CN117643628A

CN117643628A - Novel coronavirus neutralizing antibody pharmaceutical composition and application thereof

Info

Publication number: CN117643628A
Application number: CN202311077459.7A
Authority: CN
Inventors: 刘沛想; 冯辉; 姚盛; 刘洪川; 苏凯迪; 王静; 李理; 宋涛
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: 2022-09-02
Filing date: 2023-08-25
Publication date: 2024-03-05

Abstract

The application provides a novel coronavirus neutralizing antibody pharmaceutical composition and application thereof. The pharmaceutical composition contains a novel coronavirus antibody or antigen binding fragment HUR33 variant and/or CB6 variant thereof, and optional buffers and the like, wherein the HUR33 variant 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/or LCDR1, LCDR2 and LCDR3 with amino acid sequences shown as SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6 respectively; CB6 variants comprise HCDR1, HCDR2 and HCDR3 having amino acid sequences shown as SEQ ID NO. 18, SEQ ID NO. 17 and SEQ ID NO. 15, respectively, and/or LCDR1, LCDR2 and LCDR3 having amino acid sequences shown as SEQ ID NO. 21, SEQ ID NO. 16 and SEQ ID NO. 22, respectively, or as SEQ ID NO. 19, SEQ ID NO. 16 and SEQ ID NO. 20, respectively. The present application also provides liquid formulations containing the pharmaceutical compositions, as well as the use of the pharmaceutical compositions and liquid formulations in the manufacture of a medicament for the prevention, treatment, detection or diagnosis of diseases associated with novel coronavirus infections.

Description

Novel coronavirus neutralizing antibody pharmaceutical composition and application thereof

The present application claims priority from the chinese patent office, application No. 202211074840.3, entitled "novel coronavirus neutralizing antibody pharmaceutical composition and use thereof," filed on month 2, 9, 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 neutralizing antibody pharmaceutical composition and application thereof.

Background

The International Commission on classification of viruses (International Committee on Taxonomy of Viruses, ICTV) announced that the formal classification of novel coronaviruses was named Severe acute respiratory syndrome coronavirus 2 (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2). The novel coronavirus SARS-CoV-2 causes a great deal of disease in the global accumulation of definite cases, which poses a serious threat to the life and health of the public.

Currently, scientists have developed a variety of neutralizing antibodies for the treatment of novel coronavirus (SARS-CoV-2) infection, but there are few stable pharmaceutical compositions specifically adapted for such antibodies. Therefore, there is a need to develop a pharmaceutical composition comprising a novel coronavirus neutralizing antibody with high stability.

Disclosure of Invention

The aim of the application is to provide a pharmaceutical composition containing a novel coronavirus neutralizing antibody pharmaceutical composition, which has the advantages of long-term stability, no aggregation and the like.

In a first aspect the present application provides a pharmaceutical composition comprising a novel coronavirus neutralizing antibody or antigen binding fragment thereof selected from at least one of a HUR33 variant and a CB6 variant; the HUR33 variant 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/or LCDR1, LCDR2 and LCDR3 with amino acid sequences shown as SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6 respectively; the CB6 variant comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences shown as SEQ ID NO. 18, SEQ ID NO. 17 and SEQ ID NO. 15 respectively, and/or LCDR1, LCDR2 and LCDR3 with amino acid sequences shown as SEQ ID NO. 21, SEQ ID NO. 16 and SEQ ID NO. 22 respectively or as SEQ ID NO. 19, SEQ ID NO. 16 and SEQ ID NO. 20 respectively.

In some embodiments of the present application, the HUR33 variant comprises a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO. 8 or SEQ ID NO. 9, and/or a light chain variable region having an amino acid sequence as set forth in SEQ ID NO. 7; the CB6 variant comprises a heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 23 and/or a light chain variable region with an amino acid sequence shown as SEQ ID NO. 25 or SEQ ID NO. 24.

In some embodiments of the present application, the HUR33 variant further comprises a heavy chain constant region having an amino acid sequence as set forth in SEQ ID NO. 13 and a light chain constant region having an amino acid sequence as set forth in SEQ ID NO. 14.

