Antibody formulation

Abstract

The present application relates to an antibody formulation, comprising a bispecific antibody that serves as an active ingredient, and further comprising a buffer system, a stabilizer, and a surfactant.

Description

antibody preparations

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

[0002] This application belongs to the field of immunology. Specifically, it relates to a formulation of a bispecific antibody. Background Technology

[0003] Hemophilia A is a genetic disorder of blood clotting caused by mutations in the genes encoding clotting factors, leading to dysfunction of these factors. The main symptom is a persistent bleeding risk, which can be life-threatening in severe cases. Current treatments primarily use clotting factor VIII extracted from normal human blood and recombinant human clotting factor VIII. However, these drugs have drawbacks, including the risk of introducing viruses during the manufacturing process, the inconvenient need for frequent dosing (every 1-7 days), and the potential for the formation of inhibitors that affect efficacy. [Shuyoulile] @ It is a novel drug for treating hemophilia A. Its active ingredient is emicizumab, which has dual-target binding properties. It can restore the patient's coagulation function by simultaneously binding to coagulation factor IX and coagulation factor X, thereby mimicking coagulation factor VIII. It has a convenient dosing method with no risk of virus introduction, up to 4 weeks / dose, and an extremely low inhibitor production rate. This drug has brought good news to patients, especially those who have developed inhibitors.

[0004] Existing drug formulations containing emecizumab as the active ingredient typically generate antibody aggregates, low-molecular-weight degradation fragments, antibody charge variants, and insoluble particles. Among these, antibody charge variants are mainly acidic peak regions with greatly reduced activity, which are more likely to be generated under the high-temperature conditions that are likely to be encountered in reality. In addition, the drug formulations also contain high concentrations of aspartic acid, which has neurotoxicity. All of these factors affect the stability and safety of the drug formulations. Summary of the Invention

[0005] The antibody formulation developed in this application contains the active ingredient emecizumab, and is used to treat diseases related to congenital FVIII deficiency or dysfunction. To provide antibody drugs with better activity stability and safety, it is necessary to develop formulations that facilitate stable antibody preservation, enabling the long-term maintenance of antibody function and structure, particularly reducing components involved in aggregate formation, fragment formation, and / or charge variant formation.

[0006] To maintain the stability of emecizumab, this application developed an antibody formulation that significantly inhibits the formation of charge variants (acid peaks), dimers / polymers, degradation products, and insoluble microparticles during freeze / thaw cycles, long-term storage, and temperature changes. Specifically, in the aforementioned formulation, emecizumab maintains stability after at least three freeze-thaw cycles, is stably stored at 40°C for at least two weeks, at room temperature for at least six months, and at 4°C for at least 24 months. Therefore, the antibody formulation of this application can be used for the long-term stable storage of emecizumab and meets more stringent transportation and usage conditions, which is of great significance for the treatment of diseases caused by congenital FVIII secretion insufficiency or dysfunction.

[0007] Specifically, the antibody formulation of this application contains the following components:

[0008] (1) A bispecific antibody or its antigen-binding fragment at a concentration of 20–200 mg / mL, 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 containing 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 containing 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 containing 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 containing the heavy chain variable region amino acid sequence shown in SEQ ID NO:13; the second polypeptide comprises an H chain containing the heavy chain variable region amino acid sequence shown in SEQ ID NO:14; and the third and fourth polypeptides contain the common light chain variable region amino acid sequence shown in SEQ ID NO:15. More preferably, the first polypeptide comprises an H chain containing the amino acid sequence of SEQ ID NO:10; the second polypeptide comprises an H chain containing the amino acid sequence of SEQ ID NO:11; and the third and fourth polypeptides contain the common L chain of SEQ ID NO:12.

[0010] (2) A buffer solution with a concentration of 5-40 mM, selected from histidine-acetic acid buffer or histidine-aspartic acid buffer.

