Antibody pharmaceutical composition for treating rabies and preparation method thereof

Abstract

Pharmaceutical formulations, such as liquid formulations, comprising bispecific antibodies against rabies virus G protein and antioxidants are provided. The bispecific antibody comprises two antigen binding fragments that bind to epitope I and epitope III of rabies virus G protein, respectively, and has the activity of neutralizing rabies virus. The preparation can be used for prevention after suspected rabies virus exposure, and is administrated through injection.

Description

[0001] This application is a divisional application of Chinese patent application No. 202210357290.X, filed on April 2, 2022, with the invention name “Pharmaceutical preparation comprising bispecific antibodies against rabies virus G protein and preparation method thereof”. Technical Field

[0002] The present application generally relates to the field of pharmaceutical preparations containing antibodies. Specifically, the present application relates to a stable pharmaceutical preparation containing a bispecific antibody against rabies virus G protein and a method for preparing the same. Background Art

[0003] Rabies virus is a bullet-shaped, enveloped, single-stranded RNA virus belonging to the family Rhabdoviridae and the genus Lyssavirus. The genome of rabies virus encodes five viral proteins: RNA-dependent RNA polymerase (L), nucleoprotein (N), phosphorylated protein (P), matrix protein (M) located on the inner side of the viral protein envelope, and outer surface glycoprotein (G). The glycoprotein (G protein) of rabies virus can bind to acetylcholine, which determines the neurotropic nature of rabies virus. G protein (62-67kDa) is a type I glycoprotein composed of 505 amino acids. G protein forms protrusions covering the outer surface of the viral particle envelope. Studies have confirmed that G protein can induce virus neutralizing antibodies. G protein has at least 5 neutralizing epitopes, among which epitope II is a discontinuous spatial epitope, including amino acid residues 34-42 and 198-200, and epitope III is located at 330-338, which is a linear epitope. About 97% of the reported antibodies recognize epitopes II and III, and the rabies virus neutralizing antibody CR4098 binds to epitope III. There are very few antibodies that recognize epitopes I and IV. The rabies virus neutralizing antibody CR57 recognizes linear epitope I, i.e., positions 218-240, and the core binding domain is KLCGVL at positions 226-231. Epitope IV contains residues 251 and 264. There is also an epitope, a micro-epitope a, which is separated from epitope III by 3 amino acid residues and does not overlap with epitope III, and has only two amino acid residues 342-343.

[0004] For the prevention and treatment of rabies virus, WHO recommends that for Category III exposure and Category II or higher exposure to wild animal bites, active and passive immunotherapy should be carried out simultaneously to obtain rapid protection. Currently, bispecific antibodies against rabies virus G protein have been developed for passive immunotherapy.

[0005] As with any protein, the biological activity of antibodies depends on the conformational integrity of at least the core amino acid sequence remaining intact while protecting multiple functional groups of the protein from degradation. Both chemical and physical instabilities can lead to antibody degradation. Because antibodies are larger and more complex than traditional organic and inorganic drugs, special problems may occur in the preparation of such antibodies. Antibody stability can be affected by a variety of factors, such as pH, temperature, repeated freezing / thawing cycles and shear forces, etc. Active antibodies can be lost as a result of physical instability and chemical instability, including denaturation, aggregation (soluble and insoluble aggregate formation), precipitation and adsorption, and chemical instability including, for example, racemization, hydrolysis and deamidation, etc. Any of these instabilities can potentially lead to the formation of antibody byproducts or derivatives with reduced biological activity, increased toxicity and / or increased immunogenicity.

[0006] Although the prior art indicates many examples of excipients that may be suitable for antibody formulations that produce a particular antibody, it is not possible to predict which excipients should be added and in what amounts to overcome a particular instability problem that a particular antibody may have. Furthermore, it is difficult to find the optimal conditions for maintaining the chemical and biological stability of a particular antibody within a particular formulation. Given all the factors that can vary, finding the right excipients and optimal conditions for formulating an antibody is fraught with challenges.

[0007] Therefore, there is still a need in the art for a stable pharmaceutical preparation containing antibodies against rabies virus G protein that can be stored for a long time. SUMMARY OF THE INVENTION

[0009] On the one hand, the present application provides a pharmaceutical composition comprising about 0.2 mg / ml to about 20 mg / ml of a bispecific antibody against rabies virus G protein (hereinafter referred to as "bispecific antibody"), an osmotic pressure regulator selected from trehalose and mannitol, a metal ion chelating antioxidant, a non-ionic surfactant and the remainder water, wherein the pH value of the pharmaceutical composition is about 5.0-about 6.2, wherein the bispecific antibody comprises an antigen-binding fragment that binds to epitope I of rabies virus G protein and an antigen-binding fragment that binds to epitope III of rabies virus G protein, and the bispecific antibody has the activity of neutralizing rabies virus.

[0010] On the one hand, the present application provides a pharmaceutical composition comprising about 0.2 mg / ml to about 20 mg / ml of a bispecific antibody against rabies virus G protein (hereinafter referred to as "bispecific antibody"), a buffer salt, an osmotic pressure regulator selected from trehalose and mannitol, a metal ion chelating antioxidant, a non-ionic surfactant and the remainder water, wherein the pH value of the pharmaceutical composition is about 5.0-about 6.2, wherein the bispecific antibody comprises an antigen-binding fragment that binds to epitope I of rabies virus G protein and an antigen-binding fragment that binds to epitope III of rabies virus G protein, and the bispecific antibody has the activity of neutralizing rabies virus.

[0011] In some embodiments, the present application provides a pharmaceutical composition comprising about 0.2 mg / ml to about 20 mg / ml of the bispecific antibody, about 10 mM to about 50 mM of a buffer salt, about 100 mM to about 400 mM of an osmotic pressure regulator selected from trehalose and mannitol, about 0.2 mM to about 5 mM of a metal ion chelating antioxidant, about 0.05 mg / ml to about 1.0 mg / ml of a nonionic surfactant, and the remainder water, wherein the pH value of the pharmaceutical composition is about 5.0 to about 6.2.

[0012] On the other hand, the present application provides the use of a pharmaceutical composition in the preparation of a medicament for preventing or treating rabies, wherein the pharmaceutical composition comprises about 0.2 mg / ml to about 20 mg / ml of the bispecific antibody, an osmotic pressure regulator selected from trehalose and mannitol, a metal ion chelating antioxidant, a non-ionic surfactant and the remainder water, wherein the pH value of the pharmaceutical composition is about 5.0-about 6.2.

