CN112972674B

CN112972674B - Stable pharmaceutical formulation comprising CD147 monoclonal antibody

Info

Publication number: CN112972674B
Application number: CN202110262504.0A
Authority: CN
Inventors: 陈小春; 唐浩; 孙生柏
Original assignee: Jiangsu Pacific Meinuoke Biopharmaceutical Co ltd
Current assignee: Jiangsu Pacific Biopharmaceutical Co ltd
Priority date: 2021-03-10
Filing date: 2021-03-10
Publication date: 2022-02-15
Anticipated expiration: 2041-03-10
Also published as: CN112972674A

Abstract

The application discloses a pharmaceutical formulation comprising a CD147 monoclonal antibody, a buffer, a protein protectant, a lyophilized scaffold agent, and a surfactant. The pharmaceutical formulation is capable of maintaining the stability of the CD147 monoclonal antibody over a long period of time. The application also discloses the application of the pharmaceutical preparation in preventing and/or treating CD147 related diseases, in particular malaria prevention and treatment, and a method for preparing the pharmaceutical preparation.

Description

Stable pharmaceutical formulation comprising CD147 monoclonal antibody

Technical Field

The present invention relates to pharmaceutical formulations, and in particular to stable pharmaceutical formulations comprising a CD147 monoclonal antibody. The invention also relates to a preparation method and application of the pharmaceutical preparation.

Background

The research shows that CD147 molecule is the necessary receptor of plasmodium falciparum invading erythrocyte, and the CD147 resisting monoclonal antibody can specifically recognize and combine with CD147 molecule expressed on the surface of human erythrocyte, block the interaction of the molecule and its ligand, RAP2 expressed in merozoite of plasmodium falciparum, inhibit the formation of plasmodium nanobubble, block the invasion of plasmodium falciparum into erythrocyte and prevent and treat plasmodium falciparum, and the mechanism is different from the mechanism of other antimalarial drugs and is specific to CD147 molecule target drugs. Thus, the CD147 molecule has become a new target for Plasmodium treatment (Meng-yao Zhang et al, Blood, 131, 2018).

Since antibody molecules have a complex protein hierarchical structure and tend to physically bind together, which may lead to an undesirable immune response, or may clog syringes or pumps during administration, which is unsafe for the patient, one of the long-standing problems with liquid formulations of antibodies is stability problems caused by agglomeration, while areas of high malaria incidence to which the antibodies are directed are high-heat areas, where higher temperatures are more likely to cause aggregation of the antibodies in the liquid formulation, thereby reducing antibody stability.

Therefore, there is a need for pharmaceutical formulations of CD147 monoclonal antibodies with stability and quality consistency, especially at higher temperatures.

Summary of The Invention

The present invention provides a stable pharmaceutical formulation comprising a CD147 monoclonal antibody that remains homogeneous and stable over an extended period of time.

In one aspect, the present invention provides a pharmaceutical formulation comprising a CD147 monoclonal antibody, a buffer, a protein protectant, a lyophilized scaffold, and a surfactant, wherein the buffer is a histidine buffer at a concentration of 0.125-50 mmol/L in the pharmaceutical formulation, the protein protectant is sucrose at a concentration of 0.25-100 mg/ml in the pharmaceutical formulation, the lyophilized scaffold is mannitol at a concentration of 0.35-100 mg/ml in the pharmaceutical formulation, and the surfactant is polysorbate 20 and/or polysorbate 80 at a concentration of 0.005-2.0 mg/ml in the pharmaceutical formulation.

In some embodiments, the CD147 monoclonal antibody comprises a heavy chain as set forth in SEQ ID NO: 9 and/or the amino acid sequence shown in SEQ ID NO: 10, wherein the concentration of said CD147 monoclonal antibody in said pharmaceutical formulation is between 0.05mg/ml and 15 mg/ml.

In other embodiments, the pharmaceutical formulation has a pH of 5.0 to 8.0.

In other embodiments, the pharmaceutical formulation comprises 1-15 mg/mL of the CD147 monoclonal antibody, 20-30 mmol/L of histidine buffer, 40-90 mg/mL of sucrose, 60-80 mg/mL of mannitol, and 1.0-2.0 mg/mL of polysorbate 20 and/or polysorbate 80, and the pharmaceutical formulation has a pH of 5.0-8.0.

In other embodiments, the pharmaceutical formulation comprises 1-15 mg/mL of the CD147 monoclonal antibody, 20-30 mmol/L of histidine buffer, 40-90 mg/mL of sucrose, 30-70 mg/mL of mannitol, and 1.0-2.0 mg/mL of polysorbate 20 and/or polysorbate 80, and the pharmaceutical formulation has a pH of 5.0-8.0.

In other embodiments, the pharmaceutical formulation is stable at 2-8 ℃ for at least 12 months.

In other embodiments, the pharmaceutical formulation is capable of being diluted 20-400 fold with a diluent and is stable at a temperature of 25 ± 2 ℃ for at least 48 hours.

In other embodiments, the pharmaceutical formulation is stable for up to 60 days at an illumination of 4500lux and a temperature of 2-8 ℃.

In other embodiments, the pharmaceutical formulation is a lyophilized powder injection.

In other embodiments, the lyophilized powder for injection is suitable for intravenous injection after reconstitution.

In another aspect, the present invention provides the use of a pharmaceutical formulation as described herein for the preparation of a medicament for the prevention and/or treatment of a CD147 related disease. In some embodiments, the disease is malaria.

In another aspect, the present invention provides a method of preparing a pharmaceutical formulation, the method comprising:

1) providing a formulation vehicle and a CD147 monoclonal antibody stock solution, wherein the formulation vehicle comprises a buffer solution, a protein protectant, a lyophilized scaffold agent, and a surfactant; 2) the CD147 monoclonal antibody stock solution was filtered to exchange solutions using the formulation vehicle to obtain the pharmaceutical formulation as described herein.

In some embodiments, the methods described herein further comprise freeze-drying the pharmaceutical formulation, thereby obtaining a pharmaceutical formulation as described herein.

Drawings

FIG. 1 shows DSC scan data of CD147 monoclonal antibody at different pH values.

FIG. 2 shows DSC scan data of CD147 monoclonal antibody in different buffers.

Fig. 3 is a 48 hour appearance plot of formulations containing the CD147 monoclonal antibody and different surfactants in the shaking stability test.

Fig. 4 is the appearance of a lyophilized formulation comprising CD147 monoclonal antibody and different protein protectants and lyophilized scaffolds in a lyophilization test.

Detailed Description

The following description of the invention is intended only to illustrate various embodiments of the invention. The specific embodiments described should not be construed as limiting the scope of the invention. Various equivalent substitutions, changes or modifications may be made by those skilled in the art without departing from the spirit and substance of the invention, and it should be understood that such equivalent embodiments are also included herein. All documents, including publications, patents, and patent applications, cited herein are hereby incorporated by reference in their entirety.

Where a method referred to in this disclosure comprises two or more defined steps, the defined steps may be performed in any order or simultaneously (unless the context excludes that possibility), and the method may comprise one or more further steps, which may be performed before any defined step, between two defined steps or after all defined steps (unless the context excludes that possibility).

Antibodies

As used herein, the term "monoclonal antibody" refers to a population of antibodies that contain a single identical antibody that is homogeneous or substantially homogeneous. Monoclonal antibodies can be obtained from a single hybridoma cell clone (Milstein, C (1999). "The hybridoma revolution: an offset of basic research". BioEssays.21 (11): 966-73). An intact monoclonal antibody comprises two heavy chains and two light chains. Each heavy chain is composed of a heavy chain variable region (V)_H) And heavy chain first, second and third constant regions (C)_H1，C_H2，C_H3) Whereas each light chain is composed of a light chain variable region (V)_L) And light chain constant region (C)_L) And (4) forming. V of heavy and light chains_HAnd V_LThere are also three Complementarity Determining Regions (CDRs) in each region, separated by a contiguous portion called the Framework Region (FR) which is larger than the CDRsMore highly conserved and forms a stent supporting hyper-variable loop. The 6 CDRs of one heavy chain and one light chain together constitute the antigen binding site of an antibody, determining the specificity of the antibody. The monoclonal antibodies described herein also include fragments or derivatives of the intact monoclonal antibodies that have an antigen binding function, which fragments or derivatives have the same antigen binding specificity as the intact monoclonal antibodies, but which fragments or derivatives bind to their specific antigen with the same or different affinity as the intact monoclonal antibodies.

In some embodiments, a monoclonal antibody described herein comprises an antigen binding fragment. An antigen-binding fragment refers to one or more antibody fragments that retain the ability to specifically bind to an antigen. Examples of antigen binding fragments include, but are not limited to, (i) Fab fragments, which refers to fragments consisting of V_L、V_H、C_LAnd C_H1Monovalent fragments consisting of domains; (ii) a Fab' fragment, which refers to a Fab fragment comprising a portion of the hinge region; (iii) f (ab')₂A fragment, which refers to a bivalent fragment comprising 2 Fab fragments linked by disulfide bonds of the hinge region; (iv) from V_HAnd C_mDomain-forming Fd fragments; (v) v with one arm consisting of antibody_LAnd V_H(vii) Fv fragments consisting of domains, (vi) dAb fragments (Ward et al, Nature 341: 544-546 (1989); PCT publication WO 90/05144) comprising a single variable domain; (vii) an isolated CDR; (viii) a single chain Fv fragment, which is denoted V_LAnd V_HMonovalent fragments formed by direct linkage of domains or by linkage through a peptide chain (Huston JS et al, Proc Natl Acad Sci USA, 85: 5879 (1988)).

In some embodiments, the monoclonal antibodies described herein include chimeric monoclonal antibodies in which a portion of the heavy and/or light chains are identical or homologous to corresponding sequences of antibodies derived from or belonging to a particular antibody class or subclass, while the remaining chains are identical or homologous to corresponding sequences of antibodies derived from or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired functional activity.

In some embodiments, the monoclonal antibodies described herein include human murine chimeric monoclonal antibodies having murine heavy and light chain variable regions and human heavy and light chain constant regions.