[0011] (3) A stabilizer, wherein the stabilizer is 20–90 mM arginine and a combination of one or more substances selected from the following: mannitol, sucrose, trehalose, and sorbitol, or amino acids (such as glycine, proline), used to adjust osmotic pressure, stabilize antibodies, and / or inhibit antibody aggregation. The stabilizing arginine is sometimes also referred to as arginine-aspartic acid, where aspartic acid is primarily used to neutralize basic amino acids. The conventional osmotic pressure of antibody preparations is known in the art to be 200–600 mOsm / kg. The concentration of mannitol, sucrose, trehalose, sorbitol, or amino acids can be 25-200 mM, for example, 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;

[0012] The preferred concentration of arginine or arginine-aspartic acid 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 formulation comprises:

[0015] (1) A bispecific antibody at a concentration of 20–200 mg / mL (preferably 20–180 mg / mL, for example 20–150 mg / mL, 120–180 mg / mL, 35–150 mg / mL or 150 mg / mL), 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 comprising LCDR1-3 as shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9 respectively;

[0016] (2) Histidine-acetic acid buffer solution with a concentration of 5-40 mM;

[0017] (3) Stabilizer, wherein when the buffer solution is a histidine-acetic acid buffer, the stabilizer is >90–300 mM arginine; and

[0018] (4) 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 bispecific antibody preparations is 5.5-6.5.

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

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

[0022] (2) Contains 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, wherein reagent A is selected from sucrose, trehalose, sorbitol, mannitol, proline or glycine, pH 6.0.

[0023] (3) Contains 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, and 140 mM of reagent A, wherein reagent A is selected from sucrose, trehalose, sorbitol, mannitol, proline or glycine, pH 6.0.

[0024] (4) Contains 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) mannitol, pH 6.0;

[0025] (5) Contains 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.

[0026] (6) Contains 150 mg / mL 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) Contains 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).

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

[0029] The terms used in this application have their conventional meanings as understood by those skilled in the art. Where a term has two or more definitions as used and / or where acceptable in the art, the definitions used herein are intended to encompass all meanings.

[0030] In this application, the bispecific antibody is emecizumab. This application develops an antibody formulation by screening buffers and stabilizers, which can enhance the stability of the emecizumab formulation and prevent antibody aggregation, degradation, and the generation and / or increase of charge variants (acidic charge isomers or acidic peaks of charge isomers) and insoluble particles. For example, by reducing the arginine content, the proportion of acidic charge isomers can be reduced, or by reducing the arginine content, the aspartic acid content in the formulation can be reduced, thereby reducing neurotoxicity caused by excessive aspartic acid content. Alternatively, by selecting a specific buffer, histidine-acetate buffer, the bispecific antibody formulation can be made free of aspartic acid. In a preferred embodiment of this application, acetate is selected as the counterion of the buffer (buffer), i.e., histidine-acetate buffer is selected, which also shows better advantages in inhibiting the increase of aggregates, the generation of small molecule fragments, and the reduction of acidic peak formation.

[0031] In this document, "stability" or "stable" means that in a liquid formulation containing an antibody (including its antigen-binding fragment), the antibody (including its antigen-binding fragment) does not aggregate, or only minimally aggregates, degrades, or fragments under given manufacturing, preparation, transport, and / or storage conditions. A "stable" formulation retains its biological activity under given manufacturing, preparation, transport, and / or storage conditions. The stability of the antibody (including its antigen-binding fragment) can be assessed by measuring the degree of aggregation, degradation, or fragmentation of the formulation using techniques such as SEC-HPLC, CEX-HPLC, and CE-SDS.

[0032] It should be noted that in this application, if the formulation contains a buffer solution or buffer system, it refers to the formulation containing a buffer agent, and the buffer agent forms a buffer system in the formulation.

[0033] As used herein, aspects referred to by the expression “contains” include those referenced by the expression “consistently of…” as well as those referenced by the expression “consisting of…”.

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

[0035] The emecizumab described in this article 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. The first polypeptide contains an H chain, which contains the amino acid sequence of SEQ ID NO:10; the second polypeptide contains an H chain, which contains the amino acid sequence of SEQ ID NO:11; and the third polypeptide and the fourth polypeptide contain a common L chain of SEQ ID NO:12.