[0013] On the other hand, the present application provides a method for preventing or treating rabies, comprising administering a pharmaceutical composition to an individual in need thereof, wherein the pharmaceutical composition comprises about 0.2 mg / ml to about 20 mg / ml of the bispecific antibody, an osmotic pressure regulator selected from trehalose and mannitol, a metal ion chelating antioxidant, a non-ionic surfactant, and the remainder water, wherein the pH value of the pharmaceutical composition is about 5.0-about 6.2.

[0014] On the other hand, the present application provides a pharmaceutical composition for preventing or treating rabies, comprising about 0.2 mg / ml to about 20 mg / ml of the bispecific antibody, an osmotic pressure regulator selected from trehalose and mannitol, a metal ion chelating antioxidant, a non-ionic surfactant and the remainder water, wherein the pH value of the pharmaceutical composition is about 5.0-about 6.2.

[0015] In one embodiment of the present application, the pharmaceutical composition further comprises a buffer salt, such as about 10 mM to about 50 mM buffer salt.

[0016] In one embodiment of the present application, the metal ion chelating antioxidant is ethylenediaminetetraacetate.

[0017] In some embodiments, the pharmaceutical composition of the present application is used to prevent or treat rabies by implementing passive immunization after suspected rabies virus exposure. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 A-1F illustrates the effect of an osmotic pressure regulator on the stability of a pharmaceutical formulation comprising the S2E3-SCFV-FCH+C34M-IGG1K antibody.

[0019] Figure 2 A-2F illustrates the effect of antioxidants on the stability of pharmaceutical formulations containing low concentrations of S2E3-SCFV-FCH+C34M-IGG1K antibody.

[0020] Figure 3 A-3F illustrates the effect of antioxidants on the stability of pharmaceutical formulations containing high concentrations of S2E3-SCFV-FCH+C34M-IGG1K antibodies.

[0021] Figure 4 A-4F illustrates the effect of pH on the stability of a pharmaceutical formulation comprising the S2E3-SCFV-FCH+C34M-IGG1K antibody in the absence of an antioxidant.

[0022] Figure 5 A-5F illustrates the effect of pH on the stability of a pharmaceutical formulation comprising the S2E3-SCFV-FCH+C34M-IGG1K antibody in the presence of an antioxidant.

[0023] Figure 6 A-6F illustrates the effect of buffer salt on the stability of a pharmaceutical formulation comprising the S2E3-SCFV-FCH+C34M-IGG1K antibody.

[0024] Figure 7 A-7E illustrates the effect of polysorbate 80 concentration on the amount of insoluble particulates and the level of SEC aggregates in a drug formulation comprising the S2E3-SCFV-FCH+C34M-IGG1K antibody in a shaking experiment.

[0025] Figure 8A-8F illustrates the effect of the presence of polysorbate 80 on the stability of a pharmaceutical formulation comprising the S2E3-SCFV-FCH+C34M-IGG1K antibody.

[0026] Fig. 9 A-9F illustrates the effect of the concentration of S2E3-SCFV-FCH+C34M-IGG1K antibody on the stability of a pharmaceutical formulation comprising S2E3-SCFV-FCH+C34M-IGG1K antibody in an accelerated stability experiment.

[0027] Fig.10 A-10F illustrates the effect of the concentration of the S2E3-SCFV-FCH+C34M-IGG1K antibody on the stability of a pharmaceutical formulation comprising the S2E3-SCFV-FCH+C34M-IGG1K antibody in a long-term stability experiment. DETAILED DESCRIPTION OF THE INVENTION

[0029] Bispecific antibodies against rabies virus G protein

[0030] The bispecific antibody against rabies virus G protein of the present application comprises two antigen-binding fragments that bind to different epitopes of rabies virus G protein, particularly comprising an antigen-binding fragment that binds to epitope I of rabies virus G protein and an antigen-binding fragment that binds to epitope III of rabies virus G protein, and the bispecific antibody has the activity of neutralizing rabies virus. Chinese Patent Application No. 201910706710.9 has described the structure, amino acid sequence, preparation method and biological activity of the bispecific antibody against rabies virus G protein of the present application, and the entire contents are hereby incorporated herein by reference in their entirety.

[0031] In some embodiments, the antigen-binding fragment that binds to epitope I of rabies virus G protein comprises:

[0032] HCDR1 with an amino acid sequence of RYTIN (SEQ ID NO: 13), HCDR2 with an amino acid sequence of GIIPIFGTANYAQRFQG (SEQ ID NO: 8), HCDR3 with an amino acid sequence of ENLDNSGTYYYFSGWFDP (SEQ ID NO: 9), LCDR1 with an amino acid sequence of TGTSSDIGAYDYVS (SEQ ID NO: 14), LCDR2 with an amino acid sequence of DATKRPS (SEQ ID NO: 11), and LCDR3 with an amino acid sequence of CSYAGDYTPGVV (SEQ ID NO: 12); or

[0033] HCDR1 with an amino acid sequence of RYSIN (SEQ ID NO: 15), HCDR2 with an amino acid sequence of GIIPIFGTANYAQRFQG (SEQ ID NO: 8), HCDR3 with an amino acid sequence of ENLDNSGTYYYFSGWFDP (SEQ ID NO: 9), LCDR1 with an amino acid sequence of TGTSSDIDGYDFVS (SEQ ID NO: 16), LCDR2 with an amino acid sequence of DATKRPS (SEQ ID NO: 11), and LCDR3 with an amino acid sequence of CSYAGDYTPGVV (SEQ ID NO: 12); or

[0034] HCDR1 with an amino acid sequence of GYTIN (SEQ ID NO: 7), HCDR2 with an amino acid sequence of GIIPIFGTANYAQRFQG (SEQ ID NO: 8), HCDR3 with an amino acid sequence of ENLDNSGTYYYFSGWFDP (SEQ ID NO: 9), LCDR1 with an amino acid sequence of TGTSSDLGGYDFVS (SEQ ID NO: 10), LCDR2 with an amino acid sequence of DATKRPS (SEQ ID NO: 11), and LCDR3 with an amino acid sequence of CSYAGDYTPGVV (SEQ ID NO: 12);

[0035] The HCDR and LCDR amino acid sequences are based on the Kabat definition.