In some embodiments, the monoclonal antibodies described herein comprise humanized monoclonal antibodies. Humanized forms of antibodies of non-human origin (e.g., murine) are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (e.g., Fv, Fab ', F (ab')₂Or other antigen-binding sequence of an antibody). In some examples, the humanized antibody may be a CDR-grafted antibody in which the amino acid sequence of a human CDR is introduced into a non-human V_HAnd V_LTo replace the amino acid sequence of the corresponding non-human CDR. In other instances, a substantial portion of the amino acid sequence of a humanized antibody may be derived from a human-derived immunoglobulin (recipient antibody) in which amino acid residues from a CDR of the recipient are replaced by amino acid residues from a CDR of a non-human-derived (e.g., mouse, rat, rabbit) antibody having the desired specificity, affinity, and capacity. Generally, humanized antibodies will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to sequences of an immunoglobulin of non-human origin, and all or substantially all of the Framework (FR) regions are sequences of an immunoglobulin of human origin. In some instances, framework region residues of the variable region of a human-derived immunoglobulin are substituted with corresponding non-human-derived residues. Furthermore, humanized antibodies may include residues that are not found in either the recipient antibody or the imported CDR or framework sequences.

The CD147 monoclonal antibody described herein refers to a monoclonal antibody capable of specifically binding to the CD147 protein.

The CD147 protein is single transmembrane glycoprotein belonging to an immunoglobulin superfamily, the full-length sequence of the single transmembrane glycoprotein has 269 amino acid residues, the first 21 amino acid residues at the beginning of the N terminal are signal peptide, 22-205 amino acid residues form an extracellular region, 206-229 amino acid residues form a transmembrane region, the single transmembrane region has a typical leucine zipper structure, and 230-269 amino acid residues close to the C terminal form the intracellular region. The extracellular 4 cysteines form 2 disulfide bonds, constituting two Ig-like domains. Representative sequences of CD147 proteins can be, for example, as shown in GenBank number BAC76828.1, and also referenced, for example, Zhang DW et al, J hepatol.2012; chen YK et al, PLoS One, 7(7), 2012; tang J et al, Cell Death Differ, 19(11), 2012; li Y et al, J Biol Chem, 287(7), (2012); wu J et al, Oncogene, 2011; zhao P et al, Hepatology, 54(6), 2011; zhao P et al, Cancer sci.101(2), 2010; gou XC et al, Cancer Sci.100(5), 2009; chen YK et al, Cancer Letters, 278(1), 2009.

In some embodiments, the CD147 monoclonal antibody described herein comprises a heavy chain CDR sequence selected from the group consisting of SEQ ID NOs: 1.2 and 3. In some embodiments, the CD147 monoclonal antibody described herein comprises a light chain CDR sequence selected from the group consisting of SEQ ID NOs: 4.5 and 6.

In some embodiments, the CD147 monoclonal antibody described herein comprises a heavy chain variable region V_HSaid heavy chain variable region V_HComprising the heavy chain CDR sequences shown below: as shown in SEQ ID NO: 1, CDR1 of the amino acid sequence set forth in SEQ ID NO: 2, and/or a CDR2 of the amino acid sequence as set forth in SEQ ID NO: 3, CDR3 of the amino acid sequence set forth in seq id no.

In some embodiments, the CD147 monoclonal antibody described herein comprises a light chain variable region V_LSaid light chain variable region V_LComprises light chain CDRs as shown below: as shown in SEQ ID NO: 4, CDR1 of the amino acid sequence set forth in SEQ ID NO: 5, and/or the amino acid sequence as set forth in SEQ ID NO: 6, CDR3 of the amino acid sequence set forth in seq id no.

In some embodiments, the CD147 monoclonal antibody described herein comprises a heavy chain variable region V_HAnd light chain variable region V_LSaid heavy chain variable region V_HComprises a polypeptide having the sequence shown in SEQ ID NO: 1, a CDR1 having the amino acid sequence set forth as SEQ ID NO: 2, and/or a CDR2 having an amino acid sequence as set forth in SEQ ID NO: 3, the light chain amino acid sequence of CDR3Variable region V_LComprises a polypeptide having the sequence shown in SEQ ID NO: 4, a CDR1 having the amino acid sequence set forth as SEQ ID NO: 5, and/or a CDR2 having the amino acid sequence set forth as SEQ ID NO: 6, CDR3 of the amino acid sequence set forth in seq id no.

In some embodiments, the CD147 monoclonal antibody described herein comprises a polypeptide having the amino acid sequence as set forth in SEQ ID NO: 7, and a heavy chain variable region V having the amino acid sequence shown in SEQ ID No. 7_H. In some embodiments, the CD147 monoclonal antibody described herein comprises a polypeptide having the amino acid sequence as set forth in SEQ ID NO: 8 in a light chain variable region_L. In some embodiments, the CD147 monoclonal antibody described herein comprises a polypeptide having the amino acid sequence as set forth in SEQ ID NO: 7, and a heavy chain variable region V having the amino acid sequence shown in SEQ ID No. 7_HAnd as shown in SEQ ID NO: 8 in a light chain variable region_L。

In some embodiments, the CD147 monoclonal antibodies described herein further comprise an immunoglobulin constant region. In some embodiments, the immunoglobulin constant region comprises a heavy chain and/or light chain constant region. The heavy chain constant region comprises C_H1、C_H1-C_H2Or C_H1-C_H3A region, said light chain constant region comprising C_LAnd (4) a zone.

In some embodiments, the CD147 monoclonal antibodies described herein include chimeric CD147 monoclonal antibodies, particularly human murine chimeric CD147 monoclonal antibodies.

In some embodiments, a human murine chimeric CD147 monoclonal antibody described herein comprises an amino acid sequence as set forth in SEQ ID NO: 9 and the amino acid sequence shown in SEQ ID NO: 10, or a light chain of the amino acid sequence set forth in seq id No. 10.

In some embodiments, the CD147 monoclonal antibody described herein is a 6H8MAb monoclonal antibody comprising an amino acid sequence as set forth in SEQ ID NO: 9 and the amino acid sequence shown in SEQ ID NO: 10, or a light chain of the amino acid sequence set forth in seq id No. 10.

Exemplary amino acid sequences in some embodiments are listed in table 1 below:

table 1 exemplary amino acid sequences

The present invention relates to pharmaceutical formulations comprising a CD147 monoclonal antibody. In some embodiments, the pharmaceutical formulation of the invention may comprise said CD147 monoclonal antibody at a concentration of 0.05-15 mg/ml. In some embodiments, the concentration of the CD147 monoclonal antibody is any concentration value within the above range. For example, the CD147 monoclonal antibody can be present in the pharmaceutical formulation at a concentration of at least 0.05mg/ml, at least 0.1mg/ml, at least 0.15mg/ml, at least 0.2mg/ml, at least 0.25mg/ml, at least 0.3mg/ml, at least 0.35mg/ml, at least 0.4mg/ml, at least 0.45mg/ml, at least 0.5mg/ml, at least 0.55mg/ml, at least 0.6mg/ml, at least 0.65mg/ml, at least 0.7mg/ml, at least 0.75mg/ml, at least 0.8mg/ml, at least 0.85mg/ml, at least 0.9mg/ml, at least 0.95mg/ml, at least 1mg/ml, at least 2mg/ml, at least 3mg/ml, at least 4mg/ml, at least 5mg/ml, at least 6mg/ml, at least 7mg/ml, at least 0.65mg/ml, at least 0.7mg/ml, or a combination thereof, At least 8mg/ml, at least 9mg/ml, at least 10mg/ml, at least 11mg/ml, at least 12mg/ml or at least 13mg/ml, and/or at most 14mg/ml or at most 15 mg/ml.

Buffer solution

The term "buffer" generally refers to a buffered solution that is resistant to changes in pH by the action of its acid-base conjugate components. As used herein, "buffer" refers to a solution of a compound known to be safe for use in pharmaceutical formulations, having the ability to maintain or control the pH of the formulation within a desired range. Acceptable buffers capable of controlling the pH value from a mildly acidic pH value to a mildly alkaline pH value (e.g., pH 5.0 to 8.0) include, but are not limited to, one or a combination of any of a succinic acid buffer, a citric acid buffer, a phosphate buffer, an acetate buffer, an arginine buffer, a 2-amino-2-hydroxymethyl-1, 3-propanediol (TRIS) buffer, a histidine buffer, and the like.

The stable pharmaceutical formulation of the invention may comprise a buffer such that the pharmaceutical formulation has a pH of 5.0 to 8.0, for example a pH of 5.0 to 6.0, 6.0 to 7.0 or 7.0 to 8.0. In some embodiments, a suitable buffer provides a pharmaceutical formulation of the invention with a pH of 5.0 to 6.0. In particular, the pH of the pharmaceutical formulation of the invention may be any pH within those ranges as listed above, e.g. 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0.

Examples of buffers that can control the pH of the pharmaceutical formulation within the desired range include succinic acid buffers, histidine buffers, citric acid buffers, and other organic or inorganic acid buffers. These buffers may be used alone in 1 kind, or 2 or more kinds may be mixed and used. Preferably, the pharmaceutical formulation of the present invention comprises a succinic acid buffer, a histidine buffer or a citric acid buffer. More preferably, the pharmaceutical formulation of the present invention comprises a histidine buffer.

A "succinic acid buffer" is a buffer comprising succinate ions. The succinic acid buffer may comprise one or more of succinic acid, monosodium succinate, disodium succinate, monopotassium succinate, dipotassium succinate, sodium chloride, potassium chloride, and the like. In some embodiments, the succinic acid buffer can be a succinic acid-disodium succinate buffer. In some embodiments, the pH of the succinate buffer can be any pH in the range of 5.0 to 6.0, such as 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0.

A "histidine buffer" is a buffer comprising histidine ions. The histidine buffer may include one or more of histidine, histidine hydrochloride, histidine acetate, histidine phosphate, histidine sulfate, and the like. In some embodiments, the histidine buffer may be a histidine-histidine hydrochloride buffer. In some embodiments, the pH of the histidine buffer may be any pH in the range of 5.0 to 6.0, such as 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0.

A "citrate buffer" is a buffer that contains citrate ions. The citric acid buffer may comprise one or more of citric acid, monosodium citrate, disodium citrate, trisodium citrate, monopotassium citrate, dipotassium citrate, tripotassium citrate, sodium chloride, potassium chloride, and the like. In some embodiments, the citrate buffer is a citric acid-trisodium citrate buffer. In some embodiments, the pH of the citrate buffer can be any pH in the range of 5.0 to 6.0, such as 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0.

