CN116725960A

CN116725960A - Novel coronavirus antibody pharmaceutical composition and use thereof

Info

Publication number: CN116725960A
Application number: CN202310195384.6A
Authority: CN
Inventors: 刘沛想; 刘洪川; 渠晨曦
Original assignee: Shanghai Junshi Biosciences Co Ltd; Suzhou Junmeng Biosciences Co Ltd
Current assignee: Shanghai Junshi Biosciences Co Ltd; Suzhou Junmeng Biosciences Co Ltd
Priority date: 2022-03-11
Filing date: 2023-03-03
Publication date: 2023-09-12

Abstract

The present application provides pharmaceutical compositions of novel coronavirus antibodies and uses thereof. The pharmaceutical composition contains a novel coronavirus antibody or an antigen binding fragment thereof and a buffer solution, wherein the novel coronavirus antibody or the antigen binding fragment thereof comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3 respectively, and LCDR1, LCDR2 and LCDR3 with amino acid sequences shown as SEQ ID NO. 4, AAS and SEQ ID NO. 5 respectively. The application also provides an injection containing the pharmaceutical composition and application of the pharmaceutical composition and the injection in preparing medicines for preventing or treating novel coronavirus SARS-CoV-2 infection by eliminating, inhibiting or reducing the activity of the novel coronavirus.

Description

Novel coronavirus antibody pharmaceutical composition and use thereof

The present application claims priority from the chinese patent office, application number 202210237031.3 entitled "novel coronavirus antibody pharmaceutical composition and use thereof," filed on day 11 and 3 of 2022, the entire contents of which are incorporated herein by reference.

Technical Field

The application relates to the field of biological medicine, in particular to a novel coronavirus antibody pharmaceutical composition and application thereof.

Background

Since the advent of covd-19 in 2019, many variants have been derived from SARS-CoV-2, some of which contain amino acid substitutions at key sites of spike protein, exhibit greater transmission capacity, immune escape capacity and lower sensitivity to monoclonal antibodies, and the initial appearance of these variants is often accompanied by high infection rates in the country of origin, such as Alpha mutant (b.1.1.7), beta mutant (b.1.351) (Beta), gamma mutant (p.1) and Delta mutant (b.1.617.2), among others. The emergence of Delta mutants in india resulted in a catastrophic rise in infection rate and mortality, up to month 5 of 2021, delta mutants became dominant mutants in the uk, and 6 months of the same year, became dominant mutants worldwide.

At present, SARS-CoV-2 is mainly prevented by vaccination, and in the aspect of infection treatment, new crown vaccine neutralizing antibody medicines such as Casirvimab, sotrovimab, bamlanivimab, etesevimab (CB 6) are marketed, but when any antibody is used alone, the main Alpha (Alpha), beta (Beta), gamma (Gamma), kappa (Kappa), epsilon and Delta (Delta) mutant strains cannot be covered. There is currently no pharmaceutical composition of novel coronavirus antibodies capable of high stability against a variety of mutants.

Disclosure of Invention

The application aims to provide a pharmaceutical composition containing an antibody capable of specifically binding to a novel coronavirus, which has the advantages of long-term stability, no aggregation and the like.

The present application provides in a first aspect a pharmaceutical composition comprising:

(1) A buffer; and

(2) A novel coronavirus antibody or antigen-binding fragment thereof;

wherein the novel coronavirus antibody or antigen binding fragment thereof comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3 respectively, and LCDR1, LCDR2 and LCDR3 with amino acid sequences shown as SEQ ID NO. 4, AAS and SEQ ID NO. 5 respectively.

In some embodiments, the novel coronavirus antibody or antigen-binding fragment thereof comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO. 6 and a light chain variable region having an amino acid sequence as shown in SEQ ID NO. 7.

In some embodiments, the novel coronavirus antibody or antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence as shown in SEQ ID NO. 8 and a light chain having an amino acid sequence as shown in SEQ ID NO. 9.

In some embodiments, the concentration of the novel coronavirus antibody or antigen-binding fragment thereof in the pharmaceutical composition is 2-200 mg/mL, preferably 10-150 mg/mL, more preferably 50-100 mg/mL.

In some embodiments, the pharmaceutical composition, the buffer is selected from at least one of an acetate buffer and a histidine buffer; preferably, the buffer is a histidine buffer selected from at least one of a histidine-histidine hydrochloride buffer and a histidine-histidine acetate buffer; preferably, the concentration of the buffer is 5 to 50mM (mmol/L), preferably 10 to 30mM; preferably, the pH of the buffer is from 5.0 to 7.0, preferably from 5.5 to 6.5, more preferably from 5.9 to 6.1.

In some embodiments, the pharmaceutical composition further comprises a stabilizer selected from at least one of sucrose and trehalose; preferably, the concentration of the stabilizing agent in the pharmaceutical composition is 100 to 300mM, preferably 120 to 280mM, more preferably 200 to 250mM.

In some embodiments, the pharmaceutical composition further comprises a surfactant selected from at least one of polysorbate 80 (polysorbate 80), polysorbate 20, and poloxamer 188; preferably, in the pharmaceutical composition, the surfactant concentration is 0.01% to 0.1%, preferably 0.01% to 0.04%, calculated as w/v.

In some embodiments, the pharmaceutical composition comprises the components set forth in any one of (1) to (4) below:

(1) (a) 10-150 mg/mL of the novel coronavirus antibody or antigen-binding fragment thereof; (b) 10-30 mM acetate buffer, pH 5.0-7.0; (c) 120-280 mM sucrose; and (d) polysorbate 80 at a concentration of 0.01% -0.1% in w/v; preferably, (a) 50-100 mg/mL of the novel coronavirus antibody or antigen binding fragment thereof; (b) 10-30 mM acetate buffer, pH 5.5-6.5; (c) 200-250 mM sucrose; and (d) polysorbate 80 at a concentration of 0.01% -0.04% in w/v; more preferably, (a) 50mg/mL of the novel coronavirus antibody or antigen binding fragment thereof; (b) 20mM acetate buffer, pH 5.9-6.1; (c) 230mM sucrose; and (d) polysorbate 80 at a concentration of 0.04% w/v; or (a) 100mg/mL of the novel coronavirus antibody or antigen binding fragment thereof; (b) 20mM acetate buffer, pH 5.9-6.1; (c) 230mM sucrose; and (d) polysorbate 80 at a concentration of 0.04% w/v; or (b)

(2) (a) 10-150 mg/mL of the novel coronavirus antibody or antigen-binding fragment thereof; (b) 10-30 mM acetate buffer, pH 5.0-7.0; (c) 120-280 mM trehalose; and (d) polysorbate 80 at a concentration of 0.01% -0.1% in w/v; preferably, (a) 50-100 mg/mL of the novel coronavirus antibody or antigen binding fragment thereof; (b) 10-30 mM acetate buffer, pH 5.5-6.5; (c) 200-250 mM trehalose; and (d) polysorbate 80 at a concentration of 0.01% -0.04% in w/v; more preferably, (a) 50mg/mL of the novel coronavirus antibody or antigen binding fragment thereof; (b) 20mM acetate buffer, pH 5.9-6.1; (c) 230mM trehalose; and (d) polysorbate 80 at a concentration of 0.04% w/v; or (a) 100mg/mL of the novel coronavirus antibody or antigen binding fragment thereof; (b) 20mM acetate buffer, pH 5.9-6.1; (c) 230mM trehalose; and (d) polysorbate 80 at a concentration of 0.04% w/v; or (b)

(3) (a) 10-150 mg/mL of the novel coronavirus antibody or antigen-binding fragment thereof; (b) 10-30 mM histidine buffer, pH 5.0-7.0; (c) 120-280 mM sucrose; and (d) polysorbate 80 at a concentration of 0.01% -0.1% in w/v; preferably, (a) 50-100 mg/mL of the novel coronavirus antibody or antigen binding fragment thereof; (b) 10-30 mM histidine buffer, pH 5.5-6.5; (c) 200-250 mM sucrose; and (d) polysorbate 80 at a concentration of 0.01% -0.04% in w/v; more preferably, (a) 50mg/mL of the novel coronavirus antibody or antigen binding fragment thereof; (b) 20mM histidine buffer, pH 5.9-6.1; (c) 230mM sucrose; and (d) polysorbate 80 at a concentration of 0.04% w/v; or (a) 100mg/mL of the novel coronavirus antibody or antigen binding fragment thereof; (b) 20mM histidine buffer, pH 5.9-6.1; (c) 230mM sucrose; and (d) polysorbate 80 at a concentration of 0.04% w/v; or (b)

(4) (a) 10-150 mg/mL of the novel coronavirus antibody or antigen-binding fragment thereof; (b) 10-30 mM histidine buffer, pH 5.0-7.0; (c) 120-280 mM trehalose; and (d) polysorbate 80 at a concentration of 0.01% -0.1% in w/v; preferably, (a) 50-100 mg/mL of the novel coronavirus antibody or antigen binding fragment thereof; (b) 10-30 mM histidine buffer, pH 5.5-6.5; (c) 200-250 mM trehalose; and (d) polysorbate 80 at a concentration of 0.01% -0.04% in w/v; more preferably, (a) 50mg/mL of the novel coronavirus antibody or antigen binding fragment thereof; (b) 20mM histidine buffer, pH 5.9-6.1; (c) 230mM trehalose; and (d) polysorbate 80 at a concentration of 0.04% w/v; or (a) 100mg/mL of the novel coronavirus antibody or antigen binding fragment thereof; (b) 20mM histidine buffer, pH 5.9-6.1; (c) 230mM trehalose; and (d) polysorbate 80 at a concentration of 0.04% w/v.

In some embodiments, the pharmaceutical composition has a pH of 5.0 to 7.0, preferably 5.5 to 6.5, more preferably 5.9 to 6.1.

In some embodiments, the pharmaceutical composition has an osmotic pressure of 250 to 350mOsm/kg.

The second aspect of the present application provides a lyophilized formulation comprising the pharmaceutical composition provided in the first aspect of the present application.

A third aspect of the present application provides a liquid formulation comprising the pharmaceutical composition provided in the first aspect of the present application or a liquid formulation obtained by resuspension of the lyophilized formulation provided in the second aspect of the present application; preferably, the liquid formulation is an injection.

In some embodiments, the liquid formulation contains a dextrose solution or sodium chloride solution, and the pharmaceutical composition provided by the first aspect of the application; preferably, the concentration of the sodium chloride solution is 0.85-0.9% and the concentration of the glucose solution is 5-25% in terms of w/v; preferably, the concentration of the novel coronavirus antibody in the liquid formulation is 0.1-50 mg/mL, more preferably 0.5-30 mg/mL; preferably, the pH of the liquid formulation is from 5.0 to 7.0.

In some embodiments, the lyophilized formulation provided in the second aspect of the application or the liquid formulation provided in the third aspect of the application is stable at 2-8 ℃ for at least 3 months, at least 6 months, at least 12 months, at least 18 months or at least 24 months.

In some embodiments, the lyophilized formulation provided in the second aspect of the application or the liquid formulation provided in the third aspect of the application is stable at 40 ℃ for at least 7 days, at least 14 days, or at least 28 days.

In a fourth aspect, the present application provides the use of a pharmaceutical composition provided in accordance with the first aspect of the application, a lyophilized formulation provided in accordance with the second aspect of the application or a liquid formulation provided in accordance with the third aspect of the application for the preparation of a medicament for the prevention or treatment of an infectious disease by eliminating, inhibiting or reducing the activity of a novel coronavirus; preferably, the infectious disease is a novel coronavirus infection.

In some embodiments, the pharmaceutical composition provided in the first aspect of the application or the liquid formulation provided in the third aspect of the application is administered by intravenous injection or subcutaneous injection.

In some embodiments, the pharmaceutical composition provided in the first aspect of the application or the liquid formulation provided in the third aspect of the application is administered to the respiratory tract or mucosa.

In a fifth aspect, the application provides the use of a pharmaceutical composition provided in the first aspect, a lyophilized formulation provided in the second aspect or a liquid formulation provided in the third aspect of the application for the manufacture of a medicament for preventing or treating a novel coronavirus (SARS-CoV-2) infection by eliminating, inhibiting or reducing the activity of the novel coronavirus.

The pharmaceutical composition containing the antibody capable of specifically binding with the novel coronavirus has the advantages of long-term stability, no aggregation and the like, and can be used for preparing a medicament for eliminating, inhibiting or reducing the activity of the novel coronavirus to prevent or treat the infection of the novel coronavirus (SARS-CoV-2).

Drawings

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described, and it is apparent that the drawings in the description below are only one embodiment of the present application, and other embodiments may be obtained according to these drawings by those skilled in the art.