[0036] In some embodiments, the antigen-binding fragment that binds to epitope III of rabies virus G protein comprises:

[0037] HCDR1 with an amino acid sequence of SYGMH (SEQ ID NO: 1), HCDR2 with an amino acid sequence of TISYDGSIKDYADSVKG (SEQ ID NO: 2), HCDR3 with an amino acid sequence of GDRTGNLDY (SEQ ID NO: 3), LCDR1 with an amino acid sequence of RASQNIRNALN (SEQ ID NO: 4), LCDR2 with an amino acid sequence of DASTRQS (SEQ ID NO: 5), and LCDR3 with an amino acid sequence of QQNSEFPPT (SEQ ID NO: 6);

[0038] The HCDR and LCDR amino acid sequences are based on the Kabat definition.

[0039] In some embodiments, the amino acid sequence of the heavy chain variable region of the antigen-binding fragment that binds to epitope I of rabies virus G protein is as shown in SEQ ID NO: 22, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 23; or

[0040] The amino acid sequence of the heavy chain variable region of the antigen-binding fragment that binds to epitope I of rabies virus G protein is as shown in SEQ ID NO: 24, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 25; or

[0041] The amino acid sequence of the heavy chain variable region of the antigen binding fragment that binds to epitope I of rabies virus G protein is shown in SEQ ID NO: 26, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 27.

[0042] In some embodiments, the amino acid sequence of the heavy chain variable region of the antigen-binding fragment that binds to epitope III of rabies virus G protein is as shown in SEQ ID NO: 28, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 29.

[0043] In some embodiments, the amino acid sequence of the heavy chain variable region of the antigen-binding fragment that binds to epitope I of rabies virus G protein is as shown in SEQ ID NO: 22, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 23, the amino acid sequence of the heavy chain variable region of the antigen-binding fragment that binds to epitope III of rabies virus G protein is as shown in SEQ ID NO: 28, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 29; or

[0044] The amino acid sequence of the heavy chain variable region of the antigen-binding fragment that binds to epitope I of rabies virus G protein is as shown in SEQ ID NO: 24, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 25; the amino acid sequence of the heavy chain variable region of the antigen-binding fragment that binds to epitope III of rabies virus G protein is as shown in SEQ ID NO: 28, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 29; or

[0045] The amino acid sequence of the heavy chain variable region of the antigen-binding fragment that binds to epitope I of rabies virus G protein is shown in SEQ ID NO: 26, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 27. The amino acid sequence of the heavy chain variable region of the antigen-binding fragment that binds to epitope III of rabies virus G protein is shown in SEQ ID NO: 28, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 29.

[0046] In some embodiments, the formats of the two antigen-binding fragments are independently selected from single-chain antibodies (scFv) or Fab fragments.

[0047] In some embodiments, the antigen-binding fragment that binds to epitope I of rabies virus G protein is a single-chain antibody (scFv), and the antigen-binding fragment that binds to epitope III of rabies virus G protein is a Fab fragment.

[0048] In some embodiments, the bispecific antibody comprises the amino acid sequence shown in one of SEQ ID NOs: 17, 20, and 21. In some embodiments, the bispecific antibody comprises the amino acid sequence shown in SEQ ID NO: 18 and SEQ ID NO: 19.

[0049] Pharmaceutical composition

[0050] On the one hand, the present application provides a pharmaceutical composition comprising about 0.2 mg / ml to about 20 mg / ml of the bispecific antibody, an osmotic pressure regulator selected from trehalose and mannitol, a metal ion chelating antioxidant, a nonionic surfactant, and the remainder water, wherein the pH value of the pharmaceutical composition is about 5.0 to about 6.2.

[0051] On the one hand, the present application provides a pharmaceutical composition comprising about 0.2 mg / ml to about 20 mg / ml of the bispecific antibody, a buffer salt, an osmotic pressure regulator selected from trehalose and mannitol, a metal ion chelating antioxidant, a nonionic surfactant, and the remainder water, wherein the pH value of the pharmaceutical composition is about 5.0 to about 6.2.

[0052] In some embodiments, the present application provides a pharmaceutical composition comprising about 0.2 mg / ml to about 20 mg / ml of the bispecific antibody, about 10 mM to about 50 mM of a buffer salt, about 100 mM to about 400 mM of an osmotic pressure regulator selected from trehalose and mannitol, about 0.2 mM to about 5 mM of a metal ion chelating antioxidant, about 0.05 mg / ml to about 1.0 mg / ml of a nonionic surfactant, and the remainder water, wherein the pH value of the pharmaceutical composition is about 5.0 to about 6.2.

[0053] In some embodiments, the present application provides a pharmaceutical composition comprising about 0.5 mg / ml to about 2.0 mg / ml of the bispecific antibody, about 10 mM to about 50 mM of a buffer salt, about 100 mM to about 400 mM of an osmotic pressure regulator selected from trehalose and mannitol, about 0.2 mM to about 5 mM of a metal ion chelating antioxidant, about 0.05 mg / ml to about 1.0 mg / ml of a nonionic surfactant, and the remainder water, wherein the pH value of the pharmaceutical composition is about 5.2 to about 6.2.

[0054] In some embodiments, in the pharmaceutical composition, the content of the bispecific antibody is about 0.2 mg / ml to about 20 mg / ml, about 0.5 mg / ml to about 2.0 mg / ml, about 0.5 mg / ml to about 1.0 mg / ml, about 1.0 mg / ml to about 2.0 mg / ml, about 0.5 mg / ml, about 1.0 mg / ml, about 1.5 mg / ml or about 2.0 mg / ml. Alternatively, those skilled in the art can select a suitable content of the bispecific antibody according to practical needs.

[0055] In some embodiments, the buffer salt may be a commonly used buffer salt in the art for preparing liquid preparations, examples of which include but are not limited to phosphate / phosphate buffer pairs, histidine / histidine inorganic salt buffer pairs, acetate / acetate buffer pairs, citric acid / citrate buffer pairs, etc. In some embodiments, the buffer salt is a histidine / histidine inorganic salt buffer pair or an acetate / acetate buffer pair, such as a histidine / histidine hydrochloride buffer pair or an acetate / sodium acetate buffer pair. In some embodiments, the buffer salt is a histidine / histidine hydrochloride buffer pair.