The concentration of the buffer as described herein refers to the concentration of the buffer ion in the buffer. In some embodiments, suitable buffers for use in the pharmaceutical formulations of the present invention may have a concentration of 0.125 to 50 mmol/L. In some embodiments, the concentration of the buffer is any concentration value within the above range. For example, the buffer can be at a concentration of at least 0.125mmol/L, at least 0.25mmol/L, at least 0.5mmol/L, at least 1mmol/L, at least 1.5mmol/L, at least 2mmol/L, at least 2.5mmol/L, at least 3mmol/L, at least 3.5mmol/L, at least 4mmol/L, at least 4.5mmol/L, at least 5mmol/L, at least 6mmol/L, at least 7mmol/L, at least 8mmol/L, at least 9mmol/L, at least 10mmol/L, at least 11mmol/L, at least 12mmol/L, at least 13mmol/L, at least 14mmol/L, at least 15mmol/L, at least 16mmol/L, at least 17mmol/L, at least 18mmol/L, at least 19mmol/L, at least 20mmol/L, at least 21mmol/L, a, At least 22mmol/L, at least 23mmol/L, at least 24mmol/L, at least 25mmol/L, at least 30mmol/L, at least 35mmol/L, at least 40mmol/L, or at least 45mmol/L, and/or at most 50mmol/L, at most 45mmol/L, at most 40mmol/L, at most 35mmol/L, at most 30mmol/L, at most 25mmol/L, at most 24mmol/L, at most 23mmol/L, at most 22mmol/L, at most 21mmol/L, at most 20mmol/L, at most 19mmol/L, at most 18mmol/L, at most 17mmol/L, at most 16mmol/L, at most 15mmol/L, at most 14mmol/L, at most 13mmol/L, at most 12mmol/L, at most 11mmol/L, Up to 10mmol/L, up to 9mmol/L, up to 8mmol/L, up to 7mmol/L, or up to 6mmol/L, depending on the particular buffer and the desired stability of the pharmaceutical formulation.

In some embodiments, the buffer used in the pharmaceutical formulation of the present invention is a histidine buffer, including histidine-histidine hydrochloride buffer, which may be at a concentration of 0.125 to 50 mmol/L. In some embodiments, the concentration of histidine buffer may be 11-25 mmol/L, 12-25 mmol/L, 13-25 mmol/L, 14-25 mmol/L, 15-25 mmol/L, 16-25 mmol/L, 17-25 mmol/L, 18-25 mmol/L, 19-25 mmol/L, 20-25 mmol/L, 21-25 mmol/L, 22-25 mmol/L, or 23-25 mmol/L. In some embodiments, the concentration of histidine buffer is 20-30 mmol/L, 21-29 mmol/L, 22-28 mmol/L, 23-27 mmol/L, 24-26 mmol/L or 25 mmol/L.

Protein protective agent

As used herein, the term "protein protectant" refers to an agent that, when bound to a protein of interest, prevents or reduces chemical and/or physical instability of the protein. Examples of protein protectants include sugars, alcohols, acids, salts, polymers, and the like. Examples of sugars include glucose, sucrose, trehalose, lactose, dextran, and the like. Examples of alcohols include sorbitol and the like. Examples of acids include citric acid, phosphoric acid, tartaric acid, amino acids, ethylenediaminetetraacetic acid, and the like. Examples of salts include sodium sulfate, sodium glutamate, sodium chloride, potassium chloride, ammonium acetate, and the like. Examples of polymers include polyethylene glycol, povidone, and the like.

In some embodiments, the protein protectant used in the pharmaceutical formulation of the invention is selected from the group consisting of carbohydrates. In some embodiments, the protein protectant used in the pharmaceutical formulation of the invention is selected from sucrose, trehalose, or a combination thereof.

In some embodiments, the concentration of the protein protectant used in the pharmaceutical formulations of the invention in the pharmaceutical formulation may be 0.25-100 mg/ml. In some embodiments, the concentration of the protein protectant is any concentration value within the above-described ranges. For example, the concentration of the protein protectant in the pharmaceutical formulation can be at least 0.25mg/ml, at least 0.5mg/ml, at least 1mg/ml, at least 2mg/ml, at least 3mg/ml, at least 4mg/ml, at least 5mg/ml, at least 6mg/ml, at least 7mg/ml, at least 8mg/ml, at least 9mg/ml, at least 10mg/ml, at least 20mg/ml, at least 30mg/ml, at least 40mg/ml, at least 50mg/ml, at least 60mg/ml, at least 70mg/ml, at least 80mg/ml, at least 90mg/ml, and/or at most 100mg/ml, at most 90mg/ml, at most 80mg/ml, at most 70mg/ml, at most 60mg/ml, at most 50mg/ml, at least 50mg/ml, Up to 40mg/ml, up to 30mg/ml, up to 20mg/ml, or up to 10mg/ml, depending on the particular protein protectant and the desired stability of the pharmaceutical formulation.

In some embodiments, the protein protectant used in the pharmaceutical formulations of the invention is sucrose, which may be present in the pharmaceutical formulation at a concentration of 0.35-100 mg/ml. In some embodiments, the concentration of sucrose in the pharmaceutical formulation may be 10-100 mg/ml, 20-80 mg/ml, 70-90 mg/ml, 20-90 mg/ml, 30-80 mg/ml, 40-70 mg/ml, or 50-60 mg/ml. In some embodiments, the concentration of sucrose in the pharmaceutical formulation is 20-40 mg/ml, 40-60 mg/ml, 50mg/ml, 70-90 mg/ml, 60-80 mg/ml, or 80-100 mg/ml.

Freeze-drying support agent

As used herein, the term "lyophilized scaffold" refers to an agent that can provide a lyophilized powder for injection with sufficient mechanical strength to maintain its good shape. In some embodiments, the collapse temperature of the lyophilized scaffold is high, collapse does not occur during lyophilization, and its crystallization can form a porous structure, facilitating sublimation of water. Examples of lyophilized scaffolds include, but are not limited to, polyols/sugars such as mannitol, xylitol, sucrose, trehalose, lactose, glucose, maltose and inositol, dextran; polymers such as polysaccharides, povidone, polyethylene glycol, albumin; amino acids such as glycine, L-serine, sodium glutamate, alanine, arginine, and sarcosine.

In some embodiments, the concentration of the lyophilized scaffold in the pharmaceutical formulation of the present invention may be 0.35-100 mg/ml. In some embodiments, the concentration of the lyophilized scaffold is any concentration value within the above range. For example, the concentration of the scaffold agent in the pharmaceutical formulation may be at least 0.35mg/ml, at least 0.7mg/ml, at least 1mg/ml, at least 2mg/ml, at least 3mg/ml, at least 4mg/ml, at least 5mg/ml, at least 6mg/ml, at least 7mg/ml, at least 8mg/ml, at least 9mg/ml, at least 10mg/ml, at least 20mg/ml, at least 30mg/ml, at least 40mg/ml, at least 50mg/ml, at least 60mg/ml, at least 70mg/ml or at least 80mg/ml, and/or at most 100mg/ml, at most 90mg/ml, at most 80mg/ml, at most 70mg/ml, at most 60mg/ml, at most 50mg/ml or at most 40mg/ml, depending on the particular lyoprotectant and the desired stability of the pharmaceutical formulation.

In some embodiments, the lyophilized scaffold of the pharmaceutical formulation of the present invention is mannitol, which may be present in the pharmaceutical formulation at a concentration of 0.35-100 mg/ml, 20-80 mg/ml, 20-40 mg/ml, 30-70 mg/ml, 40-60 mg/ml, or 60-80 mg/ml. In some embodiments, the concentration of mannitol in the pharmaceutical formulation may be 60-80 mg/ml, 61-79 mg/ml, 62-79 mg/ml, 63-78 mg/ml, 64-77 mg/ml, 65-76 mg/ml, 66-75 mg/ml, 67-74 mg/ml, 68-73 mg/ml, 69-72 mg/ml or 70-71 mg/ml, 40-100 mg/ml, 40-90 mg/ml, 40-80 mg/ml, 50-80 mg/ml or 60-80 mg/ml. In some embodiments, the concentration of mannitol in the pharmaceutical formulation may be 60-80 mg/ml, 65-75 mg/ml, or 70 mg/ml.

Surface active agent

As used herein, the term "surfactant" refers to organic substances that have both hydrophilic and hydrophobic amphoteric structures, i.e., they contain groups with opposite solubility tendencies, e.g., oil-soluble hydrocarbon chains and water-soluble ionic groups. Surfactants can be classified into anionic, cationic and nonionic surfactants, depending on the charge of the surface active moiety.

Examples of surfactants include polysorbates (e.g., polysorbate 20 or 80); poloxamers (e.g., poloxamer 188); triton; and polyethylene glycol, polypropylene glycol, and copolymers of ethylene glycol and propylene glycol (e.g., Pluronics, PF68, etc.). In some embodiments, the surfactant of the pharmaceutical formulation of the invention is selected from polysorbate 20(polysorbate 20, PS 20 or tween 20), polysorbate 80(polysorbate 80, PS80 or tween 80) or a combination thereof.

In some embodiments, the concentration of surfactant in the pharmaceutical formulation of the present invention may be 0.005-2.0 mg/ml. In some embodiments, the concentration of the surfactant is any concentration value within the above range. For example, the concentration of the surfactant in the pharmaceutical formulation may be at least 0.005mg/ml, at least 0.05mg/ml, at least 0.06mg/ml, at least 0.07mg/ml, at least 0.08mg/ml, at least 0.09mg/ml, at least 0.1mg/ml, at least 0.2mg/ml, at least 0.4mg/ml, at least 0.6mg/ml, at least 0.8mg/ml, at least 1.0mg/ml, at least 1.2mg/ml, at least 1.4mg/ml, at least 1.6mg/ml, at least 1.8mg/ml, and/or at most 2.0mg/ml, at most 1.8mg/ml, at most 1.6mg/ml, at most 1.4mg/ml, at most 1.2mg/ml, at most 1.0mg/ml, at most 0.8mg/ml, at most 0.6mg/ml, or at most 0.4mg/ml, depending on the particular surfactant and the desired stability of the pharmaceutical formulation.

In some embodiments, the surfactant of the pharmaceutical formulation of the present invention is polysorbate 20 and/or polysorbate 80, which may be present in the pharmaceutical formulation at a concentration of 0.005-2.0 mg/ml. In some embodiments, the concentration of polysorbate 20 and/or polysorbate 80 in the pharmaceutical formulation may be 0.005-1.0 mg/ml or 1.0-2.0 mg/ml.

Other materials

The pharmaceutical formulations of the present invention may also optionally comprise other materials such as, but not limited to, isotonic agents, diluents, and the like.

The term "isotonic agent" refers to a compound or composition that can impart a suitable osmotic tension to a drug to prevent the net flow of water across the cell membrane that contacts the drug. In some embodiments, the formulations of the invention have an osmolality substantially the same as human blood. In some embodiments, the formulations of the present invention have an osmolality of 400 to 800 mOsmol/kg. Suitable isotonic agents include, but are not limited to: glycerol, amino acids or proteins (such as glycine or albumin), salts (such as sodium chloride) and sugars (such as glucose, mannitol, sucrose and lactose).