[0036] In one embodiment of this application, the concentration of emecizumab in the antibody preparation is approximately 20-200 mg / mL; as a preferred embodiment, the concentration of emecizumab 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 this application, the effects of different buffer systems such as phosphate buffer, histidine buffer, citrate buffer, succinate buffer, histidine-glutamate buffer, histidine + acetate buffer, histidine-hydrochloride buffer, and histidine-aspartate buffer on antibody stability were evaluated. Among these buffers, histidine-acetate buffer, histidine-glutamate buffer, histidine-hydrochloride buffer, or histidine-aspartate buffer are preferred, with histidine-aspartate buffer and histidine-acetate buffer being more preferred. In other embodiments, the effects of adding appropriate stabilizers to the buffer, such as sucrose, trehalose, sorbitol, mannitol, amino acids (e.g., arginine, proline, glycine), and sodium chloride, on antibody stability were evaluated. Formulations combining arginine with sugars, alcohols, and amino acids are more advantageous in reducing antibody aggregation and charge heterogeneity. In other embodiments, the effects of adding appropriate surfactants such as polysorbate 20, polysorbate 80, and poloxamer 188 to formulations containing appropriate buffers and stabilizers under high-temperature conditions were evaluated. Poloxamer 188 was found to be more stable and could reduce the generation of insoluble particles in the formulation during storage.

[0038] In a preferred embodiment of this application, the formulation contains a reduced aspartic acid concentration or is substantially free of aspartic acid as a counterion in the buffer solution (buffer). "Substantially free of aspartic acid" means that the aspartic acid concentration 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-acetic acid buffer. (Shuyoulile) @@The formulation contains approximately 175 mM aspartic acid primarily as a pH adjuster. High doses of aspartic acid have been reported to cause hypothalamic neuronal necrosis in newborn mice and headaches in humans (Toxicology, 29(1983), pp. 109-119; NEUROLOGY 1990, 401582-1586). Several marketed products using high doses of aspartic acid have exhibited headache as an adverse reaction. In the antibody formulation of this application, 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 in this application 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.

[0039] Therefore, this application also 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), preferably mannitol; 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.

[0040] This application also 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.

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

[0042] If desired, the formulations of this application 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

[0043] The embodiments of this application 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 construed as limiting the scope of this application. 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 on the market.

[0044] Example 1: Screening of pH / buffer system for emecizumab formulation I

[0045] 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.

[0046] 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.

[0047] 3. Detection method for emecizumab multimers

[0048] The polymers in the sample were detected by size exclusion chromatography (SEC-HPLC) using a G3000SWXL (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.

[0049] Among the detected peaks, the peak with the largest area and height is identified as a monomer, and the peaks detected earlier than the monomers are collectively referred to as polymers or high molecular weight species (HMWS). The percentage of peak area for polymers and monomers is recorded.

[0050] 4. Detection method for non-reducing purity of CE-SDS

[0051] 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).

[0052] 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.

[0053] 5. Experimental Results

[0054] 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.

[0055] Example 2 Screening of pH / buffer system for emecizumab formulation II

[0056] 1. Experimental objective: Based on the results of screening I of pH / buffer systems for emecizumab formulation, this study aims to evaluate the effect of different buffer salts on the stability of emecizumab.

[0057] 2. Materials and Methods: The buffer system used for stability testing had a pH range of 5.8-6.2 and contained 20 mM citrate-sodium citrate buffer, 20 mM succinate-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-aspartate buffer (His / Asp). The buffer salt system contained 150 mg / mL of emecizumab antibody, 150 mM of arginine, and 0.5 mg / mL of poloxamer 188.

[0058] The study investigated the trends in the changes of aggregates and small molecule fragments of various formulations after being placed at a high temperature of 40°C for 28 days.

[0059] 3. Detection method for CE-SDS reduction purity

[0060] 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).

[0061] Record the retention time of each peak and the percentage of the corrected peak area for the sample, and report the purity (sum of the percentages of LC and HC peak areas) and the percentages of other peak areas.

[0062] 4. Experimental Results

[0063] After being placed at 40℃ for 28 days, 20mM histidine-hydrochloride, 20mM histidine-glutamate, 20mM histidine-acetate and 20mM histidine-aspartate showed good protective effects in inhibiting the increase of aggregates and the generation of small molecular fragments, among which histidine-acetate showed the most significant protective effect.

[0064] Example 3: Screening of Stabilizers

[0065] 1. Experimental objective: To screen for stabilizers that can stabilize proteins.