[0056] In some embodiments, the concentration of the buffer salt in the pharmaceutical composition is about 10mM to about 50mM, about 15mM to about 30mM, about 15mM to about 25mM, about 15mM to about 20mM, about 20mM to about 25mM, or about 20mM. In some embodiments, the buffer salt is a histidine / histidine hydrochloride buffer pair or an acetate / sodium acetate buffer pair having a concentration of about 10mM to about 50mM, about 15mM to about 30mM, about 15mM to about 25mM, about 15mM to about 20mM, about 20mM to about 25mM, or about 20mM. Alternatively, those skilled in the art can select a suitable concentration of histidine / histidine hydrochloride buffer pair or an acetate / sodium acetate buffer pair according to the needs of practice. When the histidine / histidine hydrochloride buffer pair and the acetic acid / sodium acetate buffer pair are selected, the bispecific antibody in the pharmaceutical composition aggregates slowly, and the buffer salt can maintain the pH of the pharmaceutical composition and can be quickly neutralized to a physiological pH when entering the human body during clinical use.

[0057] In some embodiments, the concentration of the osmotic pressure regulator in the pharmaceutical composition is about 100mM-about 400mM, about 200mM-about 300mM, about 200mM-about 250mM, about 230mM-about 260mM or about 250mM-about 300mM, for example, about 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300mM. The osmotic pressure regulator is selected from trehalose and mannitol, preferably trehalose. Trehalose and mannitol have a solvent exclusion effect in the pharmaceutical preparation comprising the bispecific antibody of the present application, which can improve the stability of the bispecific antibody in aqueous solution and inhibit the aggregation of the bispecific antibody.

[0058] In some embodiments, in the pharmaceutical composition, examples of the metal ion chelating antioxidant include, but are not limited to, ethylenediaminetetraacetate, ethylene glycol tetraacetic acid (EGTA), thiamine tetrahydrogen disulfide (TTFD), 2,3-dithioethanediol (DMSA), diethyltriamine pentaacetic acid or dimercaprol. In some embodiments, the ethylenediaminetetraacetate may be an alkali metal salt or alkaline earth metal salt of ethylenediaminetetraacetic acid, such as disodium ethylenediaminetetraacetate or sodium calcium ethylenediaminetetraacetate. In some embodiments, the concentration of the metal ion chelating antioxidant (e.g., ethylenediaminetetraacetate) is about 0.2mM-about 5mM, about 0.5mM-about 5mM, about 0.5mM-about 2.0mM, 0.5mM-about 1.5mM, about 0.5mM-about 1.0mM, about 1.0mM-about 1.5mM or about 1.0mM. In some embodiments, the concentration of the metal ion chelating antioxidant (e.g., ethylenediaminetetraacetate) is about 0.5, about 1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, or 5.0 mM. Alternatively, those skilled in the art can select a suitable concentration of a metal ion chelating antioxidant (e.g., ethylenediaminetetraacetate) according to practical needs.

[0059] In some embodiments, in the pharmaceutical composition, the nonionic surfactant may be a polysorbate surfactant, such as polysorbate 20 or polysorbate 80. In some embodiments, the content of the nonionic surfactant (e.g., a polysorbate surfactant, such as polysorbate 20 or polysorbate 80) is about 0.05 mg / ml to about 1.0 mg / ml, about 0.1 mg / ml to about 1.0 mg / ml, about 0.2 mg / ml to about 0.5 mg / ml, about 0.3 mg / ml to about 1.0 mg / ml, or about 0.5 mg / ml to about 1.0 mg / ml, for example, about 0.3 mg / ml, 0.34 mg / ml, about 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml, or 1.0 mg / ml. As a protein, the bispecific antibody may be denatured at the interface (air / liquid interface, liquid / glass interface, etc.) due to hydrophobic interaction, resulting in protein aggregation or even precipitation. The use of a nonionic surfactant (e.g., a polysorbate surfactant, such as polysorbate 20 or polysorbate 80) can prevent protein aggregation and precipitation during shaking, stirring or freeze-thawing.

[0060] In some embodiments, the pH of the pharmaceutical composition is about 5.0 to about 6.2, about 5.0 to about 6.0, about 5.2 to about 6.2, about 5.5 to about 6.0, or about 5.5 to about 5.8, e.g., about 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0.

[0061] In some embodiments, the water in the pharmaceutical composition can be sterile pyrogen-free water, such as water for injection.

[0062] In some embodiments, the present application provides a pharmaceutical composition comprising about 0.5 mg / ml to about 2.0 mg / ml of the bispecific antibody, about 10 mM to about 50 mM of a histidine / histidine hydrochloride buffer pair or an acetic acid / sodium acetate buffer pair, about 100 mM to about 400 mM of an osmotic pressure regulator selected from trehalose and mannitol, about 0.2 mM to about 5 mM of ethylenediaminetetraacetate, about 0.05 mg / ml to about 1.0 mg / ml of polysorbate 80, and the remainder of water, wherein the pH value of the pharmaceutical composition is about 5.5 to about 6.0.

[0063] In some embodiments, the present application provides a pharmaceutical composition comprising about 0.5 mg / ml to about 2.0 mg / ml of the bispecific antibody, about 15 mM to about 25 mM of a histidine / histidine hydrochloride buffer pair or an acetic acid / sodium acetate buffer pair, about 200 mM to about 300 mM of an osmotic pressure regulator selected from trehalose and mannitol, about 0.5 mM to about 5 mM of ethylenediaminetetraacetate, about 0.3 mg / ml to about 1.0 mg / ml of polysorbate 80, and the remainder of water, wherein the pH value of the pharmaceutical composition is about 5.5 to about 6.0.

[0064] In some embodiments, the present application provides a pharmaceutical composition comprising about 0.5 mg / ml to about 2.0 mg / ml of the bispecific antibody, about 10 mM to about 50 mM of histidine / histidine hydrochloride buffer pair, about 100 mM to about 400 mM of trehalose, about 0.2 mM to about 5 mM of ethylenediaminetetraacetate, about 0.05 mg / ml to about 1.0 mg / ml of polysorbate 80, and the remainder of water, wherein the pH value of the pharmaceutical composition is about 5.5 to about 6.0.

[0065] In some embodiments, the present application provides a pharmaceutical composition comprising about 0.5 mg / ml to about 2.0 mg / ml of the bispecific antibody, about 15 mM to about 25 mM of histidine / histidine hydrochloride buffer pair, about 200 mM to about 300 mM of trehalose, about 0.5 mM to about 5 mM of ethylenediaminetetraacetate, about 0.3 mg / ml to about 1.0 mg / ml of polysorbate 80, and the remainder of water, wherein the pH value of the pharmaceutical composition is about 5.5 to about 6.0.