In some embodiments of the present application, the HUR33 variant comprises a heavy chain having an amino acid sequence as set forth in SEQ ID NO. 10 or SEQ ID NO. 11, and/or a light chain having an amino acid sequence as set forth in SEQ ID NO. 12; the CB6 variant comprises a heavy chain with an amino acid sequence shown as SEQ ID NO. 26 and/or a light chain with an amino acid sequence shown as SEQ ID NO. 27 or SEQ ID NO. 28.

In some embodiments of the present application, the HUR33 variant comprises: (1) The amino acid sequences are shown as HCDR1, HCDR2 and HCDR3 shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3 respectively, and the amino acid sequences are shown as LCDR1, LCDR2 and LCDR3 shown as SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6 respectively; or (2) a heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 8 and a light chain variable region with an amino acid sequence shown as SEQ ID NO. 7; or (3) a heavy chain with an amino acid sequence shown as SEQ ID NO. 10 and a light chain with an amino acid sequence shown as SEQ ID NO. 12; the CB6 variant comprises: (1) The amino acid sequences are shown as HCDR1, HCDR2 and HCDR3 shown as SEQ ID NO. 18, SEQ ID NO. 17 and SEQ ID NO. 15 respectively, and the amino acid sequences are shown as LCDR1, LCDR2 and LCDR3 shown as SEQ ID NO. 21, SEQ ID NO. 16 and SEQ ID NO. 22 respectively; or (2) a heavy chain variable region having an amino acid sequence shown as SEQ ID NO. 23 and a light chain variable region having an amino acid sequence shown as SEQ ID NO. 25; or (3) a heavy chain with an amino acid sequence shown as SEQ ID NO. 26 and a light chain with an amino acid sequence shown as SEQ ID NO. 27.

In some embodiments of the present application, the mass ratio of the HUR33 variant and the CB6 variant is (1-10): (10-1), about 1: 1. about 1: 2. about 1: 3. about 1: 4. about 1: 5. about 2: 1. about 2: 3. about 3: 1. about 3: 2. about 4:1 or about 5:1.

in some embodiments of the present application, the novel coronavirus neutralizing antibody or antigen-binding fragment thereof has a concentration of 1-300 mg/mL, 10-200 mg/mL, 20-150 mg/mL, 30-120 mg/mL, 30-100 mg/mL, 20-100 mg/mL, 5-90 mg/mL, 5-85 mg/mL, 10-90 mg/mL, 10-85 mg/mL, 20-90 mg/mL, 20-85 mg/mL, 30-90 mg/mL, 30-85 mg/mL, 40-90 mg/mL, 40-85 mg/mL, 50-90 mg/mL, 50-85 mg/mL, 60-90 mg/mL, 10-80 mg/mL, 20-80 mg/mL, 30-80 mg/mL, 40-80 mg/mL, 50-80 mg/mL, 10-60 mg/mL, 20-60 mg/mL, 30-60 mg/mL, 10-50 mg/mL, about 40-50 mg, about 40mg/mL, about 40-40 mg/mL, about 40-80 mg/mL, about 40-80 mg/mL.

In some embodiments of the present application, the pharmaceutical composition further comprises a buffer selected from at least one of an acetate buffer, a citrate buffer, and a histidine buffer; preferably, the histidine buffer is histidine-histidine hydrochloride buffer; preferably, the concentration of the buffer is 1 to 100mM, 5 to 50mM, 10 to 40mM, 10 to 30mM, 15 to 25mM or about 20mM; preferably, the buffer is selected from histidine-histidine hydrochloride buffer at a concentration of about 20 mM.

In some embodiments of the present application, the pH of the buffer is from 5.0 to 6.5, from 5.5 to 6.0, about 5.0, about 5.5, about 6.0, or about 6.5.

In some embodiments of the present application, the pharmaceutical composition further comprises a stabilizer selected from at least one of arginine, arginine salt, proline, glycine, sodium chloride, mannitol, sorbitol, sucrose, maltose, xylitol, and trehalose; preferably, the concentration of the stabilizing agent in the pharmaceutical composition is 10 to 400mM, 50 to 300mM, 150 to 300mM, 200 to 300mM, 160 to 300mM, 180 to 280mM or about 230mM.