The term "diluent" is an agent that is pharmaceutically acceptable and can be used to dilute the pharmaceutical formulation of the present invention. Typical diluents include water, physiological saline, bacteriostatic agents for injection, pH buffers, sterile saline solutions, ringer's solution or dextrose solution. In some embodiments, the diluent used in the present invention is a 0.9% NaCl injection.

Preparation

In one aspect, the present invention provides a stable pharmaceutical formulation comprising a CD147 monoclonal antibody, a buffer, a protein protectant, a lyophilized scaffold, and a surfactant. The pharmaceutical formulation has a pH of 5.0 to 8.0, and in some embodiments, 5.0 to 6.0, to achieve sufficient stability.

In some embodiments, the pharmaceutical formulation of the present invention comprises:

the CD147 monoclonal antibody has the concentration of 0.05-15 mg/ml, preferably 1-15 mg/ml, 1-5 mg/ml, 5-15 mg/ml or 1-10 mg/ml;

a buffer solution, preferably a succinic acid buffer solution, a histidine buffer solution or a citric acid buffer solution, most preferably a histidine buffer solution, wherein the concentration of the buffer solution in the pharmaceutical preparation is 0.125-50 mmol/L, preferably 20-30 mmol/L;

the protein protective agent is preferably sucrose or trehalose, most preferably sucrose, and the concentration of the protein protective agent in the pharmaceutical preparation is 0.25-100 mg/ml, preferably 20-80 mg/ml, 60-80 mg/ml, 70-90 mg/ml, 80-100 mg/ml or 40-60 mg/ml;

the freeze-dried scaffold is preferably sucrose or mannitol, most preferably mannitol, and the concentration of the freeze-dried scaffold in the pharmaceutical preparation is 0.35-100 mg/ml, preferably 20-80 mg/ml, 30-70 mg/ml, 20-40 mg/ml, 40-60 mg/ml or 60-80 mg/ml;

a surfactant, preferably polysorbate 80 or polysorbate 20, most preferably polysorbate 80, and the concentration of the surfactant in the pharmaceutical formulation is 0.005-2.0 mg/ml, preferably 0.005-1.0 mg/ml or 1.0-2.0 mg/ml; and is

Wherein the pharmaceutical formulation has a pH of 5.0 to 6.0.

In some embodiments, the pharmaceutical formulation of the present invention comprises CD147 monoclonal antibody at a concentration of about 0.05-15 mg/ml, histidine buffer at a concentration of about 0.125-50 mmol/L, wherein the pH of the pharmaceutical formulation is 5.0-8.0. In some embodiments, the above pharmaceutical formulation comprises CD147 monoclonal antibody at a concentration of about 0.05-10 mg/ml. In some embodiments, the above pharmaceutical formulations comprise the CD147 monoclonal antibody at a concentration of about 1mg/ml or about 10 mg/ml.

In some embodiments, the pharmaceutical formulation of the present invention comprises CD147 monoclonal antibody at a concentration of about 0.05-15 mg/ml, histidine buffer, succinic buffer, or citric buffer at a concentration of about 0.125-50 mmol/L, wherein the pH of the pharmaceutical formulation is 5.0-6.0. In some embodiments, the above pharmaceutical formulation comprises CD147 monoclonal antibody at a concentration of about 0.05-10 mg/ml. In some embodiments, the above pharmaceutical formulations comprise the CD147 monoclonal antibody at a concentration of about 1mg/ml or about 10 mg/ml.

In some embodiments, the pharmaceutical formulation of the present invention comprises CD147 monoclonal antibody at a concentration of about 0.05-15 mg/ml, histidine buffer at a concentration of about 0.125-50 mmol/L, sucrose at a concentration of about 0.25-100 mg/ml, and polysorbate 20 at a concentration of about 0.005-2.0 mg/ml or polysorbate 80 at a concentration of about 0.005-2.0 mg/ml, wherein the pharmaceutical formulation has a pH of 5.0-6.0. In some embodiments, the above pharmaceutical formulation comprises CD147 monoclonal antibody at a concentration of about 0.05-15 mg/ml. In some embodiments, the above pharmaceutical formulation comprises CD147 monoclonal antibody at a concentration of about 0.05-10 mg/ml. In some embodiments, the above pharmaceutical formulations comprise the CD147 monoclonal antibody at a concentration of about 1mg/ml or about 10 mg/ml.

In some embodiments, the pharmaceutical formulation of the present invention comprises CD147 monoclonal antibody at a concentration of about 0.05-15 mg/ml, histidine buffer at a concentration of about 0.125-50 mmol/L, sucrose at a concentration of about 0.25-100 mg/ml, and mannitol at a concentration of about 0.35-100 mg/ml, wherein the pH of the pharmaceutical formulation is 5.0-6.0. In some embodiments, the above pharmaceutical formulation comprises CD147 monoclonal antibody at a concentration of about 0.05-10 mg/ml. In some embodiments, the above pharmaceutical formulations comprise the CD147 monoclonal antibody at a concentration of about 1mg/ml or about 10 mg/ml.

In some embodiments, the pharmaceutical formulation of the present invention comprises CD147 monoclonal antibody at a concentration of about 0.05-15 mg/ml, histidine buffer at a concentration of about 0.125-50 mmol/L, sucrose at a concentration of about 0.25-100 mg/ml, mannitol at a concentration of about 0.35-100 mg/ml, and polysorbate 20 at a concentration of about 0.005-2.0 mg/ml or polysorbate 80 at a concentration of about 0.005-2.0 mg/ml, wherein the pharmaceutical formulation has a pH of 5.0-6.0. In some embodiments, the above pharmaceutical formulation comprises CD147 monoclonal antibody at a concentration of about 0.05-10 mg/ml. In some embodiments, the above pharmaceutical formulations comprise the CD147 monoclonal antibody at a concentration of about 1mg/ml or about 10 mg/ml.

In some embodiments, the pharmaceutical formulation of the present invention comprises CD147 monoclonal antibody at a concentration of about 0.05-15 mg/ml, histidine buffer at a concentration of about 20-30 mmol/L, sucrose at a concentration of about 40-90 mg/ml, mannitol at a concentration of about 30-70 mg/ml, and polysorbate 20 at a concentration of about 1.0-2.0 mg/ml or polysorbate 80 at a concentration of about 1.0-2.0 mg/ml, wherein the pH of the pharmaceutical formulation is 5.0-6.0. In some embodiments, the above pharmaceutical formulation comprises CD147 monoclonal antibody at a concentration of about 0.05-10 mg/ml. In some embodiments, the above pharmaceutical formulations comprise the CD147 monoclonal antibody at a concentration of about 1mg/ml or about 10 mg/ml.

In some embodiments, the pharmaceutical formulation of the present invention comprises CD147 monoclonal antibody at a concentration of about 0.05-15 mg/ml, histidine buffer at a concentration of about 0.125-50 mmol/L, sucrose at a concentration of about 0.25-100 mg/ml, and polysorbate 20 at a concentration of about 0.005-2.0 mg/ml or polysorbate 80 at a concentration of about 0.005-2.0 mg/ml, wherein the pharmaceutical formulation has a pH of 5.0-6.0.

In some embodiments, the pharmaceutical formulation of the present invention comprises CD147 monoclonal antibody at a concentration of about 0.05-15 mg/ml, histidine buffer at a concentration of about 0.125-50 mmol/L, sucrose at a concentration of about 0.25-100 mg/ml, mannitol at a concentration of about 0.35-100 mg/ml, and polysorbate 80 at a concentration of about 0.005-2.0 mg/ml, wherein the pharmaceutical formulation has a pH of 5.0-6.0.

In some embodiments, the pharmaceutical formulation of the present invention comprises CD147 monoclonal antibody at a concentration of about 0.05-15 mg/ml, histidine buffer at a concentration of about 5-50 mmol/L, sucrose at a concentration of about 40-60 mg/ml or about 70-90 mg/ml, mannitol at a concentration of about 30-70 mg/ml, and polysorbate 80 at a concentration of about 1.0-2.0 mg/ml, wherein the pH of the pharmaceutical formulation is 5.0-6.0.

In some embodiments, the pharmaceutical formulation of the present invention comprises CD147 monoclonal antibody at a concentration of about 0.05-5.0 mg/ml or 5.0-15.0 mg/ml, histidine buffer at a concentration of about 23-27 mmol/L, sucrose at a concentration of about 40-60 mg/ml or about 70-90 mg/ml, mannitol at a concentration of about 60-80 mg/ml, and polysorbate 80 at a concentration of about 0.9-1.1 mg/ml or 1.9-2.1 mg/ml, wherein the pH of the pharmaceutical formulation is 5.0-6.0.

Chemical degradation of the antibody molecule, or aggregation to form a polymer, or deglycosylation, glycosylation modification, oxidation, or other structural modification of the antibody molecule that may result in a reduction in at least one functional activity of the monomeric protein, etc., may cause instability of the antibody preparation. For pharmaceutical formulations comprising the CD147 monoclonal antibody, the CD147 monoclonal antibody may chemically degrade during storage of the pharmaceutical formulation, resulting in a decrease in the concentration of the antibody; the CD147 monoclonal antibody may also aggregate to form a polymer that is sometimes insoluble in the form of a multimeric molecule containing multiple antibody molecules, resulting in a reduction in the monomer content of the individual antibody molecules. Thus, the content of the polymer antibody increases, which results in a decrease in the purity of the monomer antibody. Moreover, the turbidity of the pharmaceutical formulation may increase due to the formation of insoluble polymers.

In some embodiments, the pharmaceutical formulations of the present invention comprising a CD147 monoclonal antibody can maintain stability over an extended period of time, wherein the physical and/or chemical stability and/or functional activity, etc., of the CD147 monoclonal antibody remains relatively constant over time. In some embodiments, antibody protein concentration, protein purity, protein activity, pH of the formulation, osmolality of the formulation, appearance of the formulation, insoluble particulates in the formulation, moisture of the formulation, and the like may be used as an indicator of the stability of the pharmaceutical formulation. There are a number of analytical techniques in the art for determining Protein stability, as described in Peptide and Protein Drug Delivery, 247-: 29-90 (1993). In some embodiments, the stability of a pharmaceutical formulation can be measured by methods known in the art under selected conditions and times.