[0066] In some embodiments, the present application provides a pharmaceutical composition comprising about 0.5 mg / ml to about 2.0 mg / ml of the bispecific antibody, about 20 mM histidine / histidine hydrochloride buffer pair, about 240 mM trehalose, about 1.0 mM ethylenediaminetetraacetate, about 0.5 mg / ml-about 1.0 mg / ml polysorbate 80, and the remainder water, wherein the pH value of the pharmaceutical composition is about 5.5-about 5.8.

[0067] The pharmaceutical composition of the present application is a liquid preparation, which can be in the form of a solution, emulsion or suspension, preferably a solution. The pharmaceutical composition of the present application can be administered to an individual in need by a parenteral administration route, such as an injection administration route. The injection administration route includes but is not limited to subcutaneous injection (e.g., wound subcutaneous infiltration injection), intramuscular injection and intradermal injection, etc.

[0068] After the pharmaceutical composition comprising the bispecific antibody of the present application is placed at 5°C±3°C for 6 months, the SEC-HPLC monomer peak area%, CEX-HPLC acidic peak area%, CE-SDS (non-reducing method) and activity level are substantially unchanged. The pharmaceutical composition comprising the bispecific antibody of the present application can be stored for a long time at 5°C±3°C under light-proof conditions, and can maintain stable quality, thereby ensuring the safety, effectiveness and uniformity of the drug.

[0069] It should be understood that the above detailed description is only for the purpose of enabling those skilled in the art to more clearly understand the content of the present application, and is not intended to be limiting in any aspect. Those skilled in the art can make various modifications and changes to the embodiments.

[0070] The following examples are only for the purpose of illustrating and not limiting the scope of the present application. In the following examples, the S2E3-scFv-FcH+C34m-IgG1K antibody disclosed in CN201910706710.9 is taken as an example of the bispecific antibody of the present application. Example

[0071] Example 1: Effect of osmotic pressure regulator on the stability of a pharmaceutical formulation containing S2E3-SCFV-FCH+C34M-IGG1K antibody

[0072] Take an appropriate amount of S2E3-SCFV-FCH+C34M-IGG1K antibody sample, replace the solution with pure water by ultrafiltration, and dilute to 2 mg / ml with purified water to obtain S2E3-SCFV-FCH+C34M-IGG1K antibody aqueous solution. Prepare 2× preparation buffer mother solution (containing 2 times the concentration of buffer salt and osmotic pressure regulator, pH is about 6.0) of each formula shown in Table 1. Then, take 7ml of S2E3-SCFV-FCH+C34M-IGG1K antibody aqueous solution, 7ml of 2× preparation buffer mother solution and 28μL of 10% polysorbate 80 solution, mix well, and prepare preparation samples with osmotic pressure regulators of trehalose, mannitol, sucrose, arginine hydrochloride, glycine and proline, as well as preparation samples without osmotic pressure regulators.

[0073] Table 1 Liquid formulation composition

[0074]

[0075] √: Indicates that the prescription contains the corresponding substance; -: Indicates that the prescription does not contain the corresponding substance;

[0076] 20mM Histidine Salt is a buffer system consisting of histidine and histidine hydrochloride containing 20mM histidine / histidine ions

[0077] The samples of each formulation were stored at high temperature (37±2°C) for stability testing to accelerate the degradation of the S2E3-SCFV-FCH+C34M-IGG1K antibody, and samples were taken at day 0, 2 weeks, 4 weeks, 6 weeks and 8 weeks. The purity of the formulation samples at each time point was determined by SEC-HPLC, CEX-HPLC, non-reduced CE-SDS and reduced CE-SDS methods as described below. The Tm value of each formulation sample was determined using the DSF method to determine its conformational stability properties. The test results are shown in Figure 1 A to Figure 1 F.

[0078] The SEC-HPLC (size exclusion chromatography) purity was determined using a TSKgel G3000SWXL column and a high performance liquid chromatograph. The mobile phase was 50 mM PB / 300 mM NaCl, pH 7.0 solution. The sample was diluted and injected, and isocratic elution was performed at a flow rate of 1.0 ml / min for 15 min. The detection was performed at an ultraviolet wavelength of 280 nm, and the peak area percentages of monomers and aggregates were obtained by peak area normalization.

[0079] CEX-HPLC (cation exchange chromatography) purity determination using MabPac TM SCX-10BioLC TM Analytical chromatographic column and high performance liquid chromatograph, mobile phase A is 20mM MES, pH 5.6 solution, mobile phase B is 20mM MES+0.5MNaCl, pH5.6 solution, the sample is diluted and injected, gradient elution is performed at a flow rate of 0.5ml / min, elution is 50min, detection is performed at 280nm ultraviolet wavelength, and the peak area percentages of acidic peak, main peak and basic peak are obtained by peak area normalization method.

[0080] The non-reducing CE-SDS (sodium dodecyl sulfate capillary electrophoresis) and reducing CE-SDS methods were determined using a capillary electrophoresis instrument, using an uncoated capillary column with an effective length of 20 cm and SDS-MW Gel separation gel, a column temperature of 25°C, a separation voltage of 15 kV, and a detection wavelength of 214 nm. The purity of the samples was determined under non-reducing and reducing conditions, respectively.

[0081] DSF (differential scanning fluorescence) was measured using a Real-Time PCR instrument. The sample was diluted and mixed with a fluorescent dye ( Orange Protein gel stain, Life) was mixed and transferred to a PCR tube, and a heating program (25°C to 95°C, 1°C / min) was set. Fluorescence was collected in real time and data was recorded. The fluorescence-temperature curve was derived, and the highest peak of the derived curve was the Tm value.

[0082] Table 2 Effect of osmotic pressure regulator on Tm value of drug formulation containing S2E3-SCFV-FCH+C34M-IGG1K antibody

[0083] Preparation No. H60T H60 H60M H60S H60R H60G H60P Tm value (℃) 61.1 59.9 60.6 60.9 58.8 60.6 60.2

[0084] From the above table and Figure 1 A to Figure 1 As can be seen in F, different osmotic pressure regulators have an effect on the T of S2E3-SCFV-FCH+C34M-IGG1K antibody. m The value was less affected, with a change of less than 2°C, all around 60°C. The preparation T of arginine hydrochloride m The value is slightly lower. Under high temperature storage conditions, sucrose, arginine hydrochloride, glycine and proline increase the level of SEC-HPLC aggregates and CEX-HPLC basic peaks in the liquid preparation containing S2E3-SCFV-FCH+C34M-IGG1K antibody at a relatively fast rate, and the level of non-reducing CE-SDS purity decreases at a relatively fast rate. Therefore, these osmotic pressure regulators are not suitable for preparing liquid preparations containing S2E3-SCFV-FCH+C34M-IGG1K antibody. The preparation samples using trehalose and mannitol are relatively stable under high temperature storage conditions.