In some embodiments of the present application, the stabilizer is sodium chloride; in the pharmaceutical composition, the concentration of the sodium chloride is 50-200 mM; or the stabilizer is mannitol; in the pharmaceutical composition, the concentration of mannitol is 100-300 mM or 200-300 mM; or the stabilizer is sucrose; the concentration of sucrose in the pharmaceutical composition is 100-300 mM, 150-300 mM, 200-300 mM or about 230mM; or the stabilizer is trehalose; in the pharmaceutical composition, the concentration of trehalose is 100-300 mM, 150-300 mM, 200-300 mM, 160-300 mM, 180-280 mM or about 230mM.

In some embodiments of the present application, the pharmaceutical composition further comprises a surfactant selected from at least one of polysorbate 80, polysorbate 20, and poloxamer 188; preferably, in the pharmaceutical composition, the concentration of the surfactant is 0.01% to 0.1%, 0.01% to 0.08%, 0.01% to 0.06%, 0.01% to 0.05%, 0.01% to 0.04%, 0.02% to 0.08%, 0.02% to 0.06%, 0.02% to 0.05%, 0.02% to 0.04%, about 0.02%, about 0.03%, about 0.04%, or about 0.05% in w/v; preferably, the surfactant is selected from polysorbate 80, and the concentration of polysorbate 80 in the pharmaceutical composition is 0.02% or 0.04% in w/v.

In some embodiments of the present application, the pharmaceutical composition comprises the components shown below:

(a) 5-90 mg/mL of said HUR33 variant and 5-90 mg/mL of said CB6 variant; (b) 10-30 mM histidine buffer, pH 5.5-6.5; (c) 150-300 mM trehalose; and (d) polysorbate 80 at a concentration of 0.01% -0.06% in w/v;

preferably, (a) 5 to 90mg/mL of said HUR33 variant and 5 to 90mg/mL of said CB6 variant; (b) 15-25 mM histidine buffer, pH 5.5-6.0; (c) 160-300 mM trehalose; and (d) polysorbate 80 at a concentration of 0.02% to 0.04% w/v;

More preferably, (a) 10 to 80mg/mL of said HUR33 variant and 10 to 80mg/mL of said CB6 variant; (b) 15-25 mM histidine buffer, pH 5.5-6.0; (c) 180-280 mM trehalose; and (d) polysorbate 80 at a concentration of 0.02% to 0.04% w/v;

still more preferably, (a) about 15mg/mL of said HUR33 variant and about 15mg/mL of said CB6 variant; (b) about 20mM histidine buffer, pH about 5.5; (c) about 230mM trehalose; and (d) polysorbate 80 at a concentration of about 0.04% w/v; or (b)

(a) About 25mg/mL of the HUR33 variant and about 25mg/mL of the CB6 variant; (b) about 20mM histidine buffer, pH about 5.5; (c) about 230mM trehalose; and (d) polysorbate 80 at a concentration of about 0.04% w/v; or (b)

(a) About 40mg/mL of the HUR33 variant and about 40mg/mL of the CB6 variant; (b) about 20mM histidine buffer, pH about 5.5; (c) about 230mM trehalose; and (d) polysorbate 80 at a concentration of about 0.04% w/v; or (b)

(a) About 10mg/mL of the HUR33 variant and about 10mg/mL of the CB6 variant; (b) about 20mM histidine buffer, pH about 5.5; (c) about 230mM trehalose; and (d) polysorbate 80 at a concentration of about 0.04% w/v; or (b)

(a) About 30mg/mL of the HUR33 variant and about 30mg/mL of the CB6 variant; (b) about 20mM histidine buffer, pH about 5.5; (c) about 230mM trehalose; and (d) polysorbate 80 at a concentration of about 0.04% w/v; or (b)

(a) About 20mg/mL of the HUR33 variant and about 20mg/mL of the CB6 variant; (b) about 20mM histidine buffer, pH about 5.5; (c) about 230mM trehalose; and (d) polysorbate 80 at a concentration of about 0.04% w/v.

A second aspect of the present application provides a liquid formulation comprising a pharmaceutical composition as described in the first aspect of the present application.

In some embodiments of the present application, the liquid formulation contains a dextrose solution or sodium chloride solution, and the pharmaceutical composition described in the first aspect of the present 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 neutralizing antibody or antigen binding fragment thereof 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.