[0066] 2. Materials and Methods: A mixed solution containing 150 mg / mL emecizumab, pH 6.0, was prepared. This solution contained 20 mM histidine-aspartate buffer or 20 mM histidine-acetate buffer, 0.5 mg / mL poloxamer 188, and liquid compositions of various stabilizers, including 150 mM sodium chloride, 150 mM arginine (or arginine-aspartate) or 80 mM arginine (or arginine-aspartate), and reagent A, which consisted of 140 mM sucrose, 140 mM trehalose, 140 mM sorbitol, 140 mM mannitol, and 140 mM proline or 140 mM glycine. The trends of aggregate (SEC-HPLC) and charge variants (acidic peak charge isomers) changes were investigated after 28 days of storage at 40°C for various formulations.

[0067] 3. Methods for measuring and calculating the charge variant of emecizumab.

[0068] 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 morpholine ethanesulfonic acid (pH 6.0) and 20 mM morpholine ethanesulfonic acid + 500 mmol / L sodium chloride (pH 6.0) as the mobile phase. Separation was achieved by gradient elution in liquid chromatography (detection wavelength: 280 nm, flow rate: 0.5 mL / min, column temperature: 40 °C).

[0069] Among the detected peaks, the peak with the largest area and height is identified as the main peak. Peaks detected earlier than the main peak are collectively referred to as acidic peaks, and peaks detected later than the main peak are collectively referred to as basic peaks. Record the results of acidic peaks, main peaks, and basic peaks.

[0070] 4. Experimental Results

[0071] The results are shown in the table below. Antibodies containing 150 mM arginine, or combinations of 80 mM arginine (or arginine-aspartic acid) with sugars, alcohols, or amino acids, showed better advantages in reducing protein aggregation and acid peak formation. In particular, the combination of arginine and mannitol showed lower aggregates and acid peaks.

[0072] Example 4: Effect of mannitol concentration on aggregate and acid peak suppression

[0073] 1. Experimental objective: Based on the results of stabilizer screening, the combination of arginine and mannitol was selected as a stabilizer, and the effect of mannitol concentration on emetics aggregates and acid peaks was investigated.

[0074] 2. Materials and Methods: Liquid compositions containing 150 mg / mL emecizumab, 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 trends in aggregate (SEC-HPLC) and the acidic peaks of the charge isomers were investigated after 28 days of storage at 40°C.

[0075] 3. Experimental Results

[0076] Different concentrations of mannitol effectively inhibited changes in aggregates and acid peaks.

[0077] Example 5: Effect of arginine concentration on the inhibition of aggregates and acid peaks

[0078] 1. Experimental objective: Based on the results of stabilizer screening, the combination of arginine and mannitol was selected as a stabilizer to investigate the effect of arginine concentration on emetic benzyl aggregates and acid peaks.

[0079] 2. Materials and Methods: Liquid compositions containing 150 mg / mL emecizumab, 20 mM histidine-acetate buffer, 90 mM mannitol, 20-90 mM arginine, and 0.5 mg / mL poloxamer 188 were prepared. The changes in aggregate (SEC-HPLC) and the acidic peaks of charge isomers of various formulations after being stored at 40°C for 28 days were investigated.

[0080] 3. Experimental Results

[0081] The formulation containing 20-90 mM arginine in combination with mannitol showed comparable acid peak suppression and better aggregate suppression compared to the formulation containing only 150 mM arginine.

[0082] Example 6: Effect of histidine concentration on the inhibition of aggregates and acid peaks

[0083] 1. Experimental objective: Based on the results of stabilizer screening, the combination of arginine and mannitol was selected as a stabilizer to investigate the effect of histidine concentration on emetics aggregates and acid peaks.

[0084] 2. Materials and Methods: Liquid compositions containing 150 mg / mL emecizumab, 5-40 mM histidine-acetate buffer, 90 mM mannitol, 80 mM arginine, and 0.5 mg / mL poloxamer 188 were prepared. The changes in aggregate (SEC-HPLC) and the acidic peaks of charge isomers of various formulations after being stored at 40°C for 28 days were investigated.

[0085] 3. Experimental Results

[0086] Buffer solutions containing 5 mM or more histidine-acetic acid showed better aggregate inhibition.

[0087] Example 7: Effect of pH on the suppression of aggregates and acid peaks

[0088] 1. Experimental objective: To investigate the effect of pH on emetic benzyl aggregates and acid peaks during high-temperature storage.