[0085] Example 2. Effect of antioxidants on the stability of pharmaceutical formulations containing S2E3-SCFV-FCH+C34M-IGG1K antibodies

[0086] Liquid formulations H60T, H60TE and H60TM were prepared according to the formulation compositions shown in the following table using a preparation method similar to that described in Example 1, except that 1 mM disodium ethylenediaminetetraacetate and 1 mM methionine were also added as antioxidants.

[0087] Table 3 Liquid formulation composition

[0088]

[0089] √: Indicates that the prescription contains the corresponding substance; -: Indicates that the prescription does not contain the corresponding substance;

[0090] 20mM Histidine Salt is a buffer system consisting of histidine and histidine hydrochloride containing 20mM histidine / histidine ions

[0091] The purity of each preparation sample was tested using storage conditions, sampling time points and test items similar to those described in Example 1. The test results are shown in the following table and Figure 2 A to Figure 2F.

[0092] Table 4 Effect of antioxidants on the Tm value of the drug formulation containing S2E3-SCFV-FCH+C34M-IGG1K antibody

[0093] Preparation No. H60T H60TE H60TM Tm value (℃) 61.1 61.1 61.2

[0094] From the above table and Figure 2 A to Figure 2 As can be seen in F, the addition of antioxidants does not affect the Tm value of S2E3-SCFV-FCH+C34M-IGG1K antibody. Both disodium EDTA and methionine can reduce the rate of increase of SEC aggregates, and the trend of CEX purity decrease will not change, but methionine will accelerate the rate of decrease of non-reduced CE-SDS purity.

[0095] The above experimental steps were repeated using the following formulation to further investigate the effect of antioxidants on the stability of the drug formulation containing S2E3-SCFV-FCH+C34M-IGG1K antibody. Figure 3 A to Figure 3 F.

[0096] Table 5 Liquid formulation composition

[0097]

[0098] √: Indicates that the prescription contains the corresponding substance;

[0099] 20mM Histidine Salt is a buffer system consisting of histidine and histidine hydrochloride containing 20mM histidine / histidine ions

[0100] from Figure 3 A to Figure 3 As can be seen from F, in the drug preparation containing a higher concentration of S2E3-SCFV-FCH+C34M-IGG1K antibody, when L-methionine was used as an antioxidant, the purity of SEC-HPLC, CEX-HPLC and non-reduced CE-SDS decreased faster under high temperature (37±2°C) conditions.

[0101] It can be seen that disodium EDTA is more suitable than L-methionine as an antioxidant in a pharmaceutical preparation containing S2E3-SCFV-FCH+C34M-IGG1K antibody.

[0102] Example 3. Effect of pH on the stability of a pharmaceutical formulation containing S2E3-SCFV-FCH+C34M-IGG1K antibody

[0103] Liquid formulations H55T, H58T, H60T and H62T were prepared according to the formulation compositions shown in the following table using a preparation method similar to that described in Example 1, except that the final pH of the formulation was adjusted to 5.5-6.2.

[0104] Table 6 Liquid formulation composition

[0105]

[0106] √: Indicates that the prescription contains the corresponding substance;

[0107] 20mM Histidine Salt is a buffer system consisting of histidine and histidine hydrochloride containing 20mM histidine / histidine ions

[0108] The purity of each preparation sample was tested using storage conditions, sampling time points and test items similar to those described in Example 1. The test results are shown in the following table and Figure 4 A to Figure 4 F.

[0109] Table 7 Effect of pH on Tm value of drug formulations containing S2E3-SCFV-FCH+C34M-IGG1K antibody

[0110] Preparation No. H55T H58T H60T H62T Tm value (℃) 60.5 60.9 61.1 60.9

[0111] From the above table and Figure 4 A to Figure 4 As can be seen in F, the Tm value of the S2E3-SCFV-FCH+C34M-IGG1K antibody is basically unaffected by changes in the pH value of the preparation. In the pH range of 5.5 to 6.2, under high temperature storage conditions, except for the rapid decrease in purity of the preparation at pH 6.2, the increase in aggregates of the preparation at pH 5.8 was the slowest, while the preparations at pH 5.5, 5.8, and 6.0 showed almost no difference in the decreasing trend of purity by CEX-HPLC and non-reduced CE-SDS.

[0112] The above experimental steps were repeated using the following formulation to further investigate the effect of pH changes on the stability of the pharmaceutical preparation containing S2E3-SCFV-FCH+C34M-IGG1K antibody in the presence of antioxidants. Figure 5 A to Figure 5 F.

[0113] Table 8 Liquid formulation composition

[0114]

[0115]

[0116] √: Indicates that the prescription contains the corresponding substance;

[0117] 20mM Histidine Salt is a buffer system consisting of histidine and histidine hydrochloride containing 20mM histidine / histidine ions

[0118] from Figure 5 A to Figure 5 As can be seen in F, in the pH range of 5.2 to 6.2, there is basically no difference in the trend of decreasing purity between SEC-HPLC and non-reduced CE-SDS. The CEX alkaline peak increases slightly faster in the preparation samples at pH 5.2 and pH 5.5. Therefore, in the pH range of 5.2 to 6.2, the stability of the drug preparation containing S2E3-SCFV-FCH+C34M-IGG1K antibody will not be significantly affected.

[0119] Example 4. Effect of buffer system on the stability of a pharmaceutical formulation containing S2E3-SCFV-FCH+C34M-IGG1K antibody

[0120] Liquid formulations H58T, A50T, A52T, A55T and A58T were prepared using a preparation method similar to that described in Example 1 and according to the formulation compositions shown in the table below, except that an acetate buffer system was used instead of a histidine buffer system and the final pH of the formulation was adjusted to 5.0-5.8.

[0121] Table 9 Liquid formulation composition

[0122]

[0123] √: Indicates that the prescription contains the corresponding substance;

[0124] 20 mM histidine salt is a buffer system composed of histidine and histidine hydrochloride containing 20 mM histidine / histidine ions;

[0125] 20mM Acetate: A buffer system consisting of glacial acetic acid and sodium acetate containing 20mM acetic acid / acetate.

