggregation and charge heterogeneity. In other embodiments, the changes in the addition of appropriate surfactants such as polysorbate 20, polysorbate 80 and poloxamer 188 to a formulation containing an appropriate buffer and stabilizer under high temperature conditions were evaluated. Poloxamer 188 is more stable and can reduce the generation of insoluble particles in the formulation during storage.

[0038] In a preferred embodiment of the present application, the concentration of aspartic acid is reduced in the preparation of the present application or aspartic acid is substantially not contained as the counter ion of the buffer (buffer). "Substantially free of aspartic acid" means that the concentration of aspartic acid is, for example, 5 mM or lower, preferably 2 mM or lower, more preferably 1 mM or lower. Preferably, the buffer system is a 10-40 mM histidine-acetate buffer. @@About 175 mM aspartic acid is added to the prescription mainly as a pH regulator. The toxicity of large doses of aspartic acid causing hypothalamic neuronal necrosis in newborn mice and headache in humans has been reported (Toxicology, 29 (1983), p109-119; NEUROLOGY 1990, 401582-1586). Many marketed products that use large doses of aspartic acid in their formulas all show adverse reactions of headache. In the antibody preparation of the present application, aspartic acid is used as a counter ion of arginine and as a pH regulator, and its dosage is substantially the same as that of arginine. For example, the antibody preparation provided in the present application reduces the concentration of arginine, thereby reducing the concentration of aspartic acid; more preferably, a buffer solution that does not contain aspartic acid is selected to reduce the risk of neuronal toxicity.

[0039] Thus, the present application provides a method for reducing the neuronal toxicity of a bispecific antibody preparation on the other hand, which comprises reducing the content of aspartic acid in the bispecific antibody preparation or making the bispecific antibody free of aspartic acid, and adding 20-200mM of one or more selected from the following to the bispecific antibody preparation: mannitol, sucrose, trehalose, sorbitol, amino acids (such as proline, glycine), wherein mannitol is preferred; the bispecific antibody is as defined above. Preferably, the bispecific antibody preparation also comprises a buffer as defined above, and preferably the bispecific antibody preparation also comprises a surfactant, preferably a surfactant as defined above.

[0040] In another aspect, the present application also provides a method for preparing the bispecific antibody preparation, which comprises adding a buffer, a surfactant and a stabilizer as defined above to a solution containing the bispecific antibody.

[0041] The solution pH of the preparation of the present application is preferably 5.5 to 6.5, more preferably 6.

[0042] If necessary, the preparation of the present application may further contain appropriate cryoprotectants, suspending agents,

[0043] Solubilizers, isotonic agents, preservatives, adsorption inhibitors, diluents, excipients, pH adjusters,

[0044] Analgesics, sulfur-containing reducing agents, antioxidants, etc. These are conventional choices in the art. DETAILED DESCRIPTION

[0045] The following will be described in detail with reference to the embodiments of the present application. It will be appreciated by those skilled in the art that the following examples are only used to illustrate the present application and should not be considered as limiting the scope of the present application. The specific techniques or conditions not specified in the examples are carried out according to the techniques or conditions described in the literature in this area or according to the product specification. The reagents used or the instruments not specified by the manufacturer are conventional products that can be purchased from the market.

[0046] Example 1: pH / buffer system screening of emicizumab preparation formulation I

[0047] 1. Experimental purpose: To study antibody stability in the pH range of 5.0-7.0 and to select a pH value that can reduce the formation of soluble aggregates.

[0048] 2. Materials and methods: Liquid compositions containing emicizumab were prepared, which contained 150 mg / mL emicizumab, 150 mM NaCl and 20 mM histidine / histidine hydrochloride buffer (His / His-HCl), 20 mM citric acid / sodium citrate buffer (Cit / Cit-Na) and 20 mM disodium hydrogen phosphate / sodium dihydrogen phosphate buffer (PB) of different pH values. The trends of changes in aggregates (SEC-HPLC) and small molecule fragments (rCE-SDS) of various formulations were studied when placed at high temperature of 40°C for 28 days.