FIG. 1A is a graph showing the binding activity of a pharmaceutical composition comprising a novel coronavirus antibody of example 3 of the present application to wild-type SARS-CoV-2 RBD;

FIG. 1B is a graph showing the binding activity of the pharmaceutical composition comprising the novel coronavirus antibody of example 3 of the present application to the Alpha mutant SARS-CoV-2 RBD;

FIG. 1C is a graph showing the binding activity of the pharmaceutical composition comprising the novel coronavirus antibody of example 3 of the present application to the Beta mutant SARS-CoV-2 RBD;

FIG. 1D is a graph showing the binding activity of a pharmaceutical composition comprising a novel coronavirus antibody of example 3 of the present application to the Gamma mutant SARS-CoV-2 RBD;

FIG. 1E is a graph showing the binding activity of a pharmaceutical composition comprising a novel coronavirus antibody of example 3 of the present application to the Kappa mutant SARS-CoV-2 RBD;

FIG. 1F is a graph showing the binding activity of the pharmaceutical composition comprising the novel coronavirus antibody of example 3 of the present application to the Epsilon mutant SARS-CoV-2 RBD;

FIG. 1G is a graph showing the binding activity of a pharmaceutical composition comprising a novel coronavirus antibody of example 3 of the present application to the Delta mutant strain SARS-CoV-2 RBD;

FIG. 2A is a graph showing the results of competitive inhibition activity of a pharmaceutical composition comprising a novel coronavirus antibody of example 4 of the present application against the binding of wild-type SARS-CoV-2RBD to human ACE2 protein;

FIG. 2B is a graph showing the competitive inhibition activity of the pharmaceutical composition comprising the novel coronavirus antibody of example 4 of the present application on the binding of the Alpha mutant SARS-CoV-2RBD to human ACE2 protein;

FIG. 2C is a graph showing the competitive inhibition activity of the pharmaceutical composition comprising the novel coronavirus antibody of example 4 of the present application against the binding of the Beta mutant SARS-CoV-2RBD to human ACE2 protein;

FIG. 2D is a graph showing the competitive inhibition activity of the pharmaceutical composition comprising the novel coronavirus antibody of example 4 of the present application against the binding of the Gamma mutant SARS-CoV-2RBD to human ACE2 protein;

FIG. 2E is a graph showing the competitive inhibition activity of a pharmaceutical composition comprising a novel coronavirus antibody of example 4 of the present application against the binding of the Kappa mutant SARS-CoV-2RBD to human ACE2 protein;

FIG. 2F is a graph showing the competitive inhibition activity of the pharmaceutical composition comprising the novel coronavirus antibody of example 4 of the present application against the binding of the Epsilon mutant SARS-CoV-2RBD to human ACE2 protein;

FIG. 2G is a graph showing the competitive inhibition activity of the pharmaceutical composition comprising the novel coronavirus antibody of example 4 of the present application against the binding of the Delta mutant SARS-CoV-2RBD to human ACE2 protein;

FIG. 3A is a graph showing the blocking effect of a pharmaceutical composition comprising a novel coronavirus antibody of example 5 of the present application on the process of infecting 293-ACE2 cells with wild-type SARS-CoV-2 pseudovirus;

FIG. 3B is a graph showing the blocking effect of the pharmaceutical composition comprising the novel coronavirus antibody of example 5 of the present application on the infection of 293-ACE2 cells by the Alpha mutant SARS-CoV-2 pseudovirus;

FIG. 3C is a graph showing the blocking effect of a pharmaceutical composition comprising a novel coronavirus antibody of example 5 of the present application on the process of infection of 293-ACE2 cells by the Beta mutant SARS-CoV-2 pseudovirus;

FIG. 3D is a graph showing the blocking effect of a pharmaceutical composition comprising a novel coronavirus antibody of example 5 of the present application on the infection of 293-ACE2 cells by the Gamma mutant SARS-CoV-2 pseudovirus;

FIG. 3E is a graph showing the blocking effect of a pharmaceutical composition comprising a novel coronavirus antibody of example 5 of the present application on the process of infection of 293-ACE2 cells by the Kappa mutant SARS-CoV-2 pseudovirus;

FIG. 3F is a graph showing the blocking effect of a pharmaceutical composition comprising a novel coronavirus antibody of example 5 of the present application on the process of Delta mutant SARS-CoV-2 pseudovirus infection of 293-ACE2 cells.

Detailed Description

Definition and description

In order that the application may be more readily understood, certain technical and scientific terms are defined below. Unless defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. It is to be understood that this application is not limited to particular methods, reagents, compounds, compositions or biological systems, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. All references cited herein, including patents, patent applications, articles, textbooks, and the like, and to the extent that they have not been cited, are hereby incorporated by reference in their entirety. The present application is directed to the incorporated documents and/or similar materials if one or more of the incorporated documents and similar materials differs from or contradicts the present application, including but not limited to the defined terms, term usage, described techniques and the like.

As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a polypeptide" includes a combination of two or more polypeptides and the like.

The term "pharmaceutical composition" or "formulation" means a mixture comprising one or more antibodies of the application and other components, such as physiologically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote the administration to organisms, facilitate the absorption of active ingredients and thus exert biological activity.

The term "liquid formulation" refers to a formulation in a liquid state and is not intended to refer to a resuspended lyophilized formulation. The liquid formulations of the present application are stable upon storage and their stability is independent of lyophilization (or other state-change methods, such as spray drying).

The term "aqueous liquid formulation" refers to a liquid formulation using water as a solvent. In some embodiments, the aqueous liquid formulation is a formulation that does not require lyophilization, spray drying, and/or freezing to maintain stability (e.g., chemical and/or physical stability and/or biological activity).

The term "excipient" refers to an agent that may be added to a formulation to provide desired characteristics (e.g., consistency, increased stability) and/or to regulate osmotic pressure. Examples of common excipients include, but are not limited to, sugars, polyols, amino acids, surfactants, and polymers.

As used herein, "about" when referring to a measurable value (e.g., amount, duration, etc.) is intended to encompass variations of + -20% or + -10% relative to the particular value, including + -5%, + -1% and + -0.1%, as these variations are suitable for carrying out the disclosed methods.

The term "buffer pH of about 5.0 to 7.0" refers to an agent that, by the action of its acid/base conjugated components, renders a solution containing the agent resistant to pH changes. Buffers used in the formulations of the present application may have a pH in the range of about 5.0 to about 7.0, or a pH in the range of about 5.0 to about 6.5, or a pH in the range of about 5.5 to about 6.5, or a pH in the range of about 5.0 to about 6.0.

In the present application, examples of "buffers" that control pH within this range include acetic acid, acetate (e.g., sodium acetate), succinic acid, succinate (e.g., sodium succinate), gluconic acid, histidine, histamine salts (e.g., histidine hydrochloride), methionine, citric acid (citric acid), citrate (citrate), phosphate, citrate/phosphate, imidazole, combinations thereof, and other organic acid buffers.

A "histidine buffer" is a buffer comprising histidine ions. Examples of histidine buffers include histidine and histidine salts, such as histidine hydrochloride, histidine acetate, histidine phosphate and histidine sulfate, and the like, such as histidine buffers containing histidine and histidine hydrochloride; histidine buffers of the present application also include histidine buffers comprising histidine and acetate (e.g., sodium or potassium salts).

An "acetate buffer" is a buffer that includes acetate ions. Examples of acetate buffers include acetic acid-sodium acetate, acetic acid-potassium acetate, acetic acid-calcium acetate, acetic acid-magnesium acetate, and the like. The preferred acetate buffer is acetic acid-sodium acetate buffer.

The term "stabilizer" refers to a pharmaceutically acceptable excipient that protects the active pharmaceutical ingredient and/or formulation from chemical and/or physical degradation during manufacture, storage and use. Stabilizers include, but are not limited to, sugars, amino acids, salts, polyols and their metabolites as defined below, such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, sucrose, trehalose, arginine or salts thereof (e.g., arginine hydrochloride), glycine, alanine (α -alanine, β -alanine), betaine, leucine, lysine, glutamic acid, aspartic acid, proline, 4-hydroxyproline, sarcosine, γ -aminobutyric acid (GABA), opioids (opines), alanines, octopine, glycine (strombine) and the N-oxide of Trimethylamine (TMAO), human serum albumin (BSA), bovine Serum Albumin (BSA), α -casein, globulin, α -lactalbumin, lactate Dehydrogenase (LDH), lysozyme, myoglobin, ovalbumin and ribonuclease a (RNAase a). Some stabilizers, such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, sucrose, and the like, may also act to control osmotic pressure. The stabilizer used in the present application is one or more selected from the group consisting of polyhydric alcohols, amino acids, salts and saccharides. The preferred salts are sodium chloride, the preferred sugars are sucrose and trehalose, and the preferred polyols are sorbitol and mannitol. Preferred amino acids are arginine, glycine, proline, which may be present in their D-and/or L-forms, but typically in the L-form, which may be present in any suitable salt, for example the hydrochloride salt, such as arginine hydrochloride. Preferred stabilizers are sodium chloride, mannitol, sorbitol, sucrose, trehalose, arginine hydrochloride, glycine, proline, sodium chloride-sorbitol, sodium chloride-mannitol, sodium chloride-sucrose, sodium chloride-trehalose, arginine hydrochloride-mannitol, arginine hydrochloride-sucrose.

The term "surfactant" generally includes agents that protect proteins such as antibodies from air/solution interface induced stress, solution/surface induced stress to reduce aggregation of the antibodies or minimize the formation of particulates in the formulation. Exemplary surfactants include, but are not limited to, nonionic surfactants such as: polyoxyethylene sorbitan fatty acid esters (such as polysorbate 20 and polysorbate 80), polyethylene-polypropylene copolymers, polyethylene-polypropylene glycols, polyoxyethylene-stearates, polyoxyethylene alkyl ethers, such as polyoxyethylene monolauryl ether, alkylphenyl polyoxyethylene ether (Triton-X), polyoxyethylene-polyoxypropylene copolymers (poloxamers, pluronic), sodium Dodecyl Sulfate (SDS). In the present application, unless otherwise specified, the terms "concentration of polysorbate 20" and "concentration of polysorbate 80" both refer to the mass volume concentration (w/v), e.g. "0.04%" in "about 0.04% polysorbate 80", i.e. "0.04 g polysorbate 80" in 100mL of liquid.

The term "viscosity" as used herein may be "kinematic viscosity" or "absolute viscosity". "kinematic viscosity" is a measure of the resistive flow of a fluid under the influence of gravity. "absolute viscosity", sometimes referred to as dynamic viscosity or simple viscosity, is the product of the kinematic viscosity and the fluid density (absolute viscosity = kinematic viscosity X density). The dimension of the kinematic viscosity is L ² T, where L is length and T is time. Typically, kinematic viscosity is expressed in centistokes (cSt). The International units of kinematic viscosity are in mm ² S, lcSt. Absolute viscosity is expressed in centipoise (cP) units. The units in international units of absolute viscosity are millipascal-seconds (mPa-s), where 1 cp=lmpa-s.

The term "isotonic" means that the formulation has substantially the same osmotic pressure as human blood. Isotonic formulations generally have an osmotic pressure of about 250 to 350 mOsm/kg. Isotonicity can be measured using a vapor pressure or freezing point depression osmometer.

The term "stable" formulation is a formulation in which the antibody substantially retains its physical and/or chemical stability and/or biological activity during the manufacturing process and/or upon storage. Pharmaceutical formulations may be stable even if the contained antibodies fail to retain 100% of their chemical structure or biological function after storage for a period of time. In some cases, an antibody structure or function that is capable of maintaining about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% after storage for a period of time may also be considered "stable". Various analytical techniques for measuring protein stability are available in the art and reviewed in peptide and protein drug Delivery (Peptide and Protein Drug Delivery) 247-301, major editions of vincent Lee, marcel Dekker, inc., new York, n.y., pubs (1991), and Jones, a. (1993) adv. Drug Delivery rev.10: 29-90 (both incorporated by reference).

After storage of the formulation at a temperature and for a time, the stability of the formulation can be measured by determining the percentage of natural antibodies remaining therein (and other methods). The percentage of native antibodies may be measured by size exclusion chromatography (e.g., size exclusion high performance liquid chromatography [ SEC-HPLC ]), among other methods, "native" refers to unagglomerated and undegraded. In some embodiments, the stability of a protein is determined as the percentage of monomeric protein in a solution having a low percentage of degraded (e.g., fragmented) and/or aggregated protein. In some embodiments, the formulation may be stable for at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or longer, up to no more than about 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the antibody in aggregated form at room temperature, about 25-30 ℃ or 40 ℃.

Stability can be measured by determining the percentage of antibodies ("acid forms") that migrate during ion exchange in this more acidic fraction of the antibody ("primary charged form") as well as other methods, where stability is inversely proportional to the percentage of the acid form of the antibody. The percentage of "acidified" antibody may be measured by ion exchange chromatography (e.g., cation exchange high performance liquid chromatography [ CEX-HPLC ]), among other methods. In some embodiments, an acceptable degree of stability means that the antibody in its acidic form is detectable at most about 49%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% after the formulation has been stored at a temperature for a period of time. The time stored prior to measuring stability may be at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or longer. When evaluating stability, the temperature at which the pharmaceutical formulation is allowed to be stored may be any temperature in the range of about-80 ℃ to about 45 ℃, for example, stored at about-80 ℃, about-30 ℃, about-20 ℃, about 0 ℃, about 2-8 ℃, about 5 ℃, about 25 ℃, or about 40 ℃.

An antibody "retains its physical stability" in the pharmaceutical composition if it exhibits substantially no signs of aggregation, precipitation, and/or denaturation upon visual inspection of color and/or clarity, or upon scattering by Ultraviolet (UV) light, or upon measurement by aperture-exclusion chromatography. Aggregation is the process by which individual molecules or complexes associate covalently or non-covalently to form aggregates. Aggregation may proceed to the point that a visible precipitate forms.

Stability, e.g., physical stability, of the formulation can be assessed by methods well known in the art, including measuring the apparent extinction (absorbance or optical density) of the sample. Such extinction measurements are related to the turbidity of the formulation. Turbidity of a formulation is in part an inherent property of proteins dissolved in solution and is typically measured by nephelometry and measured in Nephelometry Turbidity Units (NTU).