[0126] The stability of each formulation sample was tested using storage conditions, sampling time points, and test items similar to those described in Example 1. The test results are shown in the following table and Figure 6 A to Figure 6 F.

[0127] Table 10 Effect of buffer system on Tm value of drug formulation containing S2E3-SCFV-FCH+C34M-IGG1K antibody

[0128] Preparation No. H58T A50T A52T A55T A58T Tm value (℃) 60.9 61.1 61.2 60.9 60.4

[0129] 0026]du

[0130] From the above table and Figure 6 A to Figure 6 As can be seen in F, the T of S2E3-SCFV-FCH+C34M-IGG1K antibody in acetate and histidine buffers m After 8 weeks at 37°C, the rate of increase in SEC aggregates of S2E3-SCFV-FCH+C34M-IGG1K antibody in acetate buffer was slightly higher than that in histidine buffer at pH 5.8 in the pH range of 5.0-5.8, but the decreasing trend of CEX-HPLC and non-reducing CE-SDS purity of the two was basically the same.

[0131] Example 5. Effect of polysorbate 80 on the stability of a pharmaceutical formulation containing S2E3-SCFV-FCH+C34M-IGG1K antibody

[0132] Take an appropriate amount of S2E3-SCFV-FCH+C34M-IGG1K antibody sample, replace the solution with buffer salt buffer (20mM histidine / histidine hydrochloride, pH5.8), and dilute to 2.5mg / ml with buffer salt buffer to obtain S2E3-SCFV-FCH+C34M-IGG1K antibody solution. Prepare 5× preparation buffer mother solution (containing 1 times concentration of buffer salt, 5 times concentration of trehalose and disodium ethylenediaminetetraacetic acid, pH is about 5.8) of the prescription shown in Table 11. Then, take 32ml of S2E3-SCFV-FCH+C34M-IGG1K antibody solution and 8ml of 5× preparation buffer mother solution and mix well. Take 5 ml of each solution and add a certain volume of 10% polysorbate 80 to prepare preparation samples containing 0%, 0.005%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05% and 0.10% polysorbate 80 respectively.

[0133] Table 11 Formula composition

[0134]

[0135] "%" in Table 11 means weight / volume percentage or %w / v. For example, 0.05% polysorbate 80 means that the content of polysorbate 80 in the formulation is 0.5 mg / mL. In addition, the formulation containing 0.05% polysorbate 80 in Table 11 is numbered as H58TE(2) 0.05% Tween 80, and the formulation not containing polysorbate 80 in Table 11 is numbered as H58TE-ps(2) without Tween 80.

[0136] Each formulation sample in Table 11 was subjected to a shaking test. After shaking at a rate of 1500 times / min for 3 days, the number of insoluble particles (light obscuration method) and the level of SEC-HPLC aggregates in each sample were detected. The results of the shaking test are shown in Figure 7 A-7E. When the preparation sample did not contain polysorbate 80, the number of insoluble particles and the proportion of SEC aggregates in the sample increased significantly after shaking for 3 days, indicating that the preparation containing S2E3-SCFV-FCH+C34M-IGG1K antibody is easily affected by mechanical forces such as shaking and causes protein aggregation. When 0.005% to 0.10% polysorbate 80 was added to the preparation containing S2E3-SCFV-FCH+C34M-IGG1K antibody, the number of insoluble particles in the sample did not increase after shaking for 3 days. The SEC-HPLC aggregate level of the preparation sample containing 0.005% to 0.02% polysorbate 80 increased after shaking. When the concentration of polysorbate 80 in the preparation increases to 0.03% and above, the proportion of SEC-HPLC aggregates in the preparation sample will not increase basically.

[0137] The stability of the preparation samples numbered H58TE (2) 0.05% Tween 80 and H58TE-ps (2) without Tween 80 was tested using storage conditions, sampling time points and test items similar to those described in Example 1 to investigate the effect of the degradation products of polysorbate 80 that may be produced during storage on the stability of the preparations. The experimental results are shown in Figure 8 A-8F. At 37°C, there was basically no difference in the decreasing trend of SEC-HPLC, CEX-HPLC and non-reduced CE-SDS purity between the formulation samples containing 0.5 mg / ml polysorbate 80 and the formulation samples without polysorbate 80, indicating that the stability of the drug formulation containing S2E3-SCFV-FCH+C34M-IGG1K antibody was basically not affected by the degradation products that may be produced by polysorbate 80 during storage.

[0138] Example 6. Effect of protein concentration on the stability of a pharmaceutical formulation containing S2E3-SCFV-FCH+C34M-IGG1K antibody

[0139] Take an appropriate amount of S2E3-SCFV-FCH+C34M-IGG1K antibody stock solution (containing 20 mM histidine / histidine hydrochloride, 90 mg / ml trehalose, 1 mM disodium ethylenediaminetetraacetic acid, 0.5 mg / ml polysorbate 80, pH 5.8), dilute it to 20 mg / ml, 1 mg / ml, 0.5 mg / ml, and 0.2 mg / ml using formulation buffer, mix well, and prepare formulation samples with different protein concentrations.

[0140] Table 12 Formulation

[0141]

[0142] The samples of each formulation were stored at 25±2°C for accelerated stability testing, and samples were taken at day 0, 1 month, 2 months, 3 months and 6 months, and the levels of SEC-HPLC, CEX-HPLC and non-reduced CE-SDS in the samples were tested using test conditions similar to those described in Example 1. The test results are shown in Fig. 9 A to Fig. 9 F. In addition, samples of each formulation were stored at 5±3°C for long-term stability testing, and samples were taken at day 0, 3 months, 6 months, 9 months and 12 months, and the levels of SEC-HPLC, CEX-HPLC and non-reduced CE-SDS in the samples were tested using test conditions similar to those described in Example 1. The test results are shown in Fig.10 A to Fig.10 F.

[0143] from Fig. 9 A to Fig.10 As can be seen in F, under accelerated conditions (25±2℃), the SEC aggregate ratio of the preparation samples with S2E3-SCFV-FCH+C34M-IGG1K antibody concentrations of 0.2mg / ml and 20mg / ml increased rapidly in the first 3 months, and the purity change trends of CEX-HPLC and non-reduced CE-SDS of the preparation samples with four different protein concentrations were consistent. At 5±3℃, the SEC aggregate ratio of the preparation sample with S2E3-SCFV-FCH+C34M-IGG1K antibody concentration of 0.2mg / ml was higher, while the SEC aggregate ratio change trends of the preparation samples with S2E3-SCFV-FCH+C34M-IGG1K antibody concentrations of 0.5mg / ml, 1.0mg / ml and 20mg / ml were basically the same. The purity change trends of CEX-HPLC and non-reduced CE-SDS of the preparation samples with four different protein concentrations were consistent.