[0089] 2. Materials and Methods: Liquid compositions containing 150 mg / mL emecizumab antibody, 20 mM histidine-acetate buffer, 90 mM mannitol, 80 mM arginine, and 0.5 mg / mL poloxamer 188 were prepared at pH values ​​of 5.0, 5.5, 5.7, 6.0, 6.3, and 6.5. The trends in the changes of aggregates (SEC-HPLC) and the acidic peaks of charge isomers were investigated after 28 days of storage at 40°C.

[0090] 3. Experimental Results

[0091] The samples showed better suppression of aggregates and acid peaks in the pH range of 5.5-6.5.

[0092] Example 8: Screening of Surfactant Types and Concentrations

[0093] 1. Experimental objective: To screen out the types and concentrations of surfactants that can stabilize antibodies.

[0094] 2. Materials and Methods: Liquid compositions containing 150 mg / mL emecizumab, 20 mM histidine-acetate buffer, 80 mM arginine, 90 mM mannitol, and various 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 betacyclodextrin. The changes in aggregates, surfactant content, and visible particles of various formulations after being placed at 40°C for 28 days were investigated.

[0095] 3. Method for measuring and calculating the content of polysorbate 20, polysorbate 80 and poloxamer 188 in emecizumab antibody samples

[0096] The surfactant content in the samples was detected by high-performance liquid chromatography (ELSD-HPLC) with an evaporative light scattering detector using a Waters OASIS MAX column (30 μm, 2.1 × 20 mm). The mobile phase consisted of 2% formic acid aqueous solution and 2% formic acid isopropanol, and the flow rate was 1 mL / min. The contents of poloxamer 188, polysorbate 20, and polysorbate 80 in the samples were quantitatively determined by external standard method.

[0097] 4. Experimental Results

[0098] After being placed at a high temperature of 40°C for 28 days, no significant increase in aggregates was observed, but the contents of polysorbate 20 and polysorbate 80 decreased, indicating that polysorbate 20 and polysorbate 80 were unstable and degraded in the samples. Poloxamer 188, on the other hand, showed better stability, and no obvious visible particles were generated in the samples containing surfactants.

[0099] N / A: Not detected.

[0100] Example 9: Effect of antibody concentration on formulation

[0101] 1. Experimental objective: To explore the effect of different antibody concentrations on the stability of the formulation.

[0102] 2. Materials and Methods: Mixed solutions containing 20-200 mg / mL emecizumab were prepared, which included 20 mM histidine-acetate buffer, 0.5 mg / mL poloxamer 188, 80 mM arginine, and 90 mM mannitol. The trends of changes in the aggregate (SEC-HPLC) and charge isomer acid peaks of various formulations after being stored at 40°C for 28 days were investigated.

[0103] For the method of measuring and calculating the charge variant of emecizumab, see Example 4.

[0104] 3. Experimental Results

[0105] The results showed that when the antibody concentration reached 200 mg / mL, the combination formulation containing 20 mM histidine-acetic acid buffer, 80 mM arginine and 90 mM mannitol could effectively inhibit the formation of aggregates and acid peaks.

[0106] All references to this application are incorporated herein by reference as if each reference were individually incorporated herein by reference. Furthermore, it should be understood that after reading the foregoing teachings of this application, those skilled in the art can make various alterations or modifications to this application, and these equivalent forms also fall within the scope defined by the appended claims.

[0107] Sequence List:

[0108] SEQ ID NO:1, amino acid sequence of bispecific antibody heavy chain CDR1

[0109] SEQ ID NO:2, amino acid sequence of bispecific antibody heavy chain CDR2

[0110] SEQ ID NO:3, Amino acid sequence of bispecific antibody heavy chain CDR3

[0111] SEQ ID NO:4, Amino acid sequence of bispecific antibody heavy chain CDR1

[0112] SEQ ID NO:5, Amino acid sequence of bispecific antibody heavy chain CDR2

[0113] SEQ ID NO:6, Amino acid sequence of bispecific antibody heavy chain CDR3

[0114] SEQ ID NO:7, Amino acid sequence of the bispecific antibody light chain CDR1

[0115] SEQ ID NO:8, Amino acid sequence of the bispecific antibody light chain CDR2

[0116] SEQ ID NO:9, Amino acid sequence of the light chain CDR3 of the bispecific antibody