Turbidity levels that vary with, for example, the concentration of one or more components in a solution (e.g., protein and/or salt concentration) are also referred to as the "opacifying" or "opacifying appearance" of a formulation. Turbidity levels can be calculated with reference to standard curves generated using suspensions of known turbidity. The reference standard for determining turbidity levels of pharmaceutical compositions can be based on the "European Pharmacopeia" standard (European Pharmacopeia (European Pharmacopoeia), fourth edition, "European Committee for pharmaceutical quality" (Directorate for the Quality of Medicine of the Council of Europe) (EDQM), strasbourg, france). A clear solution is defined as a solution having a turbidity lower than or equal to the turbidity of a reference suspension according to the european pharmacopoeia standard having a turbidity of about 3. Nephelometric turbidity measurements can detect Rayleigh scattering in the absence of associative or non-ideal effects, which typically vary linearly with concentration. Other methods for assessing physical stability are known in the art.

An antibody "retains its chemical stability" in a pharmaceutical composition if its chemical stability at a given point in time is such that the antibody is considered to still retain its biological activity as defined hereinafter. Chemical stability can be assessed, for example, by detecting or quantifying the chemically altered form of the antibody. Chemical changes may include dimensional changes (e.g., scissoring) that can be assessed using, for example, aperture exclusion chromatography, SDS-PAGE, and/or matrix-assisted laser desorption ionization/time of flight mass spectrometry (MALDI/TOF MS). Other types of chemical changes include charge changes (e.g., occurring as a result of deamidation or oxidation), which can be assessed by, for example, ion exchange chromatography.

An antibody in a pharmaceutical composition "retains its biological activity" in the pharmaceutical composition if the antibody is biologically active for its intended purpose. For example, a formulation of the application may be considered stable if after storage of the formulation at isothermal temperatures, e.g., 5 ℃, 25 ℃, 45 ℃ for a period of time (e.g., 1 to 12 months), the novel coronavirus antibodies contained in the formulation bind to the novel coronavirus with an affinity of at least 90%, 95% or more than 95% of the binding affinity of the antibodies prior to said storage. Binding affinity can also be determined, for example, by ELISA (enzyme-linked immunosorbent assay) or by plasma resonance techniques.

In the context of the present application, a "therapeutically effective amount" or "effective amount" of an antibody in a pharmacological sense refers to an amount that is effective in the prevention or treatment or alleviation of the symptoms of a disorder that an antibody may effectively treat. In the present application, a "therapeutically effective amount" or "therapeutically effective dose" of a drug is any amount of drug that, when used alone or in combination with another therapeutic agent, protects a subject from onset of a disease or promotes regression of a disease as evidenced by a decrease in severity of disease symptoms, an increase in frequency and duration of disease asymptomatic periods, or prevention of injury or disability caused by pain in the disease. The ability of a drug to promote disease regression can be assessed using a variety of methods known to those skilled in the art, such as in human subjects during clinical trials, in animal model systems that predict human efficacy, or by assaying the activity of the agent in an in vitro assay. A therapeutically effective amount of a drug includes a "prophylactically effective amount," i.e., any amount of drug that inhibits the progression or recurrence of a disease when administered alone or in combination with other therapeutic drugs to a subject at risk of developing or a subject with recurrence of the disease.

The term "subject" or "patient" is intended to include mammalian organisms. Examples of subjects/patients include humans and non-human mammals, such as non-human primates, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. In a particular embodiment of the application, the subject is a human.

The terms "administering," "administering," and "treating" refer to introducing a composition comprising a therapeutic agent into a subject using any of a variety of methods or delivery systems known to those of skill in the art. Routes of administration of the novel coronavirus antibodies include intravenous, intramuscular, subcutaneous, peritoneal, spinal or other parenteral routes of administration, such as injection or infusion. By "parenteral administration" is meant administration other than enteral or topical administration, typically by injection, including, but not limited to intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraframe, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, and in vivo electroporation.

The term "antibody" as used herein is to be understood to include intact antibody molecules as well as antigen-binding fragments thereof. The term "antigen binding portion" or "antigen binding fragment" of an antibody (or simply "antibody portion" or "antibody fragment") as used herein refers to one or more fragments of an antibody that retain the ability to specifically bind to a human novel coronavirus or an epitope thereof. Thus, it is used in the broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, and single domain antibodies. The basic antibody structural unit is known to comprise tetramers, each comprising two identical pairs of polypeptide chains, each pair having one "light" chain (L, about 25 kDa) and one "heavy" chain (H, about 50-70 kDa). The amino-terminal portion or fragment of each chain may include a variable region of about 100-110 amino acids or more that is primarily responsible for antigen recognition. The carboxy-terminal portion or fragment of each strand may define a constant region primarily responsible for effector function.

The term "isolated antibody" refers to a purified state of the bound compound, and in this case means that the molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, sugars, or other substances such as cell debris and growth media. The term "isolated" does not mean that such materials are completely absent or that water, buffer or salt are absent unless they are present in amounts that would significantly interfere with the experimental or therapeutic use of the binding compounds of the application.

The term "monoclonal antibody" refers to antibodies made from highly identical immune cells, which are all clones of a single parent cell. Monoclonal antibodies have monovalent affinity because they bind to the same epitope (the site where the antibody recognizes the antigen). The monoclonal antibodies may also include minor amounts of naturally occurring mutations. In contrast, the term "polyclonal antibody" binds to multiple epitopes, typically consisting of several different plasma cell (antibody secreting immune cell) lineages, and is understood to be a hybrid of multiple monoclonal antibodies. The modifier "monoclonal" is not to be construed as requiring antibody production by any particular method.

The term "murine antibody" or "hybridoma antibody" is herein a monoclonal antibody against the human novel coronavirus prepared according to the knowledge and skill in the art. The preparation is performed by injecting a test subject with a novel coronavirus antigen and then isolating hybridomas expressing antibodies having the desired sequence or functional properties.

The term "chimeric antibody" is an antibody having a variable domain of a first antibody and a constant domain of a second antibody, wherein the first antibody and the second antibody are from different species. Typically, the variable domain is obtained from an antibody ("parent antibody") of a rodent or the like, while the constant domain sequence is obtained from a human antibody, such that the resulting chimeric antibody is less likely to induce an adverse immune response in a human subject as compared to the parent rodent antibody.

The term "humanized antibody" refers to a form of antibody that contains sequences from both human and non-human (e.g., mouse, rat) antibodies. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the complementarity determining regions correspond to those of a non-human immunoglobulin and all or substantially all of the Framework (FR) regions are those of a human immunoglobulin sequence. The humanized antibody optionally may comprise at least a portion of a human immunoglobulin constant region (Fc).

The term "full length antibody" or "whole antibody molecule" refers to an immunoglobulin molecule comprising four peptide chains: two heavy (H) chains (50-70 kDa at full length) and two light (L) chains (25 kDa at full length) are linked to each other by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated as VH in the present application) and a heavy chain constant region (abbreviated as CH in the present application). The heavy chain constant region consists of 3 domains, CH1, CH2 and CH 3. Each light chain consists of a light chain variable region (abbreviated as VL in the present application) and a light chain constant region (abbreviated as CL in the present application). The light chain constant region consists of one domain CL. VH and VL regions can be further subdivided into Complementarity Determining Regions (CDRs) with high variability and Framework Regions (FR) with higher conservation that are distributed with complementarity determining regions. The domains of each VH or VL from amino terminus to carboxy terminus are arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The variable domains of the heavy and light chains each contain a binding domain that interacts with an antigen. The constant region of an antibody may mediate binding of the antibody to various cells of the host's tissue or immune system (e.g., effector cells) and the first component of the classical complement system (Clq).

The term "binding domain" or "antigen binding site" refers to a region in an antibody that is capable of specifically binding to and complementing a portion or all of an antigen. When the antigen is large, the antibody may only bind to a specific portion of the antigen, which portion is referred to as an epitope. The binding domain may comprise the variable domains of the heavy and light chains, namely the heavy chain variable region VH and the light chain variable region VL, each comprising four conserved Framework Regions (FR) and three Complementarity Determining Regions (CDRs). CDRs can vary in sequence and determine specificity for a particular antigen.

The term "CDR" refers to the complementarity determining region within an antibody variable sequence. There are 3 CDRs in each of the heavy and light chain variable regions, which are designated HCDR1, HCDR2 and HCDR3 or LCDR1, LCDR2 and LCDR3 for each of the heavy and light chain variable regions. The exact boundaries of these CDRs are defined differently for different systems.

For the precise amino acid sequence boundaries of CDRs of the complementarity determining region of an antibody, the boundaries may be defined according to well-known methods, such as Chothia (Chothia et al, (1989) Nature342:877-883, al-Lazikani et al, "Standard conformations for the canonical structures of immunoglobulins", journal of Molecular Biology,273, 927-948 (1997) based on the topology of the CDR loops and three-dimensional structure of the antibody, or Kabat (Kabat et al, sequences of Proteins ofImmunological Interest, 4 th edition, U.S. device of Health and Human Services, national Institutes of Health (1987), abM (University of Bath), contact (University College London) and International ImMunoGeneTics database (IMGT) (1999Nucleic Acids Research,27, 209-212) based on the North CDR definition of neighbor-propagating clusters (affinity propagation clustering) using a large number of crystal structures, the CDRs of an antibody in the present application may be determined according to any scheme in the art, such as the above-described optional definition method.

As used herein, an "antigen-binding fragment" includes a fragment of an antibody or a derivative of an antibody, and the antibody corresponding to the "antigen-binding fragment" may be referred to as a parent antibody. The antigen-binding fragment of an antibody typically comprises at least one fragment of the antigen-binding or variable region of the parent antibody that retains at least some of the binding specificity of the parent antibody. Examples of antigen binding fragments include, but are not limited to, fab ', F (ab') ² And single chain Fv fragments, diabodies, linear antibodies, single chain antibody molecules, such as sc-Fv; nanobodies (nanobodies) and multispecific antibodies formed from antibody fragments, and the like. The antigen binding fragment is capable of retaining at least 10%, at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the antigen binding activity of the parent antibody at the same molar concentration. Furthermore, an antigen-binding fragment of an antibody may also include conservative or non-conservative amino acid substitutions that do not significantly alter its biological activity (referred to as "conservative variants" or "functional conservative variants" of the antibody).

The terms "specific binding", "selective binding" refer to binding of an antibody to an epitope on a predetermined antigen. The term "recognition antigen" may be used interchangeably herein with the term "specific binding".

The term "affinity" or "binding affinity" refers to the inherent binding affinity that reflects the interaction between members of a binding pair (e.g., antigen and antibody). Affinity can be generally determined by equilibrium dissociation constants (K _D ) The equilibrium dissociation constant is the ratio of the dissociation rate constant to the association rate constant. Affinity can be measured by common methods known in the art, for example, using the ForteBio biological molecular interaction workstation.

Pharmaceutical preparation

The present application provides a high stability pharmaceutical composition comprising an antibody that specifically binds to a novel coronavirus comprising: (1) a buffer; (2) novel coronavirus antibodies or antigen-binding fragments thereof; wherein the novel coronavirus antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, respectively, and LCDR1, LCDR2 and LCDR3 with amino acid sequences shown as SEQ ID NO. 4, AAS and SEQ ID NO. 5, respectively.

In some embodiments, the novel coronavirus antibody or antigen-binding fragment thereof used in the pharmaceutical composition of the present application comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO. 6, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO. 7.

In some embodiments, the novel coronavirus antibody or antigen-binding fragment thereof used in the pharmaceutical composition of the present application comprises a heavy chain having an amino acid sequence as shown in SEQ ID NO. 8, and a light chain having an amino acid sequence as shown in SEQ ID NO. 9.

In some embodiments, the concentration of the novel coronavirus antibody or antigen-binding fragment thereof in the pharmaceutical composition of the application is 2-200 mg/mL, preferably 10-150 mg/mL, more preferably 50-100 mg/mL; preferably, the novel coronavirus antibody or antigen-binding fragment thereof has a concentration of 10mg/mL, 15mg/mL, 20mg/mL, 25mg/mL, 30mg/mL, 35mg/mL, 40mg/mL, 45mg/mL, 50mg/mL, 55mg/mL, 60mg/mL, 65mg/mL, 70mg/mL, 75mg/mL, 80mg/mL, 85mg/mL, 90mg/mL, 95mg/mL, 100mg/mL, 105mg/mL, 110mg/mL, 115mg/mL, 120mg/mL, 125mg/mL, more preferably 30mg/mL, 40mg/mL, 50mg/mL, 60mg/mL, 70mg/mL, 80mg/mL, 90mg/mL, 100mg/mL, 110mg/mL, 120mg/mL, or any two of the above ranges as endpoints.

In some embodiments, the buffer in the pharmaceutical composition of the present application is selected from at least one of an acetate buffer, a citrate buffer, a phosphate buffer, and a histidine buffer; preferably, the buffer is selected from at least one of an acetate buffer and a histidine buffer, preferably a histidine buffer. Preferably, the histidine buffer is selected from a histidine-histidine hydrochloride buffer or a histidine-histidine acetate buffer, preferably a histidine-histidine hydrochloride buffer. The solvent used in the buffers according to the application is water unless otherwise specified.