In some embodiments, the formulations may be subjected to Differential Scanning Calorimetry (DSC) tests, accelerated thermal stability, light stability, oscillatory stability, long term stability, dilution stability, and/or high temperature stability tests. For example, in a DSC test, the thermal transition midpoint (Tm) of an antibody in a formulation is measured using a differential scanning calorimeter. For example, in the oscillatory stability test, the pharmaceutical formulation may be oscillated at room temperature at 67 rpm for 48 hours and sampled at 0, 24 and 48 hours for measurement. For example, in the photostability test, the pharmaceutical formulation can be stored at 4500 ± 500Lux for 60 days at 5 ± 3 ℃ and sampled for measurement at days 0, 7, 14, 30, and 60. For example, in the light stability test, the pharmaceutical formulation may be stored at 5 ± 3 ℃ for 60 days under illumination of 4000Lux, and sampled for measurement at days 0, 7, 14, 30, and 60. For example, in the photostability test, the pharmaceutical formulation can be stored at 5 ± 3 ℃ for 60 days under an illumination of 4500Lux, and sampled for measurement at days 0, 7, 14, 30, and 60. For example, in the light stability test, the pharmaceutical formulation may be stored at 5 ± 3 ℃ for 60 days at a luminance of 5000Lux, and sampled for measurement at days 0, 7, 14, 30 and 60. For example, in the long-term stability test, the pharmaceutical formulation may be stored at 2-8 ℃ for 2 years and sampled at months 0, 3, 6, 9, 12, 18, and 24 for measurement. For example, in the dilution stability test, the pharmaceutical formulation is diluted with 0.9% NaCl injection, and the diluted pharmaceutical formulation may be stored at 25 ± 2 ℃ for 48 hours, and sampled at hours 0, 24, and 48 for measurement. In the high temperature stability test, the pharmaceutical formulations can be stored at 40 ± 2 ℃ for 60 days and sampled for measurement on days 0, 7, 14, 30 and 60.

In some embodiments, a stable pharmaceutical formulation refers to a pharmaceutical formulation that can obtain three Tm values of the CD147 monoclonal antibody in a DSC test.

In some embodiments, a stable pharmaceutical formulation refers to a pharmaceutical formulation in which the protein purity does not decrease by more than 5.0%, more than 4.0%, more than 3.0%, more than 2.0%, or more than 1.0% when the pharmaceutical formulation is in an accelerated thermal stability test, wherein the protein purity can be measured by CE-SDS reduction or non-reduction methods.

In some embodiments, a stable pharmaceutical formulation refers to a polymer content in the pharmaceutical formulation that increases by less than about 1%, less than about 0.9%, less than about 0.8%, less than about 0.7%, less than about 0.6%, less than about 0.5%, less than about 0.4%, less than about 0.3%, less than about 0.2%, or less than about 0.1% when the pharmaceutical formulation is in a shaking stability test, wherein the content of polymer is measurable by SE-HPLC.

In some embodiments, a stable pharmaceutical formulation refers to a protein purity of 95.0% or more, 95.5% or more, 96% or more, 96.5% or more, 97% or more, 97.5% or more, 98% or more, 98.5% or more, 99% or more, or 99.5% or more in a pharmaceutical formulation when the pharmaceutical formulation is tested for high temperature stability, light stability, long term stability, or dilution stability, wherein the protein purity can be determined by CE-SDS reduction or non-reduction.

In some embodiments, a stable pharmaceutical formulation refers to a pharmaceutical formulation wherein less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the protein in the pharmaceutical formulation is polymeric when the pharmaceutical formulation is tested for high temperature stability, light stability, long term stability, or dilution stability, wherein the level of polymer is measurable by SE-HPLC.

In some embodiments, a stable pharmaceutical formulation refers to a pharmaceutical formulation having less than 6000 particles/ml, less than 5000 particles/ml, less than 4000 particles/ml, less than 3000 particles/ml, less than 2000 particles/ml, less than 1000 particles/ml, less than 900 particles/ml, less than 800 particles/ml, less than 700 particles/ml, less than 600 particles/ml, less than 500 particles/ml, less than 400 particles/ml, less than 300 particles/ml, less than 200 particles/ml, less than 100 particles/ml, less than 90 particles/ml, less than 80 particles/ml, or less than 70 particles/ml, less than 25 particles/ml, and less than 300 particles/ml, less than 500 particles/ml, less than 400 particles/ml, when the pharmaceutical formulation is tested for dilution stability, Less than or equal to 90 grains/ml, less than or equal to 80 grains/ml, less than or equal to 70 grains/ml, less than or equal to 60 grains/ml, less than or equal to 50 grains/ml, less than or equal to 40 grains/ml, less than or equal to 30 grains/ml, less than or equal to 20 grains/ml, less than or equal to 10 grains/ml, less than or equal to 9 grains/ml, less than or equal to 8 grains/ml, less than or equal to 7 grains/ml, less than or equal to 6 grains/ml, less than or equal to 5 grains/ml, less than or equal to 4 grains/ml, less than or equal to 3 grains/ml, less than or equal to 2 grains/ml, or less than or equal to 1 grain/ml, wherein the amount of insoluble particles is determined according to the "inspection method for insoluble particles" 0903 "of the formulation Standard in Chinese pharmacopoeia 2010.

In some embodiments, a stable pharmaceutical formulation refers to a pharmaceutical formulation in which the concentration of the protein varies by no more than +/-20%, no more than +/-19%, no more than +/-18%, no more than +/-17%, no more than +/-16%, no more than +/-15%, no more than +/-14%, no more than +/-13%, no more than +/-12%, no more than +/-11%, no more than +/-10%, no more than +/-9%, no more than +/-8%, no more than +/-7%, no more than +/-6%, no more than +/-5%, no more than +/-4%, when the pharmaceutical formulation is tested for high temperature stability, light stability, long term stability, or dilution stability, No more than +/-3%, no more than +/-2%, no more than +/-1%, or no more than +/-0.5%, wherein the protein concentration can be determined by uv-vis spectrophotometry according to chinese pharmacopoeia, version 2010, third, convention 0401.

In some embodiments, a stable pharmaceutical formulation refers to a binding activity of an antibody of 65% to 150% when the pharmaceutical formulation is tested in high temperature stability, light stability, long term stability, or dilution stability, said binding activity representing a specific binding activity of the antibody to a target, which can be measured by an enzyme linked immunosorbent assay according to chinese pharmacopoeia 2010, part three, general rule 3418.

In some embodiments, the binding activity of the antibody is determined by an enzyme-linked immunoassay comprising the steps of:

1. the standard and the test sample are respectively diluted by primary anti-dilution liquid to the following 8 gradients: 0.1ng/ml, 1ng/ml, 10ng/ml, 50ng/ml, 100ng/ml, 500ng/ml, 1000ng/ml, 10000 ng/ml;

2. adding the diluted standard substance and the test solution into a 96-well plate coated with the antigen at a concentration of 100 mu l/well, reacting at 37 ℃ for 1 hour, washing for 3 times, and patting to dry;

3. diluting Ap-goat anti-human IgG (H + L) antibody 1000 times with secondary antibody diluent, adding 100 μ L/well into coated 96-well plate, reacting at 37 deg.C for 0.5 hr, washing for 3 times, and patting to dry;

4. adding a reaction substrate pNPP (p-nitrophenol phosphate), adding the reaction substrate pNPP into a coated 96-well plate at a concentration of 100 mu l/well, reacting for 20 minutes at room temperature in a dark place, and reading at 405 nm;

5. and (3) respectively carrying out four-parameter curve fitting on the standard substance and the test substance by taking the absorbance as a vertical coordinate and the concentration of the standard substance solution as a horizontal coordinate to obtain the half effective concentration (EC50) of the standard substance and the test substance, and calculating the relative binding activity of the test substance according to the following formula:

relative binding activity (%) -, EC50 of the test article/EC 50 × 100% of the standard article.

Preparation of the formulations

In another aspect, the present invention provides a method of preparing a pharmaceutical formulation comprising:

1) providing a preparation solvent and a CD147 monoclonal antibody stock solution, wherein the preparation solvent comprises a buffer solution with the concentration of 0.125-50 mmol/L or 20-30 mmol/L, a protein protective agent with the concentration of 0.25-100 mg/ml, 40-60 mg/ml or about 70-90 mg/ml, a freeze-drying support agent with the concentration of 0.35-100 mg/ml or 30-70 mg/ml and a surfactant with the concentration of 0.005-2 mg/ml or 1.0-2.0 mg/ml;

2) the CD147 monoclonal antibody stock solution was filtered to exchange solutions using the formulation vehicle to obtain the pharmaceutical formulation as described herein. In some embodiments, the filtration is ultrafiltration, diafiltration, gel filtration, and/or other filtration methods known to those of skill in the art.

As used herein, the term "ultrafiltration" refers to a process in which a mixture (e.g., a CD147 monoclonal antibody stock solution) moves through a membrane (e.g., an ultrafiltration membrane) under, for example, pressure driving, and in which different substances (e.g., solvents and solutes) are separated due to differences in their rates of filtration across the membrane in response to a given pressure driving force.

As used herein, the term "diafiltration" refers to a process in which a mixture (e.g., CD147 monoclonal antibody bulk) is separated according to the molecular size of the components therein, using, for example, a permeable membrane filter. Typically the protein stock is diafiltered through a membrane that retains the protein and allows buffer exchange, whereby over time the protein-containing stock is replaced with new buffer.

As used herein, the term "gel filtration" refers to the process of separating larger and smaller molecules by repelling molecules (e.g., proteins) larger than the resin pores with a gel, causing them to pass through the solid phase faster than smaller molecules that diffuse into the resin pores and are thus retained and move through the solid phase slower.

In some embodiments, the method further comprises freeze-drying the pharmaceutical formulation, thereby obtaining a lyophilized powder injection as described herein.

Applications of

In another aspect, the invention also provides a method of treating a disease in a subject comprising administering a therapeutically effective amount of a pharmaceutical formulation of the invention to a subject, wherein the subject has or may have a disease in need of treatment with an antibody to a CD147 molecule.

As used herein, the term "treating" refers to reducing or ameliorating the severity and/or duration of a disorder or one or more symptoms thereof, inhibiting or preventing the progression of a disorder, causing regression of a disorder, inhibiting or preventing the recurrence, development, onset, or progression of one or more symptoms associated with a disorder. Subjects in need of treatment include subjects already having the disease.

The term "therapeutically effective amount" refers to the minimum concentration required to achieve a measurable improvement or prevention of a particular condition.

The pharmaceutical formulations of the present invention are useful for treating diseases associated with the CD147 molecule, including chronic and acute diseases. Diseases associated with the CD147 molecule include malaria, cancer, inflammation, and the like. In some embodiments, the CD147 molecule associated disease is malaria. In some embodiments, the CD147 molecule associated disorder is interstitial abuse, tri-daily abuse, ovoid abuse, or malignant abuse.