A third aspect of the present application provides the use of a pharmaceutical composition according to the first aspect of the present application, a liquid formulation according to the second aspect of the present application, for the manufacture of a medicament for the prevention, treatment, detection or diagnosis of a disease associated with a novel coronavirus infection.

The pharmaceutical composition containing the novel coronavirus neutralizing antibody pharmaceutical composition has the advantages of long-term stability, no aggregation and the like, and can be used for preparing medicines for preventing, treating, detecting or diagnosing diseases related to novel coronavirus infection. The pharmaceutical composition provided by the application can contain two different neutralizing antibodies aiming at novel coronaviruses, simultaneously maintains high stability and strong high temperature tolerance, and has wide neutralizing activity on novel coronavirus wild type and six clinical variants.

Of course, not all of the above-described advantages need be achieved simultaneously in practicing any one of the products or methods of the present application.

Drawings

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

FIG. 1 shows the binding dissociation curves of HUR33 variants and SARS-COV-2 wild-type strain RBD protein in example 4 of the present application;

FIG. 2 shows the binding dissociation curves of the CB6 variant and SARS-COV-2 wild-type strain RBD protein in example 4 of the present application;

FIG. 3 shows the binding curves of the RBD his protein for the experimental and negative control groups in example 5 of the present application.

Detailed Description

For the purposes of making the objects, technical solutions, and advantages of the present application more apparent, the present application will be further described in detail with reference to the accompanying drawings and examples. It will be apparent that the described embodiments are only some, but not all, of the embodiments of the present application. All other technical solutions obtained by a person skilled in the art based on the examples in the present application fall within the scope of protection of the present application.

Definition and description

For easier understanding of the present application, some technical and scientific terms are specifically defined below. Unless otherwise defined explicitly in the present application, 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 in this application, including patents, patent applications, papers, textbooks, and the like, and to the extent that they have not been cited, are hereby incorporated by reference in their entirety. 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, the present application controls.

The term "pharmaceutical composition" or "formulation" means a mixture comprising one or more antibodies described herein 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).

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 6.5" 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 6.5, or a pH in the range of about 5.5 to about 6.0.

In this 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 herein 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 specifically used in the present application is one or more selected from polyols, 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 this application, unless otherwise specified, the terms "concentration of polysorbate 20" and "concentration of polysorbate 80" both refer to the mass volume concentration (w/v), such as "0.04%" in "about 0.04% polysorbate 80" means "0.04 g polysorbate 80" in 100mL of liquid.

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 (incorporated by reference in their entirety herein).

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 the protein dissolved in solution and is typically measured by nephelometry and measured in nephelometry turbidity units.

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 drug quality control agency Command" (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 present 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 antibody contained in the formulation binds to the novel coronavirus with an affinity that is at least 90%, 95% or more than 95% of the binding affinity of the antibody prior to said storage. Binding affinity can also be determined, for example, by ELISA (enzyme-linked immunosorbent assay) or by plasma resonance techniques.

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 human CD39 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 to 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 described herein.

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 "full length antibody" or "whole antibody molecule" refers to an immunoglobulin molecule comprising four peptide chains: two heavy (H) chains (about 50-70 kDa in full length) and two light (L) chains (about 25kDa in full length) are linked to each other by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). 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 heavy and light chain variable region. The exact boundaries of these CDRs are defined differently for different systems.

For the precise amino acid sequence boundaries of the CDRs of an antibody, they can be defined according to well-known methods, e.g., chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops (Chothia et al, nature 342:877-883, 1989; al-Lazikani et al, journal of Molecular Biology,273:927-948, 1997); or Kabat based on antibody sequence variability (Kabat et al, sequences of Proteins of Immunological Interest, 4 th edition, U.S. Pat. No. of Health and Human Services, national Institutes of Health, 1987), abM (University of Bath), contact (University College London), and IMGT (the international ImMunoGeneTics database,1999Nucleic Acids Research,27,209-212); or on the North CDR definition of a neighbor-propagated cluster (affinity propagation clustering) that utilizes a large number of crystal structures. The CDRs of the antibodies herein can be bordered by one of skill in the art according to any protocol in the art (e.g., the optional definition methods described above).

As used herein, an "antigen-binding fragment" includes a fragment of an antibody or a derivative of an antibody, and an 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, bispecific 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. In addition, antigen binding fragments of antibodies may also include unknownsConservative or non-conservative amino acid substitutions (referred to as "conservative variants" or "functional conservative variants" of an antibody) that significantly alter its biological activity.