In some embodiments, the concentration of buffer in the pharmaceutical composition is 5 to 50mM, preferably 10 to 30mM, more preferably 15 to 25mM. The concentration of the buffer may be 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM or any two of the above values, and preferably 15mM, 20mM or 25mM. The concentration of the buffer according to the present application refers to the concentration of the buffer substance in the pharmaceutical composition unless otherwise specified.

In some embodiments, the pH of the buffer in the pharmaceutical composition is 5.0 to 7.0, preferably 5.5 to 6.5, more preferably 5.9 to 6.1, more preferably 6.0. The pH of the buffer may be 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 or any two of the above values as the end points, preferably 5.9, 6.0, 6.1 or any two of the above values as the end points.

In some embodiments, the acetate buffer is an acetate-sodium acetate buffer or an acetate-potassium acetate buffer, preferably an acetate-sodium acetate buffer. In some embodiments, the acetate buffer is made with 1 to 30mM acetic acid and 1 to 30mM sodium acetate. In some embodiments, the acetate buffer is made with acetic acid and sodium acetate in a molar ratio of 1:2.1. In some embodiments, the acetate buffer is made with acetic acid and sodium acetate in a molar ratio of 1:5.7. In some embodiments, the acetate buffer is: an acetate buffer of pH 5.0 was prepared using 6.5mM acetic acid and 13.5mM sodium acetate. In some embodiments, the acetate buffer is: an acetate buffer with a pH of 5.5 was prepared using 3mM acetic acid and 17mM sodium acetate.

In some embodiments, the histidine-histidine hydrochloride buffer is made using histidine and histidine hydrochloride, preferably using L-histidine and L-histidine monohydrochloride. In some embodiments, the histidine buffer is made with 1 to 30mM L-histidine and 1 to 30mM L-histidine monohydrochloride. In some embodiments, the histidine buffer is made with a molar ratio of histidine to histidine hydrochloride of 1:1 to 1:4. In some embodiments, the histidine buffer is made with a molar ratio of 1:1 histidine to histidine hydrochloride. In some embodiments, the histidine buffer is made with a 1:3 molar ratio of histidine to histidine hydrochloride. In some embodiments, the histidine-histidine hydrochloride buffer is: histidine-histidine hydrochloride buffer at pH 5.5 was prepared using 4.5mM L-histidine and 15.5mM L-histidine monohydrochloride. In some embodiments, the histidine-histidine hydrochloride buffer is: histidine buffer at pH 5.5 was prepared using 7.5mM L-histidine and 22.5mM L-histidine monohydrochloride. In some embodiments, the histidine-histidine hydrochloride buffer is: histidine buffer pH 6.0 was prepared using 10mM histidine and 10mM histidine hydrochloride.

In some embodiments, the citrate buffer is a citrate-sodium citrate buffer. In some embodiments, the citrate buffer is made with 1-30 mM citric acid and 1-30 mM sodium citrate. In some embodiments, the citric acid buffer is made with a molar ratio of citric acid to sodium citrate of 1:1 to 1:4. In some embodiments, the citrate buffer is: a pH of 6.5 citrate buffer was prepared using 5.0mM citric acid and 15.0mM sodium citrate. In some embodiments, the citrate buffer is: a pH of 6.0 citrate buffer was prepared using 10mM citric acid and 10mM sodium citrate.

In some embodiments, the phosphate buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate buffer. In some embodiments, the phosphate buffer is made with 1 to 20mM disodium hydrogen phosphate and 1 to 20mM sodium dihydrogen phosphate. In some embodiments, the phosphate buffer is made with a molar ratio of disodium hydrogen phosphate to sodium dihydrogen phosphate of 1:1 to 1:4. In some embodiments, the phosphate buffer is: phosphate buffer pH 7.0 was prepared using 10mM disodium hydrogen phosphate and 10mM sodium dihydrogen phosphate.

In some embodiments, the pharmaceutical composition of the present application further comprises a stabilizer selected from at least one of arginine, arginine salt, sodium chloride, mannitol, sorbitol, sucrose, glycine, and trehalose, preferably at least one of sucrose and trehalose. Preferably, the concentration of the stabilizing agent is 10 to 400mM, preferably 100 to 300mM, preferably 120 to 280mM, preferably 130 to 250mM, more preferably 200 to 250mM. The stabilizer concentration may be 100mM, 110mM, 120mM, 130mM, 140mM, 150mM, 160mM, 170mM, 180mM, 190mM, 200mM, 210mM, 220mM, 230mM, 240mM, 250mM or any two of the above values, and is preferably 210mM, 220mM, 230mM, 240mM or any two of the above values.

In some embodiments, the stabilizing agent is trehalose. The concentration of trehalose is 100 to 300mM, preferably 120 to 280mM, more preferably 200 to 250mM. The concentration of trehalose may be 180mM, 200mM, 210mM, 220mM, 230mM, 240mM, 250mM, 260mM, 270mM, 280mM or any two of the above values, and preferably 220mM, 230mM, 240mM or any two of the above values.

In some embodiments, the stabilizing agent is sucrose. The concentration of sucrose is 100 to 300mM, preferably 120 to 280mM, more preferably 200 to 250mM; the concentration of sucrose may be 180mM, 200mM, 210mM, 220mM, 230mM, 240mM, 250mM, 260mM, 270mM, 280mM or any two of the above values, and preferably 220mM, 230mM, 240mM or any two of the above values.

In some embodiments, the stabilizer is arginine or an arginine salt; preferably, the arginine salt is arginine hydrochloride. In some embodiments, the concentration of arginine or arginine salt is 100 to 300mM, preferably 120 to 280mM, more preferably 120 to 160mM. The concentration of arginine or arginine salt may be 120mM, 125mM, 130mM, 135mM, 140mM, 145mM, 150mM, 155mM, 160mM or any two of the above values, and preferably 135mM, 140mM, 145mM, 150mM, 155mM or any two of the above values.

In some embodiments, the stabilizer is sodium chloride. The concentration of sodium chloride is 100 to 300mM, preferably 100 to 200mM, more preferably 120 to 180mM, and still more preferably 130 to 150mM. The concentration of sodium chloride may be 120mM, 125mM, 130mM, 135mM, 140mM, 145mM, 150mM, 155mM, 160mM, 165mM, 170mM, 175mM or any two of the above values, and is preferably 135mM, 140mM, 145mM, 150mM, 155mM or any two of the above values.

In some embodiments, the stabilizer is mannitol. The concentration of mannitol is 100 to 300mM, preferably 200 to 300mM, and more preferably 220 to 250mM. The concentration of mannitol may be 205mM, 210mM, 215mM, 220mM, 225mM, 230mM, 235mM, 240mM, 245mM, 250mM, 255mM, 260mM, 265mM, 270mM, 275mM or any two of the above values, and preferably 235mM, 240mM, 245mM, 250mM, 255mM or any two of the above values.

In some embodiments, the stabilizer is sorbitol. The concentration of sorbitol is 100 to 300mM, preferably 200 to 300mM, preferably 220 to 250mM. The concentration of mannitol may be 205mM, 210mM, 215mM, 220mM, 225mM, 230mM, 235mM, 240mM, 245mM, 250mM, 255mM, 260mM, 265mM, 270mM, 275mM or any two of the above values, and preferably 235mM, 240mM, 245mM, 250mM, 255mM or any two of the above values.

In some embodiments, the stabilizer is a combination of sodium chloride and mannitol. In some embodiments, the stabilizer is a combination of 20-200 mM sodium chloride and 20-200 mM mannitol, preferably 30-100 mM sodium chloride and 100-180 mM mannitol, more preferably 30-70 mM sodium chloride and 120-160 mM mannitol. The stabilizer may be a combination of 50mM sodium chloride with 140mM, 145mM or 150mM mannitol.

In some embodiments, the stabilizer is arginine hydrochloride in combination with sucrose. In some embodiments, the stabilizer is a combination of 20 to 200mM arginine hydrochloride and 20 to 200mM sucrose, preferably 20 to 80mM arginine hydrochloride and 100 to 180mM sucrose, more preferably 30 to 70mM arginine hydrochloride and 100 to 160mM sucrose. The stabilizer may be a combination of 50mM arginine hydrochloride with 120mM, 125mM, 130mM or 135mM sucrose.

In some embodiments, the stabilizer is a combination of arginine hydrochloride and glycine. In some embodiments, the stabilizer is a combination of 20 to 200mM arginine hydrochloride and 20 to 200mM glycine, preferably 20 to 80mM arginine hydrochloride and 100 to 180mM glycine, more preferably 30 to 70mM arginine hydrochloride and 100 to 120mM glycine. The stabilizer may be a combination of 50mM arginine hydrochloride with 100mM, 105mM, 110mM or 115mM glycine.

In some embodiments, the stabilizer is a combination of sodium chloride and sucrose. In some embodiments, the stabilizer is a combination of 20 to 200mM sodium chloride and 20 to 200mM sucrose, preferably 20 to 100mM sodium chloride and 100 to 180mM sucrose, more preferably 30 to 70mM sodium chloride and 120 to 160mM sucrose. The stabilizer may be a combination of 50mM sodium chloride with 120mM, 125mM, 130mM or 135mM sucrose.

In some embodiments, the stabilizing agent is a combination of sodium chloride and trehalose. In some embodiments, the stabilizer is a combination of 20 to 200mM sodium chloride and 20 to 200mM trehalose, preferably 20 to 80mM sodium chloride and 100 to 180mM trehalose, more preferably 30 to 70mM sodium chloride and 120 to 160mM trehalose. The stabilizer may be a combination of 50mM sodium chloride with 120mM, 130mM or 140mM trehalose.

In some embodiments, the stabilizing agent is trehalose at a concentration of 120 to 280mM, preferably 200 to 250mM; or the stabilizer is sucrose with the concentration of 120-280 mM, preferably 200-250 mM; or the stabilizer is a combination of arginine hydrochloride with the concentration of 20-80 mM and sucrose with the concentration of 100-180 mM, preferably a combination of arginine hydrochloride with the concentration of 30-70 mM and sucrose with the concentration of 120-160 mM; or the stabilizer is a combination of sodium chloride with the concentration of 20-80 mM and sucrose with the concentration of 100-180 mM, preferably a combination of sodium chloride with the concentration of 30-70 mM and sucrose with the concentration of 120-160 mM; or the stabilizer is a combination of sodium chloride with the concentration of 20-80 mM and mannitol with the concentration of 100-180 mM, preferably a combination of sodium chloride with the concentration of 30-70 mM and mannitol with the concentration of 120-160 mM; or the stabilizer is a combination of arginine hydrochloride with the concentration of 20-80 mM and glycine with the concentration of 80-180 mM, preferably a combination of arginine hydrochloride with the concentration of 30-70 mM and glycine with the concentration of 80-140 mM; or the stabilizer is arginine or arginine salt with the concentration of 120-280 mM, preferably 120-160 mM.

In some embodiments, the pharmaceutical compositions of the present application further comprise a surfactant selected from at least one of polysorbate 80, polysorbate 20, and poloxamer 188. Preferably, the concentration of the surfactant in the pharmaceutical composition is 0.001% to 0.1%, preferably 0.01% to 0.1%, more preferably 0.01% to 0.08%, more preferably 0.01% to 0.04% in w/v. The concentration of the surfactant may be 0.01%, 0.02%, 0.04%, or a range formed by any two of the above values, and is preferably 0.04%. The concentration of the surfactant according to the present application refers to the concentration of the surfactant in the pharmaceutical composition unless otherwise specified.