[0049] 3. Detection method of emicizumab multimers

[0050] The polymers in the samples were detected by size exclusion chromatography (SEC-HPLC) using a G3000SWXL (Tosoh) column with citrate buffer (50 mmol / L, pH 6.0) containing 100 mmol / L sodium chloride as the mobile phase at a flow rate of 0.5 mL / min.

[0051] Among the detected peaks, the peak with the largest area and height is determined as the monomer, and the peaks detected earlier than the monomer are collectively referred to as polymers or high molecular weight species (HMWS). The peak area percentages of polymer and monomer peaks are recorded.

[0052] 4. CE-SDS non-reduced purity detection method

[0053] The sample purity 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 μm, outer diameter 375 μm), wherein the filling liquid was SDS-MW Gel Buffer (pH 8, 0.2% SDS), the buffer was SDS-MW Sample buffer (0.05 mol / L Tris-HCl, 1% SDS, pH 7.5), and capillary electrophoresis (capillary temperature 25°C, sample chamber temperature 8°C, detection wavelength 214 nm) was used for separation and detection.

[0054] Record the retention time of each peak in the sample and the ratio of the corrected peak area, and report the peak area percentage of the main peak and the peak area percentage of other peaks.

[0055] 5. Experimental Results

[0056] After being placed at 40℃ for 4 weeks, the increase in aggregate content and small molecule fragments in citric acid / sodium citrate buffer pH 6.0 and histidine / histidine hydrochloride buffer pH 6.0 was small, showing good physical and chemical stability. The higher the pH in the same buffer, the more obvious the increase in small molecule fragments.

[0057] 40℃4WΔAggregate% 40℃4WΔFragment% Cit / Cit-Na pH5.0 4.92 0.28 Cit / Cit-Na pH5.5 1.85 1.58 Cit / Cit-Na pH 6.0 1.31 1.93 His / His-HCl pH5.5 2.99 0.89 His / His-HCl pH 6.0 1.25 2.24 His / His-HCl pH 6.5 1.19 3.88 PB pH6.5 1.93 3.78 PB pH 7.0 2.67 5.33

[0058] Example 2: pH / buffer system screening of emicizumab preparation formulation II

[0059] 1. Experimental purpose: Based on the results of pH / buffer system screening I of emicizumab formulation, the experiment was designed to evaluate the effect of different buffer salts on the stability of emicizumab.

[0060] 2. Materials and methods: The buffer system for the stability test had a pH range of 5.8-6.2 and included 20 mM citric acid-sodium citrate buffer, 20 mM succinic acid-sodium succinate buffer (SA / SA-Na), 20 mM histidine-acetate buffer (His / Ace), 20 mM histidine-glutamate buffer (His / Glu), 20 mM histidine-hydrochloric acid buffer (His / HCl), and 20 mM histidine-aspartic acid buffer (His / Asp). In the buffer salt system, it contained 150 mg / mL of emicizol antibody, 150 mM arginine, and 0.5 mg / mL of poloxamer 188.

[0061] The trends of aggregates and small molecule fragments changes were studied after various formulations were placed at a high temperature of 40°C for 28 days.

[0062] 3. CE-SDS reduction purity detection method

[0063] The sample purity 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 μm, outer diameter 375 μm), wherein the filling liquid was SDS-MW Gel Buffer (pH 8, 0.2% SDS), the buffer was SDS-MW Sample buffer (0.05 mol / L Tris-HCl, 1% SDS, pH 7.5), and capillary electrophoresis (capillary temperature 25°C, sample chamber temperature 8°C, detection wavelength 214 nm) was used for separation and detection.

[0064] Record the retention time and corrected peak area ratio of each peak in the sample, and report the purity (the sum of the LC and HC peak area percentages) and the peak area percentages of other peaks.