[0117] SEQ ID NO:10, Amino acid sequence of the bispecific antibody heavy chain

[0118] SEQ ID NO:11, Amino acid sequence of the bispecific antibody heavy chain

[0119] SEQ ID NO:12, Amino acid sequence of the light chain of the bispecific antibody

[0120] SEQ ID NO:13, Amino acid sequence of the variable region of the heavy chain of the bispecific antibody

[0121] SEQ ID NO:14, Amino acid sequence of the variable region of the heavy chain of the bispecific antibody

[0122] SEQ ID NO:15, Amino acid sequence of the variable region of the light chain of the bispecific antibody

Claims

1. A bispecific antibody preparation, comprising: (1) A bispecific antibody at a concentration of 20–200 mg / mL (preferably 20–180 mg / mL, for example 20–150 mg / mL, 120–180 mg / mL, 35–150 mg / mL or 150 mg / mL), 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 comprising LCDR1-3 as shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9 respectively; (2) A buffer solution with a concentration of 5-40 mM, selected from histidine-acetic acid buffer or histidine-aspartic acid buffer. (3) A stabilizer, wherein the stabilizer is 20-90 mM arginine and one or more of the following reagents: mannitol, sucrose, trehalose, sorbitol, or amino acids (glycine, proline); and (4) 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 includes: (1) A bispecific antibody at a concentration of 20–200 mg / mL (preferably 20–180 mg / mL, for example 20–150 mg / mL, 120–180 mg / mL, 35–150 mg / mL or 150 mg / mL), 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 comprising LCDR1-3 as shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9 respectively; (2) Histidine-acetic acid buffer solution with a concentration of 5-40 mM; (3) Stabilizer, which is 90mM~300mM arginine; and (4) 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 formulation of claim 1, wherein the first polypeptide comprises an H chain containing the heavy chain variable region amino acid sequence shown in SEQ ID NO:13; the second polypeptide comprises an H chain containing the heavy chain variable region amino acid sequence shown in SEQ ID NO:14; and the third and fourth polypeptides contain the common light chain variable region amino acid sequence shown in SEQ ID NO:15, preferably wherein the first polypeptide comprises an H chain containing the amino acid sequence shown in SEQ ID NO:10; the second polypeptide comprises an H chain containing the amino acid sequence shown in SEQ ID NO:11; and the third and fourth polypeptides contain the common L chain shown 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 preparation according to any one of claims 1-2, wherein the concentration of arginine or arginine-aspartic acid 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 according to 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 according to any one of claims 1-4, wherein the surfactant is selected from poloxamer 188; more 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 according to any one of claims 1-4, wherein when the buffer solution is 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 lower, preferably 2 mM or lower, more preferably 1 mM or lower, or does not contain aspartic acid. Preferably, the bispecific antibody preparation is selected from the following: (1) Contains 150 mg / mL bispecific antibody, 20 mM histidine-acetate buffer, 150 mM arginine and 0.5 mg / mL poloxamer 188, pH 6.0; (2) Contains 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, wherein reagent A is selected from sucrose, trehalose, sorbitol, mannitol, proline or glycine, pH 6.

0. (3) Contains 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, and 140 mM of reagent A, wherein reagent A is selected from sucrose, trehalose, sorbitol, mannitol, proline or glycine, pH 6.

0. (4) Contains 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) mannitol, pH 6.0; (5) Contains 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) Contains 150 mg / mL 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) Contains 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 for reducing the neurotoxicity of a bispecific antibody preparation, comprising adding 5-90 mM of arginine or arginine-aspartic acid to the bispecific antibody preparation to reduce the aspartic acid content in the bispecific antibody preparation or to make the bispecific antibody aspartic acid-free, while adding 20-200 mM of one or more of the following reagents: 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 formulation further comprises a buffer solution 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 for inhibiting the formation of aggregates and charge variants in a preparation containing a bispecific antibody, comprising adding arginine (or arginine-aspartic acid) and one or more substances selected from sugars, alcohols or amino acids to the solution, preferably the preparation comprising a combination of arginine (or arginine-aspartic acid) and mannitol, wherein the concentration of arginine (or arginine-aspartic acid) is in the range of 20-90 mM and the concentration of mannitol is in the range of 80-200 mM, wherein the bispecific antibody is as defined in any one of claims 1-2.

10. A method for preparing a bispecific antibody formulation according to any one of claims 1-6, comprising adding a buffer solution 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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