In some embodiments, the pharmaceutical compositions each comprise a component as set forth in any one of (1) to (4) below:

(1) (a) 10-150 mg/mL of the novel coronavirus antibody or antigen-binding fragment thereof; (b) 10-30 mM acetate buffer, pH 5.0-7.0; (c) 120-280 mM sucrose; and (d) polysorbate 80 at a concentration of 0.01% -0.1% in w/v;

preferably, (a) 50-100 mg/mL of the novel coronavirus antibody or antigen binding fragment thereof; (b) 10-30 mM acetate buffer, pH 5.5-6.5; (c) 200-250 mM sucrose; and (d) polysorbate 80 at a concentration of 0.01% -0.04% in w/v;

More preferably, (a) 50mg/mL of the novel coronavirus antibody or antigen binding fragment thereof; (b) 20mM acetate buffer, pH 5.9-6.1; (c) 230mM sucrose; and (d) polysorbate 80 at a concentration of 0.04% w/v; or (b)

(a) 100mg/mL of the novel coronavirus antibody or antigen binding fragment thereof; (b) 20mM acetate buffer, pH 5.9-6.1; (c) 230mM sucrose; and (d) polysorbate 80 at a concentration of 0.04% w/v; or (b)

(2) (a) 10-150 mg/mL of the novel coronavirus antibody or antigen-binding fragment thereof; (b) 10-30 mM acetate buffer, pH 5.0-7.0; (c) 120-280 mM trehalose; and (d) polysorbate 80 at a concentration of 0.01% -0.1% in w/v;

preferably, (a) 50-100 mg/mL of the novel coronavirus antibody or antigen binding fragment thereof; (b) 10-30 mM acetate buffer, pH 5.5-6.5; (c) 200-250 mM trehalose; and (d) polysorbate 80 at a concentration of 0.01% -0.04% in w/v;

more preferably, (a) 50mg/mL of the novel coronavirus antibody or antigen binding fragment thereof; (b) 20mM acetate buffer, pH 5.9-6.1; (c) 230mM trehalose; and (d) polysorbate 80 at a concentration of 0.04% w/v; or (b)

(a) 100mg/mL of the novel coronavirus antibody or antigen binding fragment thereof; (b) 20mM acetate buffer, pH 5.9-6.1; (c) 230mM trehalose; and (d) polysorbate 80 at a concentration of 0.04% w/v; or (b)

(3) (a) 10-150 mg/mL of the novel coronavirus antibody or antigen-binding fragment thereof; (b) 10-30 mM histidine buffer, pH 5.0-7.0; (c) 120-280 mM sucrose; and (d) polysorbate 80 at a concentration of 0.01% -0.1% in w/v;

preferably, (a) 50-100 mg/mL of the novel coronavirus antibody or antigen binding fragment thereof; (b) 10-30 mM histidine buffer, pH 5.5-6.5; (c) 200-250 mM sucrose; and (d) polysorbate 80 at a concentration of 0.01% -0.04% in w/v;

more preferably, (a) 50mg/mL of the novel coronavirus antibody or antigen binding fragment thereof; (b) 20mM histidine buffer, pH 5.9-6.1; (c) 230mM sucrose; and (d) polysorbate 80 at a concentration of 0.04% w/v; or (b)

(a) 100mg/mL of the novel coronavirus antibody or antigen binding fragment thereof; (b) 20mM histidine buffer, pH 5.9-6.1; (c) 230mM sucrose; and (d) polysorbate 80 at a concentration of 0.04% w/v; or (b)

(4) (a) 10-150 mg/mL of the novel coronavirus antibody or antigen-binding fragment thereof; (b) 10-30 mM histidine buffer, pH 5.0-7.0; (c) 120-280 mM trehalose; and (d) polysorbate 80 at a concentration of 0.01% -0.1% in w/v;

preferably, (a) 50-100 mg/mL of the novel coronavirus antibody or antigen binding fragment thereof; (b) 10-30 mM histidine buffer, pH 5.5-6.5; (c) 200-250 mM trehalose; and (d) polysorbate 80 at a concentration of 0.01% -0.04% in w/v;

more preferably, (a) 50mg/mL of the novel coronavirus antibody or antigen binding fragment thereof; (b) 20mM histidine buffer, pH 5.9-6.1; (c) 230mM trehalose; and (d) polysorbate 80 at a concentration of 0.04% w/v; or (b)

(a) 100mg/mL of the novel coronavirus antibody or antigen binding fragment thereof; (b) 20mM histidine buffer, pH 5.9-6.1; (c) 230mM trehalose; and (d) polysorbate 80 at a concentration of 0.04% w/v.

In some embodiments, the pharmaceutical composition of the present application has a pH of 5.0 to 7.0, preferably 5.5 to 6.5, more preferably 5.9 to 6.1.

In some embodiments, the pharmaceutical composition of the application has an osmolality of 250 to 350mOsm/kg, preferably 260 to 320mOsm/kg, more preferably 290 to 310mOsm/kg.

In some embodiments, the novel coronavirus antibody or antigen-binding fragment thereof used in the pharmaceutical composition of the application is selected from the group consisting of murine antibody or antigen-binding fragment thereof, chimeric antibody or antigen-binding fragment thereof, humanized antibody or antigen-binding fragment thereof, preferably humanized antibody or antigen-binding fragment thereof.

In some embodiments, the novel coronavirus antibodies or antigen-binding fragments thereof used in the pharmaceutical compositions of the application are humanized antibodies or chimeric antibodies and may include human constant regions. In some embodiments, the constant region is selected from the group consisting of human IgG1, igG2, igG3, and IgG4 constant regions; preferably, the novel coronavirus antibody or antigen-binding fragment thereof suitable for use in the pharmaceutical composition of the application comprises a heavy chain constant region of the human IgG1 or IgG4 isotype, more preferably a human IgG1 constant region. In some embodiments, one or more amino acid modifications may be introduced into the Fc region of the novel coronavirus antibodies provided in the present application, thereby producing Fc region variants, such as introducing an S228P mutation in the sequence of the IgG4 heavy chain constant region of the novel coronavirus antibody or antigen binding fragment thereof, which replaces serine residues in the hinge region with proline residues normally present at the corresponding positions of the IgG1 isotype antibody.

It should be noted that the CDR in the above sequence is defined as IMGT. The CDR amino acid sequences of the novel coronavirus antibody or antigen binding fragment thereof are, respectively, if defined in KABAT: HCDR1: SYAMS (SEQ ID NO: 10); HCDR2: AIVGSGGSTYYADSVKG (SEQ ID NO: 11); HCDR3: SLIYGHYDILTGAYYFDY (SEQ ID NO: 12); LCDR1: RASQGIGNWLA (SEQ ID NO: 13); LCDR2: AASSLQS (SEQ ID NO: 14); LCDR3: QQANSFPP (SEQ ID NO: 15).

The present application is not particularly limited as long as the components in the pharmaceutical composition to be formulated are within the scope of the present application. For example, a novel coronavirus antibody stock sample is used with Millipore3 0.11m ² Ultrafiltration concentration (UF/DF) was performed to a concentration of 1-2 times the preset final concentration of the novel coronavirus antibody, followed by dialysis into a buffer without stabilizer. After dialysis, the concentration of the novel coronavirus antibody is determined, and a high concentration stabilizer stock solution (e.g., 3X to 5X stableThe stock solution of the fixative), the buffer solution without the stabilizer and the surfactant, the final concentration of the novel coronavirus antibody is adjusted to a preset final concentration, and the concentrations of other components are controlled to be the corresponding preset final concentrations, so that the pharmaceutical composition is obtained.

In yet another aspect, the application provides a lyophilized formulation comprising the pharmaceutical composition of the application.

In a further aspect, the present application provides a liquid formulation comprising the pharmaceutical composition of the present application, or a liquid formulation obtained by resuspension of a lyophilized formulation of the pharmaceutical composition of the present application; preferably, the liquid formulation is an injection.

In some embodiments, the liquid formulation contains a dextrose solution or sodium chloride solution, and the pharmaceutical composition of the application; preferably, the concentration of the sodium chloride solution is 0.85-0.9% and the concentration of the glucose solution is 5-25% in w/v; preferably, the concentration of the novel coronavirus antibody in the liquid formulation is 0.1-50 mg/mL, preferably 0.5-30 mg/mL; preferably, the pH of the liquid formulation is from 5.0 to 7.0. The concentration of the sodium chloride solution in the present application means the concentration of sodium chloride in the sodium chloride solution, and the concentration of the glucose solution means the concentration of glucose in the glucose solution unless otherwise specified.

Medical uses and methods

In a further aspect, the application also provides the use of a pharmaceutical composition, lyophilized formulation or liquid formulation according to the application for the manufacture of a medicament for the prevention or treatment of a novel coronavirus (SARS-CoV-2) infection.

In a further aspect, the application also provides the use of a pharmaceutical composition, lyophilized formulation or liquid formulation according to the application for preventing or treating a novel coronavirus (SARS-CoV-2) infection by eliminating, inhibiting or reducing the activity of the novel coronavirus.

In yet another aspect, the application also provides a method of preventing or treating a novel coronavirus (SARS-CoV-2) infection by eliminating, inhibiting or reducing the activity of the novel coronavirus comprising administering to a subject in need thereof a pharmaceutical composition, lyophilized formulation or liquid formulation as described herein.

In yet another aspect, the application also provides a combination therapy comprising administering to a subject a therapeutically effective amount of the pharmaceutical composition, lyophilized formulation or liquid formulation of any of the embodiments of the application in combination with one or more other therapies to treat a disease caused by a novel coronavirus (SARS-CoV-2) infection. In some embodiments, the therapy comprises surgical treatment and/or radiation therapy.

In yet another aspect, the application also provides a combination therapy comprising administering to a subject a therapeutically effective amount of the pharmaceutical composition, lyophilized formulation or liquid formulation of any of the embodiments of the application in combination with one or more other therapeutic agents to treat a disease caused by a novel coronavirus (SARS-CoV-2) infection. In some embodiments, the other therapeutic agent may be selected from at least one of a chemotherapeutic agent and a biologic therapeutic agent; wherein the chemotherapeutic agent is selected from at least one of interferon-alpha, ribavirin, chloroquine phosphate, and arbidol; the biologic therapeutic agent is selected from tolizumab.

In yet another aspect, the application also provides a method of reducing systemic or local viral load in a subject infected with a novel coronavirus (SARS-CoV-2) comprising administering to the subject an effective amount of the pharmaceutical composition, lyophilized formulation, or liquid formulation of any of the embodiments of the application.

The novel coronavirus comprises at least one of a novel coronavirus wild type and/or mutant strain, wherein the novel coronavirus mutant strain comprises at least one of an Alpha (Alpha) mutant strain, a Beta (Beta) mutant strain, a Gamma (Gamma) mutant strain, a Delta (Delta) mutant strain, an Epsilon mutant strain, a Zeta mutant strain, an Eta mutant strain, a Theta mutant strain, an Iota mutant strain, a Kappa (Kappa) mutant strain, a Mu (Mu) mutant strain and an Omicron mutant strain, and preferably at least one of an Alpha mutant strain, a Beta mutant strain, a Gamma mutant strain, a Kappa mutant strain, an Epsilon mutant strain and a Delta mutant strain.

Examples

The application will be illustrated by way of specific examples. It should be understood that these examples are illustrative only and are not intended to limit the scope of the application. The application has been described in detail with particular reference to certain embodiments thereof. It will be apparent to those skilled in the art that various changes and modifications can be made to the specific embodiments of the present application without departing from the spirit and scope of the application, and that any such changes, equivalents, modifications, etc. are intended to be included within the scope of the application. The methods and materials used in the examples are, unless otherwise indicated, conventional in the art.

The amino acid sequence of the heavy chain of the novel coronavirus antibody used in the following examples is shown in SEQ ID NO. 8, and the amino acid sequence of the light chain is shown in SEQ ID NO. 9. Novel coronavirus antibodies: firstly, using SARS-CoV-2RBD of mutant strain expressed by insect cell as antigen, screening out memory B cell capable of specifically binding SARS-CoV-2RBD protein from Peripheral Blood Mononuclear Cells (PBMCs) of patient after SARS-CoV-2 infection by means of flow separation, then making reverse transcription PCR (Reverse Transcription-PCR, RT-PCR) on the single B cell so as to obtain variable region sequence and fragment of antibody, and further connecting the variable region sequence and fragment with constant region into expression vector. After mammalian cell expression and purification, a series of functional tests are carried out, including the binding capacity with SARS-CoV-2RBD protein, the blocking effect of blocking the binding of SARS-CoV-2RBD and ACE2, the neutralization effect of inhibiting SARS-CoV-2 infection, etc., thus obtaining the novel coronavirus antibody 5-10 molecules for neutralizing SARS-CoV-2 infection.

In examples 2 to 4 below, pharmaceutical compositions FS1 to 5 were used as test subjects, and the pharmaceutical compositions were diluted with diluents corresponding to each example to bring the concentrations of the novel coronavirus antibodies to the corresponding values before the test.

The following abbreviations are used in the embodiments of the present application:

h represents hours;

d represents the number of days;

w represents a week;

m represents a month;

c represents the number of freeze-thawing cycles;

FT represents a freeze-thaw cycle;

RT represents room temperature;

t0 represents an initial test of a pharmaceutical composition sample prior to lofting;

kD represents the diffusion interaction coefficient;

tm represents the unfolding temperature;

OD represents optical density;

RLU denotes relative light units;

PBS represents phosphate buffer salt solution;

SEC-HPLC means size exclusion high performance liquid chromatography;

CEX-HPLC means ion exchange high performance liquid chromatography;

rCE-SDS represents reducing capillary electrophoresis;

nrCE-SDS represents non-reducing capillary electrophoresis;

RBD represents a receptor binding domain;

SDS represents sodium dodecyl sulfate;

MES represents 2-morpholinoethanesulfonic acid;

TMB represents 3,3', 5' -tetramethylbenzidine;

EC ₅₀ representing the half maximum effect concentration;

IC ₅₀ represents a half-inhibitory concentration;

FBS represents fetal bovine serum.

Example 1: stability test of pharmaceutical compositions

1.1 Experimental sample

Novel coronavirus antibody stock samples (antibodies 5-10) were prepared using Millipore3 0.11m ² Ultrafiltration concentration (UF/DF) was performed to a concentration of the novel coronavirus antibody of about 75mg/mL, 120mg/mL, respectively, followed by dialysis into a corresponding stabilizer-free buffer. Measuring the concentration of the novel coronavirus antibody, adding the corresponding 4×stabilizer stock solution, the buffer solution without stabilizer and the surfactant according to the measured concentration, and adding the novel coronavirus antibody The final concentration of the virus antibody is respectively regulated to 50mg/mL or 100mg/mL, and the contents of other components are respectively shown in table 1, so that different pharmaceutical compositions to be tested are obtained.

And filling the newly prepared different pharmaceutical compositions to be tested into a 2R penicillin bottle in an aseptic mode, and then carrying out stability lofting and detection according to stability investigation conditions. The components of the different pharmaceutical compositions to be tested are shown in table 1, wherein: acetic acid buffer refers to acetic acid-sodium acetate buffer; histidine buffer refers to L-histidine-L-histidine hydrochloride buffer.