[0065] 4. Experimental Results

[0066] After being placed at 40°C for 28 days, 20 mM histidine-hydrochloride, 20 mM histidine-glutamate, 20 mM histidine-acetate and 20 mM histidine-aspartate had a good protective effect in inhibiting the increase of aggregates and the production of small molecule fragments, among which the protective effect of histidine-acetate was outstanding.

[0067] Buffer salt type 40℃4WΔAggregate% 40℃4WΔFragment% 20mM Cit / Cit-Na 0.78 3.88 20mM SA / SA-Na 1.16 2.01 20mM His / Ace 0.58 1.27 20mM His / Glu 0.72 2.37 20mM His / HCl 0.79 2.2 20mM His / Asp 0.88 1.9

[0068] Example 3 Screening of stabilizers

[0069] 1. Experimental purpose: To screen out stabilizers that can stabilize proteins.

[0070] 2. Materials and methods: A mixed solution containing 150 mg / mL of emicizumab was prepared, pH 6.0, which contained 20 mM histidine-aspartate buffer or 20 mM histidine-acetate buffer, 0.5 mg / mL poloxamer 188 and a liquid composition of different stabilizers, the stabilizer comprising 150 mM sodium chloride, 150 mM arginine (or arginine-aspartate) or 80 mM arginine (or arginine-aspartate), and a combination of reagent A, wherein the reagent A is 140 mM sucrose, 140 mM trehalose, 140 mM sorbitol, 140 mM mannitol and 140 mM proline or 140 mM glycine. The trends of changes in aggregates (SEC-HPLC) and charge variants (acidic peak charge isomers) were studied for various formulations placed at high temperature of 40°C for 28 days.

[0071] 3. Methods for measuring and calculating charge variants of emicizumab

[0072] Charge variants in the samples were detected by ion exchange chromatography (IEX-HPLC) using a strong cation exchange column (MabPac SCX-10, 5 μm, 4×150 mm, Thermo Scientific) with 20 mmol / L morpholineethanesulfonic acid (pH 6.0) and 20 mM morpholineethanesulfonic acid + 500 mmol / L sodium chloride (pH 6.0) as mobile phases. The samples were separated by liquid chromatography (detection wavelength: 280 nm, flow rate 0.5 mL / min, column temperature: 40°C) with gradient elution.

[0073] Among the detected peaks, the peak with the largest area and height is determined as the main peak, the peaks detected earlier than the main peak are collectively called acidic peaks, and the peaks detected later than the main peak are collectively called basic peaks. Record the results of acidic peaks, main peaks, and basic peaks.

[0074] 4. Experimental Results

[0075] The results obtained are shown in the following table. Antibody formulations containing 150 mM arginine or 80 mM arginine (or arginine-aspartic acid) in combination with sugars, alcohols or amino acids showed better advantages in reducing protein aggregation and reducing acidic peak formation, especially the combination of arginine and mannitol showed lower aggregates and acidic peaks.

[0076]

[0077] Example 4 Effect of Mannitol Concentration on Aggregation and Acid Peak Inhibition

[0078] 1. Experimental purpose: Based on the experimental results of stabilizer screening, a combination of arginine and mannitol was selected as a stabilizer to investigate the effect of mannitol concentration on the aggregation and acidic peak of Emicar beads.

[0079] 2. Materials and methods: Liquid compositions containing 150 mg / mL emicizumab, 20 mM histidine-acetate buffer, 80 mM arginine, 80-200 mM mannitol and 0.5 mg / mL poloxamer 188, pH 6.0 were prepared. The trends of the aggregates (SEC-HPLC) and the acidic peaks of the charge isomers were studied after various formulations were placed at a high temperature of 40°C for 28 days.

[0080] 3. Experimental results

[0081] Different mannitol concentrations effectively inhibited the aggregation and changes in the acidic peak.

[0082]

[0083] Example 5 Effect of Arginine Concentration on Aggregation and Acidic Peak Inhibition

[0084] 1. Experimental purpose: Based on the experimental results of stabilizer screening, a combination of arginine and mannitol was selected as a stabilizer to investigate the effect of arginine concentration on the aggregation and acidic peak of Emicar beads.