The pharmaceutical formulations of the invention may be administered to a subject by any suitable route. For example, the pharmaceutical formulation may be administered to a subject by intravenous injection.

In yet another aspect, the present invention provides a use of a pharmaceutical preparation for the preparation of a medicament for the prevention and/or treatment of a disease associated with the CD147 molecule.

Embodiments of the present invention also include, but are not limited to, the following items:

item 1. a pharmaceutical formulation comprising a CD147 monoclonal antibody, a buffer, a protein protectant, a lyophilized scaffold agent, and a surfactant.

Item 2. the pharmaceutical formulation of item 1, wherein the CD147 monoclonal antibody comprises a heavy chain variable region and/or a light chain variable region, the heavy chain variable region comprising CDR sequences as shown below:

as shown in SEQ ID NO: 1, CDR1 of the amino acid sequence set forth in seq id no;

as shown in SEQ ID NO: 2 CDR2 of the amino acid sequence set forth in seq id no; and

as shown in SEQ ID NO: 3, CDR3 of the amino acid sequence set forth in seq id no;

and the light chain variable region comprises the CDR sequences shown below:

as shown in SEQ ID NO: 4 CDR1 of the amino acid sequence set forth in seq id no;

as shown in SEQ ID NO: 5 CDR2 of the amino acid sequence set forth in seq id no; and

as shown in SEQ ID NO: 6, CDR3 of the amino acid sequence set forth in seq id no.

Item 3. the pharmaceutical formulation of any one of the preceding items, wherein the CD147 monoclonal antibody is a human murine chimeric CD147 monoclonal antibody.

Item 4. the pharmaceutical formulation of any one of the preceding items, wherein the heavy chain variable region of the CD147 monoclonal antibody comprises the amino acid sequence as set forth in SEQ ID NO: 7.

Item 5. the pharmaceutical formulation of any one of the preceding items, wherein the light chain variable region of the CD147 monoclonal antibody comprises the amino acid sequence as set forth in SEQ ID NO: 8.

Item 6. the pharmaceutical formulation of any one of the preceding items, wherein the CD147 monoclonal antibody comprises an amino acid sequence as set forth in SEQ ID NO: 9 and/or the amino acid sequence shown in SEQ ID NO: 10, or a light chain of the amino acid sequence set forth in seq id No. 10.

Item 7. the pharmaceutical formulation of any one of the preceding items, wherein the pharmaceutical formulation has a pH of 5.0-8.0, 5.0-6.0, 6.0-7.0, or 7.0-8.0.

Item 8. the pharmaceutical formulation of any one of the preceding items, wherein the concentration of the CD147 monoclonal antibody in the pharmaceutical formulation is 0.. 05-40 mg/ml, 0.5-15 mg/ml, 15-25 mg/ml, 25-40 mg/ml, 0.5-1.5 mg/ml, 1-10 mg/ml, 5-15 mg/ml, 0.2-1.8 mg/ml, or 2-18 mg/ml.

Item 9. the pharmaceutical formulation according to any of the preceding items, wherein the buffer is selected from one or more of a histidine buffer, a succinic buffer, a citric buffer.

Item 10 the pharmaceutical formulation of any one of the preceding items, wherein the buffer has a concentration of 0.125-50 mmol/L, 5-11 mmol/L, 11-25 mmol/L, 25-50 mmol/L, 23-27 mmol/L, or 20-30 mmol/L.

Item 11. the pharmaceutical formulation according to any of the preceding items, wherein the pharmaceutical formulation has a pH of 5.0 to 6.0 and the buffer is a histidine buffer.

Item 12. the pharmaceutical formulation of any one of the preceding items, wherein the protein protectant is selected from one or more of sucrose, trehalose.

Item 13 the pharmaceutical formulation of any one of the preceding items, wherein the concentration of the protein protectant in the pharmaceutical formulation is 0.25-100 mg/ml, 10-60 mg/ml, 60-80 mg/ml, 80-100 mg/ml, 40-60 mg/ml, or 35-65 mg/ml.

Item 14. the pharmaceutical formulation of any one of the preceding items, wherein the protein protectant is sucrose, and the concentration of sucrose in the pharmaceutical formulation is 40-60 mg/ml.

Item 15. the pharmaceutical formulation according to any one of the preceding items, wherein the lyophilized scaffold is selected from one or more of sucrose, mannitol.

Item 16. the pharmaceutical formulation of any one of the preceding items, wherein the concentration of the lyophilized scaffold in the pharmaceutical formulation is 0.35-100 mg/ml, 10-60 mg/ml, 60-80 mg/ml, or 80-100 mg/ml.

Item 17. the pharmaceutical formulation of any one of the preceding items, wherein the lyophilized scaffold is mannitol, and the concentration of mannitol in the pharmaceutical formulation is 30-70 mg/ml.

Item 18. the pharmaceutical formulation of any one of the preceding items, wherein the surfactant is selected from one or more of polysorbate 20, polysorbate 80.

Item 19. the pharmaceutical formulation of any one of the preceding items, wherein the concentration of the surfactant in the pharmaceutical formulation is 0.005-2.5 mg/ml, 1.0-2.0 mg/ml, 0.2-0.8 mg/ml, 0.8-1.2 mg/ml, or 1.2-2.5 mg/ml.

Item 20. the pharmaceutical formulation of any one of the preceding items, wherein the surfactant is polysorbate 80 and the concentration of polysorbate 80 in the pharmaceutical formulation is 1.0-2.0 mg/ml.

Item 21. the pharmaceutical formulation of any one of the preceding items, wherein the concentration of the CD147 monoclonal antibody in the pharmaceutical formulation is 1-15 mg/ml; the buffer solution is a histidine buffer solution, and the concentration of the histidine buffer solution is 5-50 mmol/L or 20-30 mmol/L; the protein protective agent is sucrose, and the concentration of the sucrose in the pharmaceutical preparation is 10-60 mg/ml, 40-60 mg/ml or 70-90 mg/ml; the freeze-drying stent agent is mannitol, and the concentration of the mannitol in the pharmaceutical preparation is 60-80 mg/ml; the surfactant is polysorbate 80, the concentration of the polysorbate 80 in the pharmaceutical preparation is 0.9-1.1 mg/ml or 1.0-2.0 mg/ml, and the pharmaceutical preparation has a pH of 5.0-6.0.

Item 22. the pharmaceutical formulation of any one of the preceding items, wherein the concentration of the CD147 monoclonal antibody in the pharmaceutical formulation is 0.5-1.5 mg/ml or 5.0-15.0 mg/ml; the buffer solution is a histidine buffer solution, and the concentration of the histidine buffer solution is 23-27 mmol/L; the protein protective agent is sucrose, and the concentration of the sucrose in the pharmaceutical preparation is 40-60 mg/ml or 70-90 mg/ml; the freeze-drying stent agent is mannitol, and the concentration of the mannitol in the pharmaceutical preparation is 60-80 mg/ml; the surfactant is polysorbate 80, the concentration of the polysorbate 80 in the pharmaceutical preparation is 0.9-1.1 mg/ml or 1.9-2.1 mg/ml, and the pharmaceutical preparation has a pH of 5.0-6.0.

Item 23. the pharmaceutical formulation of any one of the preceding items, wherein the pharmaceutical formulation is stable for at least 12 months under cryogenic conditions.

Item 24 the pharmaceutical formulation of item 23, wherein the cryogenic conditions are at a temperature of 2-8 ℃.

Item 25. the pharmaceutical formulation of any one of the preceding items, wherein the pharmaceutical formulation is capable of being diluted 20-400 times by a diluent and is stable for at least 48 hours at a temperature of 25+2 ℃.

Item 26. the pharmaceutical formulation of item 25, wherein the diluent is a 0.9% NaCl injection.

Item 27. the pharmaceutical formulation of any one of the preceding items, wherein the pharmaceutical formulation is stable for up to 60 days at an illumination of 4500lux and a temperature of 2-8 ℃.

Item 28. the pharmaceutical formulation of any one of the preceding items, wherein the pharmaceutical formulation is a lyophilized powder injection.

Item 29 use of a pharmaceutical formulation of any one of items 1-28 in the manufacture of a medicament for treating a CD147 associated disorder.

Item 30 the use of item 29, wherein the CD147 associated disease is malaria.

Item 31. a method of making the pharmaceutical formulation of any one of items 1-28, the method comprising:

1) providing a preparation solvent of a buffer system, a protein protective agent, a freeze-dried bracket agent and a surfactant and a CD147 monoclonal antibody stock solution;

2) filtering and replacing the stock solution of the CD147 monoclonal antibody by using the preparation solvent to obtain the pharmaceutical preparation;

3) optionally, further comprising freeze-drying the pharmaceutical formulation to obtain a lyophilized powder for injection as described herein.

Item 2. the pharmaceutical formulation of item 1, wherein the CD147 monoclonal antibody comprises an amino acid sequence as set forth in SEQ ID NO: 9 and/or the amino acid sequence shown in SEQ ID NO: 10, wherein the concentration of the CD147 monoclonal antibody in the pharmaceutical preparation is 0.05-15 mg/mL, 1-10 mg/mL, 1-15 mg/mL, 1-5 mg/mL or 5-15 mg/mL.

Item 3. the pharmaceutical formulation of any one of items 1-2, wherein the pharmaceutical formulation has a pH of 5.0-8.0, 5.0-6.0, 6.0-7.0, or 7.0-8.0.

Item 4. the pharmaceutical formulation according to any one of items 1 to 3, wherein the buffer is selected from one or more of histidine buffer, succinic acid buffer, and citric acid buffer, and the concentration of the buffer is 0.125 to 50mmol/L or 20 to 30 mmol/L.

Item 5. the pharmaceutical preparation according to any one of items 1 to 4, wherein the protein protectant is one or more selected from sucrose and trehalose, and the concentration of the protein protectant in the pharmaceutical preparation is 0.25 to 100mg/ml, 20 to 80mg/ml, 40 to 60mg/ml, 70 to 90mg/ml, 60 to 80mg/ml or 80 to 100 mg/ml.

Item 6. the pharmaceutical preparation according to any one of items 1 to 5, wherein the freeze-dried scaffold is selected from one or more of sucrose and mannitol, and the concentration of the freeze-dried scaffold in the pharmaceutical preparation is 0.35 to 100mg/ml, 20 to 80mg/ml, 30 to 70mg/ml, 20 to 40mg/ml, 40 to 60mg/ml or 60 to 80 mg/ml.