TABLE 1 Components of different pharmaceutical compositions to be tested

1.2 Experimental methods and results

The stability investigation conditions included:

(1) Placed in an environment of 5.+ -. 3 ℃ for 1W, 2W and 4W, hereinafter referred to as "long term (5.+ -. 3 ℃ C.)";

(2) Placed in an atmosphere of 40.+ -. 2 ℃ at 1W, 2W and 4W, hereinafter simply referred to as "high temperature (40.+ -. 2 ℃);

(3) Placed in an environment of 25.+ -. 2 ℃ at 4W, hereinafter simply referred to as "acceleration (25.+ -. 2 ℃);

(4) Repeated freeze thawing for 3 times and 5 times between-40 ℃ and room temperature, hereinafter abbreviated as "FT (-40 ℃/RT)";

(5) Shaking was performed at 80.+ -.20 rpm for 5 days and 10 days at room temperature, and hereinafter, simply referred to as "shaking (80.+ -.20 rpm/RT)".

1.2.1 colloidal stability and structural stability

The testing method comprises the following steps: the concentration of the novel coronavirus antibody in each group of the pharmaceutical compositions is diluted to 10mg/mL, 6.25mg/mL, 5mg/mL, 2.5mg/mL respectively by adopting the buffer solution corresponding to each group of the pharmaceutical compositions, and the pharmaceutical compositions are centrifuged at 1000rpm for 5min. 150 μl of the supernatant was added to a 96-well plate. kD values were analyzed using a multiple blank direct-reading dynamic light scatterometer (DynaPro Plate Reader III).

The novel coronavirus antibody was centrifuged at 1000rpm for 5min at a concentration of 10mg/mL, 6.25mg/mL, 5mg/mL, and 2.5mg/mL, respectively. The supernatant was aspirated using a capillary tube, ensuring no air bubbles were inhaled. The capillary was placed in a sample cell of a capillary micro differential scanning fluorescent meter (Prometheus NT.48) for detection analysis. According to the detection curve, the Tm value of the protein sample is read.

Test results: the results of the colloidal stability and structural stability of the pharmaceutical compositions of each group are shown in tables 2 and 3.

TABLE 2 examination of colloidal stability and structural stability results

TABLE 3 examination of colloidal stability and structural stability results

From the results of FS1-1 to FS1-6 in Table 2, it can be seen that the kD value of the pharmaceutical composition containing sucrose as a stabilizer is significantly higher than that of the pharmaceutical composition containing trehalose under the same buffer system; in addition, the kD values of the pharmaceutical compositions (FS 1-5 and FS 1-6) comprising histidine buffer (pH 6.0) were significantly higher than those of the pharmaceutical compositions (FS 1-1 and FS 1-2) comprising acetate buffer (pH 5.5) and the pharmaceutical compositions (FS 1-3 and FS 1-4) comprising histidine buffer (pH 5.5); the above results illustrate: the pharmaceutical composition FS1-5 (containing histidine buffer, pH 6.0, with stabilizer sucrose) had optimal colloidal stability. In addition, the Tm values of the pharmaceutical compositions (FS 1-5 and FS 1-6) comprising histidine buffer (pH 6.0) were significantly higher than those of the other pharmaceutical compositions, indicating that the structural stability of the pharmaceutical compositions (FS 1-5 and FS 1-6) comprising histidine buffer (pH 6.0) was optimal.

From the results of FS1-7 through FS1-10 in Table 3, it can be seen that the kD values of the pharmaceutical compositions (FS 1-7) comprising acetate buffer (pH 5.0) and the pharmaceutical compositions (FS 1-10) comprising histidine buffer (pH 6.0) were significantly higher than those of the pharmaceutical compositions (FS 1-8) comprising acetate buffer (pH 5.5) and the pharmaceutical compositions (FS 1-9) comprising histidine buffer (pH 5.5). The above results demonstrate that the pharmaceutical compositions FS1-7 and FS1-10 have optimal colloidal stability. In addition, the Tm value of the pharmaceutical composition (FS 1-10) comprising histidine buffer (pH 6.0) is significantly higher than that of the other pharmaceutical compositions, and the results indicate that the pharmaceutical compositions FS1-10 have optimal structural stability.

1.2.2 appearance and protein content testing

The testing method comprises the following steps: visual inspection was used to examine the appearance of each pharmaceutical composition (sample). In the detection process, the illumination intensity of the clarity detector is ensured to be kept between 1000Lux and 1500 Lux. Holding the sample at the same level of the eye, gently shaking or inverting to avoid air bubbles; visual inspection was performed in front of black and white background, respectively. Visual inspection results were recorded in three aspects of color, opalescence and visible foreign matter.

Protein concentration in each pharmaceutical composition (sample) was measured using a NanoDrop one ultramicro spectrophotometer. The percent extinction coefficient (E1%) was set at 14.80 (g/100 mL) ^-1 cm ^-1 The method comprises the steps of carrying out a first treatment on the surface of the The detection wells were cleaned with ultrapure water, and 3. Mu.L of ultrapure water was used for blank correction. Each sample was assayed 3 times in parallel, and the assay wells were cleaned with ultrapure water before each measurement. The protein concentration was recorded for each measurement.

Test results: after being placed for 4 weeks under different conditions, the appearance of all samples is not changed obviously, namely no obvious visible foreign matters, no obvious opalescence and no color are found; specifically, the results are shown in Table 4.

Table 4 appearance results

Note that: "/" indicates not tested

The results of the protein content test of the pharmaceutical compositions of each group are shown in table 5.

TABLE 5 protein content test results (Unit: mg/mL)

From the results in table 5 above, no significant changes in protein content occurred in all samples after lofting under the different stability study conditions.

1.2.3SEC-HPLC purity test

The testing method comprises the following steps: SEC purity was checked by HPLC (Waters instrument) fitted with a SEC column (Waters 7.8 mm. Times.30 cm,3.5 μm). Diluting each medicine composition (sample) to 2.0mg/mL respectively by using a mobile phase, adding the medicine composition (sample) into a lining pipe after uniformly mixing, and directly carrying out sample injection analysis; the mobile phase composition was 50mM phosphate solution, 300mM sodium chloride solution and 300mM arginine hydrochloride solution, pH 6.8.+ -. 0.2. The relative percentages of main peak, high polymer and low polymer were calculated using area normalization. The components of the reference used in the test include: sodium acetate trihydrate 2.20mg/mL, glacial acetic acid 0.26mg/mL, sodium chloride 2.92mg/mL, trehalose dihydrate 56.00mg/mL and polysorbate 80.2 mg/mL; the pretreatment method and the test method of the reference product are the same as those of the sample. The test parameters are shown in table 6 below:

TABLE 6SEC-HPLC purity test parameters

Test results: the SEC-HPLC purity test results are shown in tables 7 and 8.

TABLE 7SEC-HPLC purity test results (%)

Note that: "ND" means not detected.

Table 8SEC-HPLC purity test results (%)

As is clear from the results in Table 7, all samples were allowed to stand at high temperature for 4 weeks, and the fragment content and the polymer content tended to increase, and there was no significant difference between the different pharmaceutical compositions. In addition, the SEC-HPLC purity of all samples did not change significantly over a period of 4 weeks under long term conditions.

From the results of Table 8, it is clear that all samples were allowed to stand at high temperature for 4 weeks, and that the fragment content and the polymer content tended to increase, and that there was no significant difference between the different pharmaceutical compositions. In addition, all samples were allowed to stand for 4 weeks under accelerated and long term conditions without significant changes in SEC-HPLC purity.

1.2.4CEX-HPLC purity test

The testing method comprises the following steps: CEX-HPLC purity detection was performed using HPLC (Waters E2695 instrument) equipped with a cation chromatography column (MabPac SCX-10, 4X 250 mm). Diluting each pharmaceutical composition (sample) to 1.0mg/mL by using a mobile phase A, then taking 500 mu L of diluted sample, uniformly mixing with 2 mu L of carboxypeptidase solution, carrying out enzymolysis for 60min at 37 ℃, and then filtering into a lining pipe by using a 0.22 mu m filter head, and carrying out sample injection analysis; the mobile phase composition is phase a: 20mM MES solution (pH 6.00.+ -. 0.05); and B phase: 20mM MES+200mM NaCl solution (pH 6.00.+ -. 0.05). And calculating the percentages of the main peak, the acid peak and the alkaline peak by adopting a peak area normalization method. The components of the reference used in the test include: sodium acetate trihydrate 2.20mg/mL, glacial acetic acid 0.26mg/mL, sodium chloride 2.92mg/mL, trehalose dihydrate 56.00mg/mL and polysorbate 80.2 mg/mL; the pretreatment method and the test method of the reference product are the same as those of the sample. The test parameters are shown in table 9 below:

TABLE 9CEX-HPLC purity test parameters

Test results: the results of the CEX-HPLC purity test are shown in tables 10 and 11.

TABLE 10 CEX-HPLC purity test results (%)

TABLE 11CEX-HPLC purity test results (%)

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As can be seen from the results in Table 10 above, all samples were left at high temperature for 4 weeks, and the main peak purity of the pharmaceutical compositions FS1-5 and FS1-6 comprising histidine buffer (pH 6.0) was significantly higher than that of the other pharmaceutical compositions. All samples were left for 4 weeks under long term conditions without significant changes in CEX purity.

From the results of Table 11 above, it can be seen that all samples were left to stand at high temperature for 4 weeks, and that the main peak purity of the pharmaceutical compositions FS1-10 comprising histidine buffer (pH 6.0) was significantly higher than that of the other pharmaceutical compositions. All samples were left under accelerated conditions for 4 weeks with a slight decrease in CEX purity, with a trend consistent with high temperature conditions. All samples were left for 4 weeks under long term conditions without significant changes in CEX purity.

1.2.5rCE-SDS purity test

The testing method comprises the following steps: the rCE-SDS purity was detected using a capillary electrophoresis apparatus (Maurice apparatus). The diluted solution (1 wt% SDS+40mM phosphate buffer solution, pH 6.5) was taken to dilute each pharmaceutical composition (sample) to 1mg/mL, then 95. Mu.L of the diluted sample was taken to mix uniformly with 5. Mu.L of 2-mercaptoethanol, centrifuged at 3000rpm at room temperature for 30s, and then incubated at 70℃for 15min. After the incubation was completed, the mixture was cooled to room temperature and centrifuged at 12000rpm for 5min at room temperature. From the sample tubes, 75. Mu.L of the sample solution was taken out to a 96-well plate, and the solution was centrifuged at 1000g for 10min at room temperature to avoid air bubbles, and detected by using a capillary electrophoresis apparatus (Maurie). The components of the reference used in the test include: sodium acetate trihydrate 2.20mg/mL, glacial acetic acid 0.26mg/mL, sodium chloride 2.92mg/mL, trehalose dihydrate 56.00mg/mL and polysorbate 80.2 mg/mL; the pretreatment method and the test method of the reference product are the same as those of the sample.

Test results: the results of the rCE-SDS purity test are shown in Table 12 and Table 13.

TABLE 12rCE-SDS purity test results (%)

TABLE 13rCE-SDS purity test results (%)

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As can be seen from the results of Table 12 above, all samples were left at high temperature for 4 weeks, and the rCE-SDS purity tended to decrease, with the decrease rate of the pharmaceutical compositions FS1-6 being significantly higher than that of the other pharmaceutical compositions. All samples were left for 4 weeks under long term conditions without significant changes in rCE-SDS purity.

As can be seen from the results in Table 13 above, all samples were left at high temperature for 4 weeks, and rCE-SDS purity tended to decrease, with FS1-10 decreasing slightly faster than other pharmaceutical compositions. All samples were left for 4 weeks under accelerated and long term conditions without significant changes in rCE-SDS purity.

1.2.6nrCE-SDS purity test

The testing method comprises the following steps: the nrCE-SDS purity was detected using a capillary electrophoresis apparatus (Maurice apparatus). The final volume of 100 mu L is calculated, the required diluent and sample amount are added when each pharmaceutical composition (sample) is diluted to 1mg/mL respectively, and then 1.5mL EP tube is added in sequence for mixing evenly according to the diluent (0.01 g/mL SDS+40mM phosphate buffer, pH 6.5), 5.0 mu L0.25M (mol/L) NEM (ethylmaleimide) and sample; ultrapure water was used as a blank, and the dilution ratio was kept consistent with the reference (dilution factor was at least 2). Centrifuge at 3000rpm at room temperature for 30s, followed by incubation at 70℃for 5min. After the incubation was completed, the mixture was cooled to room temperature and centrifuged at 12000rpm for 5min at room temperature. From the sample tubes, 75. Mu.L of the sample solution was taken out to a 96-well plate, and the solution was centrifuged at 1000g for 10min at room temperature to avoid air bubbles, and detected by using a capillary electrophoresis apparatus (Maurie). The components of the reference used in the test include: sodium acetate trihydrate 2.20mg/mL, glacial acetic acid 0.26mg/mL, sodium chloride 2.92mg/mL, trehalose dihydrate 56.00mg/mL and polysorbate 80.2 mg/mL; the pretreatment method and the test method of the reference product are the same as those of the sample.

Test results: the results of the nrCE-SDS purity test are shown in tables 14 and 15.

TABLE 14nrCE-SDS purity test results (%)

TABLE 15nrCE-SDS purity test results (%)

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As is clear from the results of Table 14, all samples were left at high temperature for 4 weeks, the purity of nrCE-SDS tended to decrease, and the purity of nrCE-SDS of the pharmaceutical compositions FS1-3 decreased relatively rapidly. All samples were left for 4 weeks under long term conditions without significant changes in nrCE-SDS purity.