[0144] It can be seen that when the S2E3-SCFV-FCH+C34M-IGG1K antibody preparation samples with concentrations of 0.2 mg / ml, 0.5 mg / ml, 1.0 mg / ml and 20 mg / ml were placed under accelerated (25±2°C) and long-term (5±3°C) conditions, there was no difference in the purity changes of CEX-HPLC and non-reduced CE-SDS, while the S2E3-SCFV-FCH+C34M-IGG1K antibody preparation sample with a concentration of 0.2 mg / ml had a higher aggregate level under long-term (5±3°C) conditions.

Claims

1. A pharmaceutical composition comprising 0.2 mg / ml to 20 mg / ml of a bispecific antibody against rabies virus G protein, 15 mM to 25 mM histidine / histidine hydrochloride buffer pair, 200 mM to 300 mM trehalose, 0.5 mM to 1.5 mM ethylenediaminetetraacetate, 0.1 mg / ml to 1.0 mg / ml of a polysorbate-type surfactant, and the remainder of water, wherein the pH value of the pharmaceutical composition is 5.5 to 6.0, wherein the bispecific antibody comprises an antigen-binding fragment that binds to epitope I of rabies virus G protein and an antigen-binding fragment that binds to epitope III of rabies virus G protein, and the bispecific antibody has the activity of neutralizing rabies virus; The antigen-binding fragment that binds to epitope III of rabies virus G protein comprises: HCDR1 with an amino acid sequence of SYGMH, HCDR2 with an amino acid sequence of TISYDGSIKDYADSVKG, HCDR3 with an amino acid sequence of GDRTGNLDY, LCDR1 with an amino acid sequence of RASQNIRNALN, LCDR2 with an amino acid sequence of DASTRQS, and LCDR3 with an amino acid sequence of QQNSEFPPT; and The antigen-binding fragment that binds to epitope I of rabies virus G protein comprises: HCDR1 with an amino acid sequence of GYTIN, HCDR2 with an amino acid sequence of GIIPIFGTANYAQRFQG, HCDR3 with an amino acid sequence of ENLDNSGTYYYFSGWFDP, LCDR1 with an amino acid sequence of TGTSSDLGGYDFVS, LCDR2 with an amino acid sequence of DATKRPS, and LCDR3 with an amino acid sequence of CSYAGDYTPGVV; in, The HCDR and LCDR amino acid sequences were according to the Kabat definition; Preferably, the antigen-binding fragment that binds to epitope III of rabies virus G protein consists of: HCDR1 with an amino acid sequence of SYGMH, HCDR2 with an amino acid sequence of TISYDGSIKDYADSVKG, HCDR3 with an amino acid sequence of GDRTGNLDY, LCDR1 with an amino acid sequence of RASQNIRNALN, LCDR2 with an amino acid sequence of DASTRQS, and LCDR3 with an amino acid sequence of QQNSEFPPT; and the antigen-binding fragment that binds to epitope I of rabies virus G protein consists of: HCDR1 with an amino acid sequence of GYTIN, HCDR2 with an amino acid sequence of GIIPIFGTANYAQRFQG, HCDR3 with an amino acid sequence of ENLDNSGTYYYFSGWFDP, LCDR1 with an amino acid sequence of TGTSSDLGGYDFVS, LCDR2 with an amino acid sequence of DATKRPS, and LCDR3 with an amino acid sequence of CSYAGDYTPGVV.

2. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition comprises 0.5 mg / ml to 2.0 mg / ml of the bispecific antibody against rabies virus G protein, 15 mM to 25 mM histidine / histidine hydrochloride buffer pair, 200 mM to 300 mM trehalose, 0.5 mM to 1.5 mM ethylenediaminetetraacetate, 0.1 mg / ml to 1.0 mg / ml of a polysorbate-type surfactant, and the remainder of water, wherein the pH value of the pharmaceutical composition is 5.5 to 6.

0.

3. The pharmaceutical composition of any one of claims 1-2, wherein the concentration of the histidine / histidine hydrochloride buffer pair is 15mM-20mM, 20mM-25mM or 20mM.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the concentration of trehalose is 230 mM-260 mM or 250 mM-300 mM.

5. The pharmaceutical composition of any one of claims 1-4, wherein the concentration of EDTA is 0.5 mM-1.0 mM, 1.0 mM-1.5 mM or 1.0 mM.

6. The pharmaceutical composition of any one of claims 1 to 5, wherein the polysorbate type surfactant is, for example, polysorbate 20 or polysorbate 80, and the content of the polysorbate type surfactant is 0.2 mg / ml-0.5 mg / ml, 0.3 mg / ml-1.0 mg / ml, 0.5 mg / ml-1.0 mg / ml, 0.3 mg / ml, 0.34 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml or 1.0 mg / ml.

7. The pharmaceutical composition of any one of claims 1-6, wherein the pH value of the pharmaceutical composition is 5.5-5.8 or 5.

8.

8. The pharmaceutical composition of claim 1, comprising 0.2 mg / ml to 20 mg / ml of a bispecific antibody against rabies virus G protein, 20 mM of histidine / histidine hydrochloride buffer pair, 240 mM of an osmotic pressure regulator selected from trehalose, 1.0 mM of ethylenediaminetetraacetate, 0.5 mg / ml-1.0 mg / ml of a polysorbate-type surfactant, and the remainder of water, wherein the pH value of the pharmaceutical composition is 5.

8.

9. The pharmaceutical composition of any one of claims 1 to 8, wherein the antigen-binding fragment that binds to epitope I of rabies virus G protein is a single-chain antibody (scFv) and the single-chain antibody comprises the amino acid sequence shown in SEQ ID NO: 17; and The antigen binding fragment that binds to epitope III of rabies virus G protein is a Fab fragment, and the Fab fragment comprises the amino acid sequences shown in SEQ ID NO:18 and SEQ ID NO:

19.

10. Use of the pharmaceutical composition according to any one of claims 1 to 9 in the preparation of a medicament for preventing or treating rabies.

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