Item 7. the pharmaceutical formulation of any one of items 1 to 6, wherein the surfactant is selected from one or more of polysorbate 20 and polysorbate 80, and the concentration of the surfactant in the pharmaceutical formulation is 0.005mg/ml to 2.0mg/ml, 0.005mg/ml to 1.0mg/ml, or 1mg/ml to 2.0 mg/ml.

Item 8. the pharmaceutical formulation of any one of items 1 to 7, which comprises 0.05 to 15mg/ml of the CD147 monoclonal antibody, 0.125 to 50mmol/L of the buffer, 0.25 to 100mg/ml of the protein protectant, 0.35 to 100mg/ml of the lyophilized scaffolding agent, and 0.005 to 2.0mg/ml of the surfactant, and which has a pH of 5.0 to 8.0.

Item 9. the pharmaceutical formulation of any one of items 1 to 8, which comprises 1 to 10mg/ml of the CD147 monoclonal antibody, 20 to 30mmol/L of a buffer solution, 40 to 60mg/ml or 70 to 90mg/ml of a protein protectant, 30 to 70mg/ml of a lyophilized scaffold agent, and 1.0 to 2.0mg/ml of a surfactant, and which has a pH of 5.0 to 6.0.

Item 10 the pharmaceutical formulation of any one of items 1-9, wherein the pharmaceutical formulation is stable at 2-8 ℃ for at least 12 months.

Item 11. the pharmaceutical formulation of any one of items 1 to 10, wherein the pharmaceutical formulation is capable of being diluted 20-400 fold with a diluent and is stable at a temperature of 25 ± 2 ℃ for at least 48 hours.

Item 12 the pharmaceutical formulation of any one of items 1 to 11, wherein the pharmaceutical formulation is stable for 60 days at an illumination of 4500lux and a temperature of 2 to 8 ℃.

Item 13 the pharmaceutical preparation of any one of items 1 to 12, wherein the pharmaceutical preparation is a lyophilized powder injection.

Item 14 the pharmaceutical formulation of item 13, wherein the lyophilized powder is suitable for intravenous injection after reconstitution.

Item 15 use of the pharmaceutical formulation of any one of items 1 to 14 in the manufacture of a medicament for the prevention and/or treatment of a CD147 associated disease, optionally wherein the disease is malaria.

Item 16. a method of preparing a pharmaceutical formulation, the method comprising:

1) providing a formulation vehicle and a CD147 monoclonal antibody stock solution, wherein the formulation vehicle comprises a buffer solution, a protein protectant, a lyophilized scaffold agent, and a surfactant;

2) and (3) filtering and replacing the CD147 monoclonal antibody stock solution by using the preparation solvent to obtain the pharmaceutical preparation of the item 8 or 9.

Item 17. the method of item 16, further comprising freeze drying the pharmaceutical formulation, thereby obtaining the pharmaceutical formulation of item 13 or 14.

Examples

The present invention may be better understood by reference to the following examples, which, however, are intended to illustrate the invention and should not be construed as limiting the scope of the invention. Many modifications and variations are possible in light of the teaching herein and are therefore within the scope of the invention.

The various reagents, devices and measurement methods used in the examples are as follows:

reagent

CD147 monoclonal antibody: a 6H8MAb monoclonal antibody comprising the amino acid sequence set forth in SEQ ID NO: 9 and the heavy chain as set forth in SEQ ID NO: 10, or a light chain as shown in figure 10.

Histidine buffer: is prepared from histidine solution and histidine hydrochloride solution.

Succinic acid buffer solution: is prepared from succinic acid solution and disodium succinate solution.

Citric acid buffer solution: is prepared from citric acid solution and trisodium citrate solution.

Test method

In studying the high temperature stability, light stability and long term stability of the formulation, the preset criteria used in the present invention to indicate the stability of the formulation are: the change of the protein concentration is not more than 2.0mg/ml (for the initial protein concentration of 10 mg/ml), the protein purity measured by a CE-SDS non-reduction method is more than or equal to 95.0 percent, the protein purity measured by a CE-SDS reduction method is more than or equal to 95.0 percent, the unglycosylated heavy chain (NGHC) is less than or equal to 5.0 percent, the monomer content in the protein purity measured by a SE-HPLC method is more than or equal to 98.00 percent, the polymer content is less than or equal to 2.00 percent, the pH of the preparation is 5.5-6.0, the osmotic pressure of the preparation is 400-800 mOsmol/kg, and the binding activity of the antibody is 65-150 percent.

In studying the stability of the formulation to shaking, the preset criteria used in the present invention to indicate the stability of the formulation are: the increase in polymer content, as measured by SE-HPLC, was less than 2.00%.

In studying the accelerated thermal stability of the formulation, the preset criteria used in the present invention to indicate the stability of the formulation are: the increase in polymer content, as measured by SE-HPLC, is less than 2.00% and the binding activity, as measured by ELISA, should be 65% to 150% of the control.

In studying the dilution stability of a formulation, the preset criteria used in the present invention to indicate the stability of the formulation are: for a preparation with the diluted protein concentration of 0.05mg/ml, after a period of time, the protein concentration of the preparation measured by an ultraviolet method is 0.05 +/-0.01 mg/ml, the protein purity measured by a CE-SDS non-reduction method is more than or equal to 95.0%, the protein purity measured by a CE-SDS reduction method is more than or equal to 95.0%, the non-glycosylated heavy chain (NGHC) is less than or equal to 5.0%, the monomer content measured by an SE-HPLC method is more than or equal to 98.00%, the polymer content is less than or equal to 2.00%, the pH value of the preparation is 5.0-6.0, the binding activity of an antibody is 65-150%, the quantity of insoluble particles with the particle size of more than 10 mu m is less than or equal to 6000 particles/ml, and the quantity of insoluble particles with the particle size of more than 25 mu m is less than or equal to 600 particles/ml.

The following table lists various test conditions for formulation stability:

protein concentration determination: ultraviolet-visible spectrophotometry (detection wavelength: 280nm)

Protein purity determination: CE-SDS method, SE-HPLC method

Determination of antibody binding Activity: enzyme linked immunosorbent assay

Example 1: CD147 single at different pH valuesStability of cloned antibodies

The CD147 monoclonal antibody was placed at a concentration of 1mg/ml in a histidine-histidine hydrochloride buffer solution (prepared from a 25mmol/L histidine solution and a 25mmol/L histidine hydrochloride solution) having a pH of 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and the thermal transition midpoint (Tm) of the antibody was measured using a Differential Scanning Calorimeter (DSC). The test results obtained are shown in table 1 and fig. 1.

TABLE 1 DSC scan data of CD147 monoclonal antibody at different pH values

pH	Tm1(℃)	Tm2(℃)	Tm3(℃)
				5.0	61.44	73.42	82.80
5.5	64.87	76.05	84.23
				6.0	70.48	79.66	84.86
6.5	72.83	/*	83.62
				7.0	/*	/*	80.88
7.5	/*	/*	79.98

Not detected

Theoretically, a DSC scan of the CD147 monoclonal antibody should obtain three Tm values, which correspond to three different domains of the antibody, respectively. However, in the DSC scan of this example, the samples at some pH values did not show three Tm values, indicating that part of the antibody domain has been denatured or destroyed under these pH conditions, in other words, these pH conditions are not favorable for antibody stability.

As can be seen from Table 1 and FIG. 1, the CD147 monoclonal antibodies all showed three Tm values at pH 5.0 to 6.0, while the CD147 monoclonal antibodies did not show three Tm values at pH higher than 6.0. This shows that the CD147 monoclonal antibody can exist stably at pH 5.0-6.0. Particularly, in the range of pH 5.0-6.0, Tml, Tm2 and Tm3 values of the CD147 monoclonal antibody at pH 5.0-6.0 are obviously higher than Tm1, Tm2 and Tm3 values at pH 6.0-7.5, which shows that the pH 5.0-6.0 is more beneficial to the stability of the CD147 monoclonal antibody. Therefore, for a pharmaceutical formulation comprising the CD147 monoclonal antibody, the pH is 5.0-6.0, the preferred pH range of the formulation, and the pH is 6.0-7.0 and the pH is 7.0-8.0, the optional pH range of the formulation.

Example 2: effect of buffer on formulation stability

The following 3 different buffers were selected in this example to formulate a pharmaceutical formulation containing CD147 monoclonal antibody at a concentration of 1mg/ml, pH 5.8:

the Tm of the antibody in the pharmaceutical formulation was measured using DSC. The DSC scan results obtained are shown in table 2 and fig. 2. Wherein the test results for each set of buffers listed in tables 2 and 3 are the average of the test results for formulations containing the relevant buffer set.

TABLE 2 DSC scan data for formulations comprising CD147 monoclonal antibody and different buffers

Buffer solution	pH	Tm1(℃)	Tm2(℃)	Tm3(℃)
					Histidine buffer	5.8	67.95	78.12	84.90
Succinic acid buffer solution	5.8	71.89	/	81.67
					Citric acid buffer	5.8	/	/	80.33

As can be seen from table 2, at pH 5.8, the CD147 monoclonal antibody in histidine buffer showed three Tm values, but the CD147 monoclonal antibody in either succinic acid buffer or citric acid buffer showed only two or one Tm value, indicating that the CD147 monoclonal antibody had better thermodynamic stability in histidine buffer than the other two buffers, theoretically having a longer useful life.

Therefore, histidine buffer may be the most preferred buffer for formulations comprising the CD147 monoclonal antibody of the present invention, while succinic buffer and citric buffer may be optional buffers.

Example 3: effect of surfactants on formulation stability

In the case of using histidine buffer, 80mg/mL of sucrose was added as a protein protectant, and the surfactant was screened as an injection. This example selects 2 different surfactants (polysorbate 80, polysorbate 20) to formulate a formulation containing CD147 monoclonal antibody at a concentration of 10.0mg/mL, histidine buffer at a concentration of 25mmol/L, and sucrose at a concentration of 80mg/mL at surfactant concentrations falling within different ranges (0-1.0 mg/mL and 1.0-2.0 mg/m 1). For comparison, a control formulation of the same other components but without surfactant was also prepared. Experimental groups of formulations containing CD147 monoclonal antibody, histidine buffer, sucrose protein protectant and different surfactants at different concentrations are shown in table 3. The above formulations were used for the oscillatory stability test. The obtained oscillation stability test experimental group and the results thereof are shown in table 3, table 4 and fig. 3.