As can be seen from the results of Table 15 above, all samples were left at high temperature for 4 weeks, and the purity of nrCE-SDS tended to decrease, with the purity of nrCE-SDS of the pharmaceutical compositions FS1-8 and FS1-10 decreasing slightly faster than that of the pharmaceutical compositions FS1-7 and FS1-9. All samples were left under accelerated conditions for 4 weeks with a slight decrease in nrCE-SDS purity. All samples were left for 4 weeks under long term conditions without significant changes in nrCE-SDS purity.

1.2.7 binding Activity test

The testing method comprises the following steps: recombinant human RBD-his (vector pCAGGS-RBD his was transiently transferred to 293F cells) was used at a concentration of 3.0. Mu.g/mLThe main elements of the vector pCAGGS-RBD his obtained by expression include in order: CMV IE (promoter of target gene expression), RBD gene, his tag, SV40 ori, SV40 polyA signal, ori p and AmpR (ampicillin resistance gene)) plates, incubated at 37℃for 90min; plates were washed and blocked with PBS containing 2% (w/v) skimmed milk; each group of pharmaceutical compositions containing the novel coronavirus antibody was added in a gradient diluted with PBS solution containing 2% (w/v) skimmed milk (the concentration of the novel coronavirus antibody was diluted to 40. Mu.g/mL with PBS solution containing 2% (w/v) skimmed milk, and then in a gradient of 4-fold to obtain 12 concentrations), and incubated at 37℃for 60min. Detection with 5000-fold dilution of goat anti-human IgG antibody (Fc-specific, coupled peroxidase) (manufacturer: sigma, cat# A0170) as detection antibody, incubation at 37℃for 60min, development with 0.1mg/mL TMB, termination of the reaction with 2M (mol/L) HCl after 15min, reading of the plate at 450nm/620nm, followed by four-parameter logistic regression (4 PL) model fitting with GraphPad Prism software to give EC ₅₀ (half maximum effect concentration) value. Binding activity (relative activity,%) =ec of reference substance ₅₀ EC of pharmaceutical composition ₅₀ ×100％。

Test results: the results of the binding activity test of the pharmaceutical compositions FS1-7, FS1-8, FS1-9, FS1-10 are shown in Table 16.

Table 16 binding Activity test results (%)

From the above results, it was found that no significant change in binding activity occurred in all samples when left at 4W under high temperature or long-term conditions.

1.2.8 ligand competitive inhibition Activity assay

The testing method comprises the following steps: recombinant human RBD-his (obtained by transiently transferring the vector pCAGGS-RBD his to 293F cells for expression), the main elements of the vector pCAGGS-RBD his include CMV IE (promoter for expression of target gene), RBD gene, his tag, SV40 ori, SV40 polyA signal, ori p and AmpR (ampicillin resistance gene) in this order) were diluted to a concentration of 5.0. Mu.g/mL of a wrapper, and incubated at 37℃for 90min. Washing plateBlocking was performed with 2% (w/v) skimmed milk. Recombinant MX2 hACE2 mFc (obtained by transiently transferring vector MX2-hACE2-ECD-FC to 293F cells) was diluted with 2% (w/v) skim milk, and the major elements of vector MX2-hACE2-ECD-FC included, in order, CMV Promoter (Promoter of target gene expression), signal peptide (signal peptide of target gene expression), hACE2-ECD (extracellular region of Human ACE 2), ori p, ampR (ampicillin resistance gene) and pMB1 ori) to a fixed concentration of 5.0. Mu.g/mL, and each group of pharmaceutical compositions containing the novel coronavirus antibody was diluted with 5.0. Mu.g/mL hACE2 mFc (initial concentration of novel coronavirus antibody was 100. Mu.g/mL, followed by 2.5-fold gradient dilution). The mixture was added to the plate for 1 hour and the plate was washed. By combining with 1:5000 dilution of peroxidase-labeled goat anti-mouse Fc fragment secondary antibody (Sigma Co., cat# A2554) was incubated for 1 hour, TMB (Sigma, cat# T2885) was added and incubated for 15 minutes, the reaction was stopped with 2M HCl, the plate was read at 450nm/620nm, and then competition inhibition curves were fitted using GraphPad Prism software to a four-parameter logistic regression (4 PL) model to give IC ₅₀ (semi-inhibitory concentration) value. Ligand competitive inhibition activity (relative activity,%) =ic of reference substance ₅₀ IC for pharmaceutical compositions ₅₀ ×100％。

Test results: the results of the ligand competitive inhibition activity test are shown in tables 17 and 18.

Table 17 ligand competitive inhibition Activity assay (%)

Table 18 ligand competitive inhibition Activity assay (%)

From the results of Table 17, it is clear that no significant change in biological activity occurred in all samples when left at 4W under high temperature or long term conditions.

From the results in Table 18 above, it is clear that no significant change in biological activity occurred in all samples when left at 4W under high temperature or long term conditions.

1.2.9 sub-visible particle results

The testing method comprises the following steps: insoluble particles in each pharmaceutical composition (sample) were statistically measured by a photoresist method using an HIAC insoluble particle counter as a detecting instrument. The Run mode uses Run counter and the set parameters are shown in table 19 below.

Table 19 sub-visible particle test parameters

Parameters (parameters)	Setting value
		Number of runs	4
Dilution factor	1
		First run flush volume (mL)	0
Whether to discard the first run result	Is that

Test results: the sub-visible particle test results are shown in tables 20 and 21. Wherein X is more than or equal to 10 and less than 25 mu m, the particle size of the particles is between 10 and 25 mu m, and X is more than 25 mu m.

Table 20 sub-visible particle test results (Unit: particles/mL)

Table 21 sub-visible particle test results (Unit: particles/mL)

From the results in Table 20 above, it was found that no significant increase in sub-visible particles occurred for all samples when left to stand for 4W at high temperature and long term.

From the results in Table 21 above, it is clear that no significant increase in sub-visible particles occurred for all samples when placed at 4W under high temperature, acceleration and long term conditions.

From the above, it is evident from the results of FS1-1 to FS1-6 that the pharmaceutical compositions FS1-5 have an optimal colloidal stability and have a superior structural stability. The main peak purity of the pharmaceutical compositions FS1-5 and FS1-6 containing histidine buffer (pH 6.0) was significantly higher than that of the other pharmaceutical compositions after 4 weeks of standing at high temperature. In addition, the protein content, appearance and sub-visible particles, biological activity of all pharmaceutical compositions were not significantly altered. Thus, the pharmaceutical composition (FS 1-5) comprising the novel coronavirus antibody (50 mg/mL) with 20mM histidine buffer (pH 6.0), 230mM sucrose and 0.04% Tween 80 (II) has optimal stability under high temperature conditions.

As can be seen from the results of FS1-7 to FS1-10, the novel coronavirus antibody or antigen binding fragment thereof with high concentration (100 mg/mL) has good stability under the condition of different pH buffer systems, and the pharmaceutical composition FS1-10 has better colloidal stability and structural stability. After 4 weeks of standing at high temperature, the main peak content of CEX of the pharmaceutical compositions FS1-10 is superior to other pharmaceutical compositions. In addition, all pharmaceutical compositions did not significantly change in appearance and sub-visible particle, biological activity. Thus, a pharmaceutical composition comprising a high concentration (100 mg/mL) of the novel coronavirus antibody with 20mM histidine buffer (ph 6.0), 230mM sucrose and 0.04% tween 80 (II) has optimal stability under high temperature conditions.

1.2.10 shaking, freezing and thawing stability test

The pharmaceutical compositions FS1-5 were subjected to stability testing under shaking, freeze thawing and white light irradiation stability study conditions, the test items including appearance, SEC-HPLC purity, CEX-HPLC purity, nrCE-SDS purity, rCE-SDS purity, binding activity, ligand competition inhibiting activity and sub-visible particles, and the specific test methods were the same as those described in 1.2.1-1.2.9 above.

The test results are shown in table 22 below. Wherein X is more than or equal to 10 and less than 25 mu m, the particle size of the particles is between 10 and 25 mu m, and X is more than 25 mu m.

Table 22 stability test results

From the above results, it was found that the pharmaceutical compositions FS1-5 were not significantly changed in appearance, SEC-HPLC purity, CEX-HPLC purity, nrCE-SDS purity, rCE-SDS purity, ligand competition inhibiting activity, binding activity and sub-visible particles after shaking or freeze thawing treatment. The above results indicate that the pharmaceutical compositions FS1-5 are well tolerated for shaking and repeated freeze thawing.

Example 2: binding and dissociation characteristics of pharmaceutical composition and SARS-COV-2 wild type and mutant RBD

2.1 Experimental methods

The pharmaceutical composition FS1-5 (concentration of novel coronavirus antibody was diluted to 1. Mu.g/mL with HBS-EP+ using molecular interaction analyzer Octet RED384 (Sartorius Fortebio Co.), was captured on the surface of Protein A probe (manufacturer Sartorius Fortebio, cat. No. 18-5010), and then immersed in gradient diluted RBD Protein solutions of different His tags to be tested, which were His tagged SARS-COV-2 wild type RBD Protein solutions (manufacturer Acro, product number SPD-C82E 8), RBD Protein solution of His-tagged Alpha mutant (manufacturer Acro, product number SPD-C82E 6), RBD Protein solution of His-tagged Beta mutant (manufacturer Acro, product number SPD-C82E 5), RBD Protein solution of His-tagged Epsilon mutant (manufacturer Acro, product number SPD-C82E 3), RBD Protein solution of His-tagged Kappa mutant (manufacturer Acro, product number SPD-C82 Ec), RBD Protein solution of His-tagged Gamma mutant (manufacturer Acro, product number SPD-room) C82E 7), RBD protein solutions of His-tagged Delta mutants (manufacturer Acro, cat. SPD-C82 Ed) with concentration gradients of 100nM, 50nM, 25nM, 12.5nM and 6.25nM for each RBD protein, and HBS-EP+ (GE medical life sciences, 10mM buffer HEPES, 150mM sodium chloride, 3mM ethylenediamine tetraacetic acid and 0.05wt% surfactant P20, pH 7.4) as diluents, and detected to obtain binding signals; dissociation in HBS-EP+, detection yields the dissociation signal. Data were analytically fitted using a kinetic model of Fortebio Analysis software (Data Analysis HT 12.0) to give affinities (K) _D ) Values.

2.2 experimental results

The experimental results are shown in Table 23, in which the association rate constant (ka), dissociation rate constant (kd) and affinity (K _D ) Values.

TABLE 23 summary of the binding and dissociation characteristics of pharmaceutical compositions FS1-5 and SARS-COV-2 wild-type and mutant RBD

Spike protein	ka(1/Ms)	kd(1/s)	K _D (M)
				Wild RBD-His	1.06E+5	2.15E-4	2.04E-9
Alpha mutant RBD-His (N501Y mutation)	2.43E+5	3.16E-4	1.30E-9
				Beta mutant RBD-His (K417N, E484K, N501Y mutation)	2.01E+5	1.76E-4	8.75E-10
Gamma mutant RBD-His (K417T, E484K, N501Y mutant)	1.96E+5	1.72E-4	8.75E-10
				Kappa mutant RBD-His (L452R, E484Q mutant)	1.93E+5	2.26E-4	1.17E-9
Epsilon mutant RBD-His (L452R mutation)	2.16E+5	2.41E-4	1.12E-9
				Delta mutant RBD-His (L452R, T478K mutant)	2.20E+5	1.61E-4	7.30E-10

The results show that the pharmaceutical compositions FS1-5 of the application can bind with high affinity to the wild type SARS-CoV-2 and RBD regions of six clinical mutants, K _D The range of values is 0.73nM to 2.04nM.

Example 3: binding Activity of pharmaceutical composition to wild-type SARS-COV-2 mutant RBD

3.1 Experimental methods

Seven His-tagged RBD protein solutions (His-tagged RBD protein solutions were His-tagged SARS-COV-2 wild-type RBD protein solutions (manufacturer: acro, cat. No. SPD-C82E 8), his-tagged Alpha mutant strain RBD protein solutions (manufacturer: ACRO, cat. No. SPD-C82E6, his-tagged Beta mutant strain RBD protein solutions (manufacturer: sino Biological, cat. No. 40592-V08H 85-B), his-tagged Epsilon mutant strain RBD protein solutions (manufacturer: ACRO, cat. No. SPD-C82E 3), his-tagged Kappa mutant strain RBD protein solutions (manufacturer: SPD-C52 Hv), his-tagged Alpha mutant strain RBD protein solutions (manufacturer: SPD-C52 Hr), his-tagged Alpha mutant strain RBD protein solutions (manufacturer: SPD-C82H 2) were diluted by a gradient of PBS (PBS-35F 1) and a gradient of a 1-35-containing antibodies were applied to a new type of dilution of the virus (PBS-35 F.1/35 F.1, 4 to a gradient of a diluted virus-containing a 1-35% of a virus carrier (PBS-35F/1, and a gradient of a diluted solution containing a 1-35F/H, respectively, the main elements of the carrier HXT1s-KLH HC LALA in turn comprise: CMV Promoter (Promoter of target gene expression), signal peptide (signal peptide of target gene expression), KLH-VH-IgG1-LALA (heavy chain of KLH antibody), ori p, ampR (ampicillin resistance gene), and pMB1ori (replication initiation); the major elements of the carrier HXT2-KLH LC comprise in order: CMV Promoter (Promoter of target gene expression), signal peptide (signal peptide of target gene expression), KLH-VL-Kappa LC (light chain of KLH antibody), ori p, ampR (ampicillin resistance gene) and pMB1ori (replication initiation)) (isotype control group, initial concentration 40. Mu.g/mL, diluted in 4-fold gradient, final concentration to 0.009537 ng/mL), incubation at 37℃for 60min. Detection was performed using a 5000-fold dilution of goat anti-human IgG antibody (Fc-specific, conjugated peroxidase) (manufacturer: sigma. Cat. No. A0170) as detection antibody, incubation at 37℃for 60min, followed by development with 0.1mg/mL TMB, After 15min, the reaction was stopped with 2M (mol/L) HCl, plates were read at 450nm/620nm, and then four-parameter logistic regression (4 PL) model fitting was performed using GraphPad Prism software to obtain EC ₅₀ (half maximum effect concentration) value.