[0085] 2. Materials and methods: Liquid compositions containing 150 mg / mL emicizumab, 20 mM histidine-acetate buffer, 90 mM mannitol, 20-90 mM arginine and 0.5 mg / mL poloxamer 188 were prepared. The changing trends of the aggregates (SEC-HPLC) and the acidic peaks of the charge isomers were studied after various formulations were placed at a high temperature of 40°C for 28 days.

[0086] 3. Experimental results

[0087] The formulations containing 20-90 mM arginine in combination with mannitol showed comparable acidic peak suppression and better aggregation inhibition effect than the formulation containing 150 mM arginine alone.

[0088]

[0089]

[0090] Example 6 Effect of histidine concentration on aggregation and acidic peak inhibition

[0091] 1. Experimental purpose: Based on the experimental results of stabilizer screening, a combination of arginine and mannitol was selected as a stabilizer to investigate the effect of histidine concentration on the aggregation and acidic peak of Emicyl beads.

[0092] 2. Materials and methods: Liquid compositions containing 150 mg / mL emicizumab, 5-40 mM histidine-acetate buffer, 90 mM mannitol, 80 mM arginine and 0.5 mg / mL poloxamer 188 were prepared. The changing trends of the aggregates (SEC-HPLC) and the acidic peaks of the charge isomers were studied after various formulations were placed at a high temperature of 40°C for 28 days.

[0093] 3. Experimental results

[0094] Buffers containing 5 mM or more histidine-acetate exhibited better aggregation inhibition effects.

[0095]

[0096] Example 7 Effect of pH on Aggregation and Acidic Peak Suppression

[0097] 1. Experimental purpose: To study the effect of pH on the aggregation and acid peak of Amylase beads during high temperature storage.

[0098] 2. Materials and methods: Liquid compositions containing 150 mg / mL emicizol antibody, 20 mM histidine-acetate buffer, 90 mM mannitol, 80 mM arginine and 0.5 mg / mL poloxamer 188 were prepared, with pH values ​​of 5.0, 5.5, 5.7, 6.0, 6.3 and 6.5, respectively. The trends of the aggregate (SEC-HPLC) and charge isomer acid peaks of various formulations were studied after being placed at a high temperature of 40°C for 28 days.

[0099] 3. Experimental results

[0100] The samples showed better aggregation and acidic peak suppression effects at pH 5.5-6.5.

[0101] pH 40℃4WΔAggregate% 40℃4WΔacidic peak% 5.0 4.62 3.78 5.5 1.34 2.32 5.7 1.02 2.17 6.0 0.92 2.44 6.3 0.89 2.24 6.5 0.96 2.68

[0102] Example 8 Screening of surfactant types and concentrations

[0103] 1. Experimental purpose: To screen out the types and concentrations of surfactants that can stabilize antibodies.

[0104] 2. Materials and methods: Liquid compositions containing 150 mg / mL emicizumab, 20 mM histidine-acetate buffer, 80 mM arginine, 90 mM mannitol and different surfactants were prepared. The surfactants included 0.5 mg / mL polysorbate 20, 0.5 mg / mL polysorbate 80, 0.5 mg / mL poloxamer 188 and hydroxypropyl beta-cyclodextrin. The trends of aggregates, surfactant content and visible particles in various formulations were studied after being placed at high temperature of 40°C for 28 days.

[0105] 3. Method for measuring and calculating the content of polysorbate 20, polysorbate 80 and poloxamer 188 in Emicarb antibody samples

[0106] The content of surfactant in the sample was detected by high performance liquid chromatography with evaporative light scattering detector (ELSD-HPLC), using Waters OASIS MAX column (30 μm, 2.1×20 mm), with 2% formic acid aqueous solution and 2% formic acid isopropanol as mobile phase, and the flow rate was 1 mL / min. The content of poloxamer 188, polysorbate 20 and polysorbate 80 in the sample was quantitatively detected by external standard method.