TABLE 3 Experimental groups of formulations containing CD147 monoclonal antibody, histidine buffer, sucrose protein protectant and different surfactants at different concentrations

TABLE 4 Polymer content data (%). by SE-HPLC in the Oscillating stability test of formulations comprising CD147 monoclonal antibody, histidine buffer, sucrose protein protectant and different surfactants at different concentrations

TABLE 5 results from appearance determination in the Shake stability test of formulations comprising CD147 monoclonal antibody, histidine buffer, sucrose protein protectant and different surfactants at different concentrations

Experimental group number	Severity of turbidity at 24h	The turbidity severity appears at 48h
			1	++	+++
2	-	-
			3	-	-
4	-	-
			5	-	-

As can be seen from tables 4 and 5 and fig. 3, the polymer contents of the experimental groups 1 to 5 have no significant difference, but the sample without any surfactant has a severe turbidity phenomenon, and after the sample with the polysorbate (polysorbate 20 or polysorbate 80) is shaken for 48h, the turbidity disappears, and the sample is in a clear state, which indicates that both polysorbate 20 and polysorbate 80 can prevent the aggregation of antibodies, reduces the generation of precipitates, and indicates that both polysorbate 80 and polysorbate 20 have the function of slowing down the protein precipitation at the concentration of 1.0-2.0 mg/ml.

From the test results, it can be seen that both polysorbate 80 and polysorbate 20 have the effect of slowing down protein precipitation at a concentration of 1.0-2.0 mg/ml. According to the description of the safety of polysorbate in a preparation auxiliary material manual, the mouse injection safe dosage of polysorbate 80 is higher than that of polysorbate 20, so that the pharmaceutical preparation of the invention preferably uses polysorbate 80 (namely, tween 80 or PS80) as a surfactant, and polysorbate 20 also has the effect of slowing down protein precipitation, so that the pharmaceutical preparation of the invention can be used as an optional surfactant.

Example 4: effect of protein protectant and lyophilized Stent on formulation stability

Using histidine buffer solution with concentration of 25mmol/L, selecting protein protective agent (sucrose) with different concentrations and freeze-drying scaffold agent (mannitol) with different concentrations, preparing preparation with pH of 5.8 and containing CD147 monoclonal antibody with concentration of 10mg/ml and different protein protective agent and freeze-drying scaffold agent with different concentrations, and preparing into freeze-dried samples. Experimental groups of formulations containing CD147 monoclonal antibody, 25mmol/L histidine buffer and different concentrations of different protein protective agents and lyophilized scaffolds are shown in Table 6.

TABLE 6 Experimental groups of formulations containing CD147 monoclonal antibody, histidine buffer and different concentrations of different protein protectants and lyophilized scaffolds

TABLE 7 formulation accelerated thermal stability data for the preferred experimental group of formulations containing CD147 monoclonal antibody, histidine buffer and 50mg/ml sucrose as protein protectant and 70mg/ml mannitol as lyophilized scaffold agent (i.e., experimental group 7)

As can be seen from fig. 4, the experimental groups 1, 2, 3, 4 shrank directly, not in the expected cake powder (not shown in the figure). While the experimental groups 5, 6, 7 added mannitol as the freeze-dried scaffolds were cakes, wherein the bottoms of the experimental groups 5 and 6 were shrunk to different degrees, and the shapes thereof were consistent with the expectation as the concentration of mannitol increased to 70mg/mL, which indicates that the mannitol concentration increased to enhance the supporting effect on the scaffold. After the freeze-drying is finished, the water content in the freeze-dried preparation is detected, and the water content is detected to be 0.6%, which shows that the freeze-drying effect is better.

Based on this, accelerated studies were conducted to examine the effect on the stability of the preparation. The test results show that the preferable protein protective agent sucrose and the freeze-drying bracket agent mannitol have better protective effect on protein and have good supporting effect on the appearance of the freeze-dried powder injection. Mannitol at a concentration of 70mg/ml is therefore most preferred for use in the lyophilized scaffolds of the pharmaceutical formulations of the present invention.

Example 5: factors influencing the stability of the pharmaceutical preparations according to the invention

The pharmaceutical formulation of the present invention was prepared wherein the concentration of the CD147 monoclonal antibody was 10.0mg/ml, the concentration of histidine buffer was 25mmol/L, the concentration of sucrose was 50mg/ml, the concentration of mannitol was 70mg/ml, the concentration of polysorbate 80 was 1.0mg/ml, and the pH of the formulation was 5.8. The resulting formulations were used for influencing factor tests including high temperature stability test, light stability test. The results of the individual tests are shown in table 8.

As can be seen from table 8, the protein concentration, pH, osmotic pressure and binding activity of the formulations of the present invention were maintained within the preset standard ranges under the high temperature, light stability test conditions. Under high temperature testing conditions, the protein purity (CE-SDS non-reduced/reduced) of the formulation of the invention did not change significantly after 60 days, increasing the polymer content from 0.47% to 0.68%; under light test conditions, the polymer content of the formulations of the invention increased from 0.47% to 0.61%, the protein purity (CE-SDS reduction) decreased from 97.5% to 96.7%, and the protein purity (SE-HPLC method) decreased from 99.53% to 99.29% after 60 days. At each test endpoint, the purity (CE-SDS) and polymer content of the formulations of the present invention met pre-set standards.

Therefore, the pharmaceutical preparation of the invention can better maintain the stability of the CD147 monoclonal antibody. In order to better maintain the stability of the antibody, it is preferred to store the pharmaceutical formulation of the present invention at a temperature of 2-8 ℃ under protection from light.

Example 6: long term stability of the formulations of the invention

3 batches of the formulation of the present invention were prepared using CD147 monoclonal antibody, histidine buffer, sucrose, mannitol and polysorbate 80, designated as group A, group B and group C, respectively, wherein the CD147 monoclonal antibody concentration was 10.0mg/ml, the histidine buffer concentration was 25mmol/L, the sucrose concentration was 50mg/ml, the mannitol concentration was 70mg/ml, the polysorbate 80 concentration was 1.0mg/ml, and the pH of the formulation was 5.8.

The long-term stability tests were performed on the formulations of groups A-C and the results are listed in Table 9. The preparation is placed in a penicillin bottle, after freeze-drying, the bottle opening is sealed by a brominated butyl rubber plug, and the bottle opening is fastened by an aluminum-plastic combined cover. The preparation is stored in a dark and sealed condition at the temperature of 2-8 ℃ for at least 12 months, and samples are taken regularly to detect the protein concentration, the purity and the like of the preparation.

As can be seen from table 9, at the end of the 12 month test, there was no significant change in protein concentration, pH, osmolality, binding activity and purity (CE-SDS reduced/non-reduced) for the group a-C formulations; the polymer content, determined by the SE-HPLC method, rose from 0.47% to 0.67% (group A), from 0.59% to 1.12% (group B) and from 0.49% to 0.71% (group C), respectively. The polymer content of the formulations of groups A-C all met the predetermined criteria. This indicates that the formulation of the present invention is stable for 12 months storage at 2-8 ℃.

Example 7: dilution stability of the formulations of the invention

After the preparation is prepared and redissolved by 1mL of water for injection, the concentration of the CD147 monoclonal antibody is 10.0mg/mL, the concentration of a histidine buffer solution is 25mmol/L, the concentration of sucrose is 50mg/mL, the concentration of mannitol is 70mg/mL, the concentration of polysorbate 80 is 1.0mg/mL, and the pH value of the preparation is 5.8. The formulation was diluted with 0.9% NaCl injection to reduce the antibody concentration in the formulation from 10.0mg/ml to 0.05 mg/ml. The stability of the diluted formulations was determined under dilution stability test conditions. This dilution stability test was performed in triplicate, A, B and group C, respectively. The results are shown in Table 10.

As can be seen from Table 10, the formulations in groups A-C all showed changes in pH, protein concentration, protein purity (CE-SDS method), monomer and polymer content (SE-HPLC method), insoluble microparticles, and binding activity after dilution to predetermined standards, indicating that the formulations of the present invention can be stably stored at 25. + -. 2 ℃ for at least 48 hours after 200-fold dilution with 0.9% NaCl injection.

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications and alterations of this invention will become apparent to those skilled in the art in light of the foregoing description. Such modifications and variations are also within the scope of the appended claims.

Claims

1. A pharmaceutical formulation comprising a CD147 monoclonal antibody, a buffer, a protein protectant, a lyophilized scaffold agent, and a surfactant, wherein,

the buffer solution is histidine buffer solution, the concentration of the histidine buffer solution in the pharmaceutical preparation is 20-30 mmol/L,

the protein protective agent is sucrose, the concentration of the protein protective agent in the pharmaceutical preparation is 40-90 mg/ml,

the freeze-dried stent agent is mannitol, the concentration of which in the pharmaceutical preparation is 30-80 mg/ml, and

the surfactant is polysorbate 20 and/or polysorbate 80, the concentration of the polysorbate 20 and/or polysorbate 80 in the pharmaceutical preparation is 1.0-2.0 mg/mL, the CD147 monoclonal antibody comprises a heavy chain of an amino acid sequence shown in SEQ ID NO. 9 and a light chain of an amino acid sequence shown in SEQ ID NO. 10, and the concentration of the CD147 monoclonal antibody in the pharmaceutical preparation is 1-15 mg/mL.

2. The pharmaceutical formulation of claim 1, wherein the pharmaceutical formulation has a pH of 5.0-8.0.

3. The pharmaceutical formulation of claim 1 or 2, wherein the pharmaceutical formulation is stable at 2-8 ℃ for at least 12 months.

4. The pharmaceutical formulation of claim 1 or 2, wherein the pharmaceutical formulation is capable of being diluted 20-400 fold by a diluent and is stable for at least 48 hours at a temperature of 25 +/-2 ℃.

5. The pharmaceutical formulation of claim 1 or 2, wherein the pharmaceutical formulation is stable for up to 60 days at an illumination of 4500lux and a temperature of 2-8 ℃.

6. The pharmaceutical preparation of claim 1 or 2, wherein the pharmaceutical preparation is a lyophilized powder injection.

7. The pharmaceutical preparation of claim 6, wherein the lyophilized powder is suitable for intravenous injection after reconstitution.

8. Use of a pharmaceutical formulation according to any one of claims 1-2 and 7 for the manufacture of a medicament for the prevention and/or treatment of a CD147 related disease.

9. A method of making a pharmaceutical formulation, the method comprising:

2) filtering and replacing the stock solution of the CD147 monoclonal antibody by using the preparation solvent to obtain the pharmaceutical preparation of claim 1 or 2.

10. The method of claim 9, further comprising freeze-drying the pharmaceutical formulation, thereby obtaining the pharmaceutical formulation of claim 6 or 7.