3.2 experimental results

The experimental results are shown in Table 24 and FIGS. 1A-1G.

Table 24 EC ₅₀ Results

Spike protein	EC ₅₀ (ng/mL)
		Wild RBD-His	14.2
Alpha mutant RBD-His (N501Y mutation)	41.6
		Beta mutant RBD-His (K417N, E484K, N501Y mutation)	11.5
Gamma mutant RBD-His (K417T, E484K, N501Y mutant)	22.7
		Kappa mutant RBD-His (L452R, E484Q mutant)	11.7
Epsilon mutant RBD-His (L452R mutation)	10.5
		Delta mutant RBD-His (L452R, T478K)Mutation	11.8

In FIGS. 1A-1G, groups FS1-5 containing the novel coronavirus antibodies were designated as "5-10", and the hIgG1 LALA isotype control group was designated as "hIgG1 LALA isotype control".

As can be seen from Table 24 and the results of FIGS. 1A-1G, the pharmaceutical compositions FS1-5 were able to specifically bind to RBD of SARS-CoV-2 wild-type and six mutants (including Alpha, beta, gamma, kappa, epsilon and Delta mutants), EC ₅₀ The values were in the range of 10.5ng/mL-41.6ng/mL, so that the pharmaceutical compositions FS1-5 all had strong in vitro binding capacity for SARS-CoV-2 wild-type and six mutant RBD regions.

Example 4: pharmaceutical composition for competitive inhibition of SARS-COV-2 wild type and mutant RBD binding to human ACE2

4.1 Experimental methods

Seven His-tagged RBD protein solutions (His-tagged RBD protein solutions were His-tagged SARS-COV-2 wild-type RBD protein solutions (manufacturer Acro, manufacturer SPD-C82E 8), his-tagged Alpha mutant RBD protein solutions (manufacturer Acro, manufacturer SPD-C82E 6), his-tagged Beta mutant RBD protein solutions (manufacturer Acro, manufacturer SPD-C82E 5), his-tagged Epsilon mutant RBD protein solutions (manufacturer Acro, manufacturer SPD-C82E 3), his-tagged Kappa mutant RBD protein solutions (manufacturer Acro, manufacturer SPD-C82 Ec), his-tagged Gamma mutant RBD protein solutions (manufacturer Acro, manufacturer SPD-C82E 7), his-tagged Delta mutant RBD protein solutions (manufacturer Acro, manufacturer SPD-C82 Ed) were incubated for 90min; washing the plate and blocking with PBS containing 2% (w/v) skimmed milk, diluting recombinant MX hACE2 mFc (vector construction method see 1.2.8 section in example 1) with PBS containing 2% (w/v) skimmed milk to 6.0 μg/mL, and diluting pharmaceutical compositions FS1-5 with PBS containing 2% (w/v) skimmed milk, respectively (the concentration of novel coronavirus antibody was first 2% (w/v) The PBS solution of skimmed milk is diluted to 100 mug/mL, and then diluted in a gradient of 2.5 times, the final concentration is 4.19 ng/mL), and hIgG1 LALA (the vector HXT1s-KLH HC LALA and HXT2-KLH LC are transiently transferred to CHOK1 cells for expression, and the main elements of the vector HXT1s-KLH HC LALA sequentially comprise: CMV Promoter (Promoter of target gene expression), signal peptide (signal peptide of target gene expression), KLH-VH-IgG1-LALA (heavy chain of KLH antibody), ori p, ampR (ampicillin resistance gene), and pMB1ori (replication initiation); the major elements of the carrier HXT2-KLH LC comprise in order: CMV Promoter (Promoter of target gene expression), signal peptide (signal peptide of target gene expression), KLH-VL-Kappa LC (light chain of KLH antibody), orip, ampR (ampicillin resistance gene) and pMB1ori (replication initiation)) (isotype control, initiation concentration 100. Mu.g/mL, dilution with 2.5-fold gradient, final concentration 4.19 ng/mL), the resulting mixture was added to the blocked plate and incubated for 60min. Detection with 5000-fold diluted goat anti-human IgG antibody (Fc-specific, coupled peroxidase) (manufacturer: sigma number: A0170) as detection antibody, incubation at 37℃for 60min, development with 0.1mg/mL TMB, termination of the reaction with 2M (mol/L) HCl after 15-20min, plate reading at 450nm/620nm, and fitting of a competition inhibition curve using GraphPad Prism software to four-parameter logistic regression (4 PL) model to give IC ₅₀ (semi-inhibitory concentration) value.

4.2 experimental results

The experimental results are shown in Table 25 and FIGS. 2A-2G.

Table 25 results of competitive inhibition of pharmaceutical compositions against SARS-CoV-2 wild-type and mutant RBD

In FIGS. 2A-2G, groups FS1-5 containing the novel coronavirus antibodies were designated as "5-10", and the hIgG1 LALA isotype control group was designated as "hIgG1 LALA isotype control".

As can be seen from Table 25 and the results of FIGS. 2A-2G, the pharmaceutical compositions FS1-5 are effective in blocking SARS-CoV-2 wild-type and RBD regions of six mutants (including Alpha, beta, gamma, kappa, epsilon and Delta mutants) and receptor ACE-2, IC ₅₀ The value is in the range of 0.26 mug/mL-1.70 mug/mL, so that the pharmaceutical compositions FS1-5 have strong in vitro competitive inhibition capability for the combination of SARS-CoV-2 wild type and six mutant strain RBD regions and human ACE-2.

Example 5: pseudo-virus neutralizing activity of pharmaceutical compositions

The blocking effect of pharmaceutical compositions FS1-5 on SARS-CoV-2 wild type (purchased from Beijing three drug technologies development Co., ltd., cat# 80033), alpha mutant (purchased from Beijing three drug technologies development Co., ltd., cat# 80043), beta mutant (purchased from Beijing three drug technologies development Co., ltd., cat# 80044), gamma mutant (purchased from Beijing three drug technologies development Co., ltd., cat# 80045), kappa mutant (purchased from Beijing three drug technologies development Co., cat# 80047) and Delta mutant (purchased from Beijing three drug technologies development Co., ltd., cat# 80048) pseudovirus infection 293-ACE2 cells (purchased from Nannunofizan biotechnology Co., ltd., product # DD 1401-01) was examined.

5.1 Experimental methods

Wild-type pseudovirus (2. Mu.L virus/well), alpha mutant (5. Mu.L virus/well), beta mutant (2. Mu.L virus/well), gamma mutant (10. Mu.L virus/well), kappa mutant (5. Mu.L virus/well) or Delta mutant (2. Mu.L virus/well) pseudovirus were diluted with pharmaceutical compositions FS1-5 (containing novel coronavirus antibodies at an antibody concentration of 100. Mu.g/mL in DMEM medium (1X) +10v/v% FBS, followed by 10-fold gradient dilution to 0.1 pg/mL) or control antibody anti-KLH hIgG1 LA (St. John's LAUnion, lot 20200507) in 96-well flat bottom whiteboards (Corning, cat. No.: 3917), respectively. Preincubation with DMEM medium (1X) +10v/v% FBS from 100. Mu.g/mL to 10-fold gradient dilution of 0.1 pg/mL) was performed for 1h at 37 ℃. The 293-ACE2 cells were then resuspended in assay buffer (DMEM medium (1X) +10v/v% FBS) and added to the pseudovirus and antibody mixture at 20000 cells per well and incubated for 24h in a 37℃incubator. After incubation, 50. Mu.L of luciferase reporter assay reagent (Bright light Luciferase substrate, available from Vazyme, cat# DD 1204) was added to each well and the mixture was digested with the enzymeThe standard instrument detects the fluorescence signal, and then uses GraphPad Prism software to carry out four-parameter logistic regression (4 PL) model fitting curve to obtain the IC ₅₀ Values.

5.2 experimental results

The experimental results are shown in table 26 and fig. 3A-3F.

TABLE 26 summary of pseudovirus neutralization activity results

Note that: NA indicates no significant activity

In FIGS. 3A-3F, groups FS1-5 containing the novel coronavirus antibodies were designated as "5-10", and the anti-KLH hIgG1 LALA isotype control group was designated as "anti-KLH hIgG1 LALA".

As can be seen from Table 26 and the results of FIGS. 3A to 3F, the pharmaceutical compositions FS1-5 not only efficiently neutralize wild-type SARS-CoV-2 pseudovirus, but also have a strong neutralizing effect on the 293-ACE2 cells infected with Alpha mutant (B.1.1.7) pseudovirus, beta mutant (B.1.315) pseudovirus, gamma mutant (P.1) pseudovirus, kappa mutant (B.1.617.1) and Delta mutant (B.1.617.2) pseudovirus, and exhibit a good virus inhibitory activity.

Claims

1. A pharmaceutical composition comprising:

(1) A buffer; and

(2) A novel coronavirus antibody or antigen-binding fragment thereof;

2. The pharmaceutical composition of claim 1, wherein the novel coronavirus antibody or antigen-binding fragment thereof comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID No. 6 and a light chain variable region having an amino acid sequence as shown in SEQ ID No. 7.

3. The pharmaceutical composition of claim 1, wherein the novel coronavirus antibody or antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence as shown in SEQ ID No. 8 and a light chain having an amino acid sequence as shown in SEQ ID No. 9.

4. The pharmaceutical composition of any one of claims 1-3, wherein the pharmaceutical composition further comprises a stabilizer and a surfactant; the stabilizer is selected from sucrose or trehalose, the surfactant is selected from polysorbate 80, polysorbate 20 or poloxamer 188, and the buffer is selected from acetic acid buffer or histidine buffer.

5. Pharmaceutical composition according to claim 4, wherein the histidine buffer is selected from a histidine-histidine hydrochloride buffer or a histidine-histidine acetate buffer, preferably a histidine-histidine hydrochloride buffer.

6. The pharmaceutical composition of any one of claims 4-5, wherein the buffer has a concentration of 10-30 mM.

7. The pharmaceutical composition of any one of claims 4-6, wherein the concentration of the novel coronavirus antibody or antigen-binding fragment thereof is 2-200 mg/mL, preferably 10-150 mg/mL, more preferably 50-100 mg/mL.

8. The pharmaceutical composition according to any one of claims 4-6, wherein the concentration of the stabilizing agent is 120-280 mM, preferably 200-250 mM.

9. The pharmaceutical composition according to any one of claims 4-6, wherein the concentration of the surfactant in the pharmaceutical composition is 0.01-0.1%, preferably 0.01-0.04% w/v.

10. The pharmaceutical composition according to any one of claims 4-6, wherein the pH of the buffer is between 5.0 and 7.0, preferably between 5.5 and 6.5, more preferably between 5.9 and 6.1.

11. The pharmaceutical composition of any one of claims 4-10, wherein the histidine buffer is histidine-histidine hydrochloride buffer, the stabilizer is sucrose, and the surfactant is polysorbate 80.

12. The pharmaceutical composition of claim 11, comprising the components shown below:

(a) 50-100 mg/mL of the novel coronavirus antibody or antigen-binding fragment thereof; (b) 10-30 mM histidine buffer, pH 5.5-6.5; (c) 200-250 mM sucrose; and (d) polysorbate 80 at a concentration of 0.01% -0.04% in w/v;

Preferably, (a) 50mg/mL of the novel coronavirus antibody or antigen binding fragment thereof; (b) 20mM histidine buffer, pH 5.9-6.1; (c) 230mM sucrose; and (d) polysorbate 80 at a concentration of 0.04% w/v; or (b)

(a) 100mg/mL of the novel coronavirus antibody or antigen binding fragment thereof; (b) 20mM histidine buffer, pH 5.9-6.1; (c) 230mM sucrose; and (d) polysorbate 80 at a concentration of 0.04% w/v.

13. The pharmaceutical composition according to any one of claims 1-12, wherein the pH of the pharmaceutical composition is between 5.0 and 7.0, preferably between 5.5 and 6.5, more preferably between 5.9 and 6.1.

14. A lyophilized formulation of the pharmaceutical composition of any one of claims 1-13.

15. A liquid formulation comprising the pharmaceutical composition of any one of claims 1-13, or a liquid formulation obtained from the resuspension of the lyophilized formulation of claim 14; preferably, the liquid formulation is an injection.

16. Use of a pharmaceutical composition according to any one of claims 1-13, a lyophilized formulation according to claim 14 or a liquid formulation according to claim 15 for the manufacture of a medicament for preventing or treating a novel coronavirus infection by eliminating, inhibiting or reducing the activity of the novel coronavirus.