[0107] 4. Experimental Results

[0108] After being placed at high temperature of 40°C for 28 days, there was no significant increase in aggregates, but the content of polysorbate 20 and polysorbate 80 decreased, indicating that polysorbate 20 and polysorbate 80 were unstable and degraded in the samples, while poloxamer 188 showed better stability, and no obvious visible particles were generated in the samples containing surfactants.

[0109]

[0110] N / A: Not tested.

[0111] Example 9 Effect of Antibody Concentration on Formulation

[0112] 1. Experimental purpose: To explore the effect of different antibody concentrations on the stability of the preparation.

[0113] 2. Materials and methods: Mixed solutions containing 20-200 mg / mL of emicizumab were prepared, which contained a combination of 20 mM histidine-acetate buffer, 0.5 mg / mL poloxamer 188, 80 mM arginine and 90 mM mannitol. The trends of changes in the acidic peaks of aggregates (SEC-HPLC) and charge isomers of various formulations were studied under high temperature conditions of 40°C for 28 days.

[0114] See Example 4 for methods of measuring and calculating charge variants of emicizumab.

[0115] 3. Experimental results

[0116] The results showed that when the antibody concentration reached 200 mg / mL, the combination formulation containing 20 mM histidine-acetate buffer, 80 mM arginine and 90 mM mannitol could effectively inhibit the formation of aggregates and acidic peaks.

[0117]

[0118]

[0119] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teaching content of this application, those skilled in the art can make various changes or modifications to this application, and these equivalent forms also fall within the scope defined by the claims attached to this application. 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 the heavy chain of the bispecific antibody QVQLVESGGGLVQPGGSLRLSCAASGFTFSYYDIQWVRQAPGKGLEWVSSISPSGQSTYYRREVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRTGREYGGGWYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVDHKPSNT KVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPS SIEKTISKAKGQPREPQVYTLPPSQKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNRYTQKSLSLSPSEQ ID NO:11,Amino acid sequence of the bispecific antibody heavy chain QVQLVQSGSELKKPGASVKVSCKASGYTFTDNNMDWVRQAPGQGLEWMGDINTRSGGSIYNEEFQDRVIMTVDKSTDTAYMELSSLRSEDTATYHCARRKSYGYYLDEWGEGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVDHKPSNTKVDKR VESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQESLSLSPSEQ ID NO: 12, the amino acid sequence of the bispecific antibody light chain DIQMTQSPSSLSASVGDRVTITCKASRNIERQLAWYQQKPGQAPELLIYQASRKESGVPDRFSGSRYGTDFTLTISSLQPEDIATYYCQQYSDPPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 13, the amino acid sequence of the bispecific antibody heavy chain variable region QVQLVESGGGLVQPGGSLRLSCAASGFTFSYYDIQWVRQAPGKGLEWVSSISPSGQSTYYRREVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRTGREYGGGWYFDYWGQGTLVTVSS SEQ ID NO: 14,The amino acid sequence of the heavy chain variable region of the bispecific antibody is QVQLVQSGSELKKPGASVKVSCKASGYTFTDNNMDWVRQAPGQGLEWMGDINTRSGGSIYNEEFQDRVIMTVDKSTDTAYMELSSLRSEDTATYHCARRKSYGYYLDEWGEGTLVTVSSSEQ ID NO: 15, the amino acid sequence of the light chain variable region of the bispecific antibody is DIQMTQSPSSLSASVGDRVTITCKASRNIERQLAWYQQKPGQAPELLIYQASRKESGVPDRFSGSRYGTDFTLTISSLQPEDIATYYCQQYSDPPLTFGGGTKVEIK,

Claims

1. A bispecific antibody preparation 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, the second polypeptide and the fourth polypeptide form a pair, 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, the third polypeptide and the fourth polypeptide comprise a common light chain, the light chain comprising LCDR1-3 as shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, respectively; (2) a buffer having a concentration of 5-40 mM, selected from a histidine-acetate buffer or a histidine-aspartate buffer; (3) a stabilizer, wherein the stabilizer is 20-90 mM arginine and one or more selected from the following agents: mannitol, sucrose, trehalose, sorbitol or amino acids (glycine, proline); and (4) a surfactant, wherein the surfactant is selected from one or more of polysorbate 20, polysorbate 80, poloxamer 188, and hydroxypropyl β-cyclodextrin; The pH of the bispecific antibody formulation is 5.5 to 6.5; or, It contains: (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, the second polypeptide and the fourth polypeptide form a pair, 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, the third polypeptide and the fourth polypeptide comprise a common light chain, the light chain comprising LCDR1-3 as shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, respectively; (2) Histidine-acetate buffer at a concentration of 5-40 mM; (3) a stabilizer, which is 90 mM to 300 mM arginine; and (4) a surfactant, wherein the surfactant is selected from one or more of polysorbate 20, polysorbate 80, poloxamer 188, and hydroxypropyl β-cyclodextrin; The pH of the bispecific antibody formulation is 5.5 to 6.

5.

2. The bispecific antibody preparation of claim 1, wherein the first polypeptide comprises an H chain comprising a heavy chain variable region amino acid sequence as shown in SEQ ID NO: 13; the second polypeptide comprises an 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, preferably, wherein the first polypeptide comprises an H chain comprising an amino acid sequence as shown in SEQ ID NO: 10; the second polypeptide comprises an H chain comprising an amino acid sequence as shown in SEQ ID NO: 11; and the third polypeptide and the fourth polypeptide comprise a common L chain as shown in SEQ ID NO: 12, preferably, the pH of the bispecific antibody preparation 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 preparation of any one of claims 1 to 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 to 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 to 4, wherein when the buffer is a histidine-acetate buffer, the formulation is substantially free of aspartic acid, wherein "substantially free of aspartic acid" means that the concentration of aspartic acid is, for example, 5 mM or less, preferably 2 mM or less, more preferably 1 mM or less, or does not contain aspartic acid. Preferably, the bispecific antibody preparation is selected from the following: (1) containing 150 mg / mL of the bispecific antibody, 20 mM histidine-acetate buffer, 150 mM arginine and 0.5 mg / mL poloxamer 188, pH 6.0; (2) comprising 150 mg / mL of a bispecific antibody, 20 mM histidine-acetate buffer, 80 mM arginine, 0.5 mg / mL poloxamer 188, and 140 mM reagent A, wherein the reagent A is selected from sucrose, trehalose, sorbitol, mannitol, proline, or glycine, pH 6.0; (3) comprising 150 mg / mL of a bispecific antibody, 20 mM histidine-aspartate buffer, 0.5 mg / mL poloxamer 188, 80 mM arginine-aspartate, 140 mM reagent A, wherein the reagent A is selected from sucrose, trehalose, sorbitol, mannitol, proline or glycine, pH 6.0; (4) comprising 150 mg / mL of the 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) mannitol, pH 6.0; (5) comprising 150 mg / mL of the bispecific antibody, 20 mM histidine-acetate buffer, 0.5 mg / mL poloxamer 188, 90 mM mannitol, and 20-90 mM (e.g., 20 mM, 50 mM, 80 mM or 90 mM) arginine, pH 6.0; (6) comprising 150 mg / mL of the 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) histidine-acetate buffer, pH 6.0; and (7) comprising 150 mg / mL of the 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 for reducing neuronal toxicity of a bispecific antibody preparation, comprising adding 5-90 mM arginine or arginine-aspartate to the bispecific antibody preparation to reduce the aspartate content in the bispecific antibody preparation or to make the bispecific antibody free of aspartate, and simultaneously adding 20-200 mM of one or more selected from the following agents: mannitol, sucrose, trehalose, sorbitol, glycine and proline and amino acids (glycine, proline) to the bispecific antibody preparation, wherein the bispecific antibody is as defined in any one of claims 1-2.

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

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

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

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