The terms "specific binding", "selective binding" refer to the 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.

The present application is based on the anti-novel coronavirus monoclonal antibodies described in chinese patent applications CN2022102308454, CN2021116585640 and their cognate applications, on the basis of which the pharmaceutical compositions of the present application were developed, the entire contents of which are incorporated herein by reference.

Pharmaceutical preparation

The present application provides a pharmaceutical composition comprising a novel coronavirus neutralizing antibody or antigen binding fragment thereof; wherein the novel coronavirus neutralizing antibody or antigen binding fragment thereof is selected from at least one of a HUR33 variant and a CB6 variant; the HUR33 variant 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/or LCDR1, LCDR2 and LCDR3 with amino acid sequences shown as SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6 respectively; the CB6 variant comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences shown as SEQ ID NO. 18, SEQ ID NO. 17 and SEQ ID NO. 15 respectively, and/or LCDR1, LCDR2 and LCDR3 with amino acid sequences shown as SEQ ID NO. 21, SEQ ID NO. 16 and SEQ ID NO. 22 respectively or as SEQ ID NO. 19, SEQ ID NO. 16 and SEQ ID NO. 20 respectively. Both the HUR33 variants and CB6 variants described herein can be targeted to bind to the novel coronavirus spike protein S1 subunit.

HUR33 variants comprise three heavy chain complementarity determining regions:

HCDR1：DYGMN(SEQ ID NO:1)；

HCDR2：WINTYSGEPTYADDFRG(SEQ ID NO:2)；

HCDR3：GGNWDWYFDV(SEQ ID NO:3)。

HUR33 variants comprise three light chain complementarity determining regions:

LCDR1：RASQSVSNFLH(SEQ ID NO:4)；

LCDR2：YASQSIS(SEQ ID NO:5)；

LCDR3：QQSNTWPLT(SEQ ID NO:6)。

the CB6 variant comprises three heavy chain complementarity determining regions:

HCDR1：GFVVQANY(SEQ ID NO:18)；

HCDR2：IYPGGST(SEQ ID NO:17)；

HCDR3：ARVLPMYGDYLDY(SEQ ID NO:15)。

the CB6 variant comprises three light chain complementarity determining regions:

LCDR1：QNIERY(SEQ ID NO:21)；

LCDR2：AAS(SEQ ID NO:16)；

LCDR3: QQSASSTPEYT (SEQ ID NO: 22); or (b)

LCDR1：QNILPY(SEQ ID NO:19)；

LCDR2：AAS(SEQ ID NO:16)；

LCDR3：QQSSSAVPEYT(SEQ ID NO:20)。

In the present application, the novel coronavirus neutralizing antibody or antigen binding fragment thereof is selected from at least one of HUR33 variant and CB6 variant; refers to novel coronavirus neutralizing antibodies or antigen binding fragments thereof selected from the group consisting of: (1) any one of the HUR33 variants; (2) any one of the CB6 variants; (3) any plurality of HUR33 variants; (4) any plurality of CB6 variants; or (5) a mixture of any one or more of the HUR33 variants with any one or more of the CB6 variants.

Specifically, the novel coronavirus neutralizing antibody or antigen binding fragment thereof is selected from at least one of HUR33 variant and CB6 variant, and at least comprises the following scheme:

(1) The novel coronavirus neutralizing antibody or antigen binding fragment thereof is HUR33 variant, and the HUR33 variant 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/or LCDR1, LCDR2 and LCDR3 with amino acid sequences shown as SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6 respectively;

(2) The novel coronavirus neutralizing antibody or antigen binding fragment thereof is a CB6 variant, wherein the CB6 variant comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences shown as SEQ ID NO. 18, SEQ ID NO. 17 and SEQ ID NO. 15 respectively, and/or LCDR1, LCDR2 and LCDR3 with amino acid sequences shown as SEQ ID NO. 21, SEQ ID NO. 16 and SEQ ID NO. 22 respectively;

(3) The novel coronavirus neutralizing antibody or antigen binding fragment thereof is a CB6 variant, wherein the CB6 variant comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences shown as SEQ ID NO. 18, SEQ ID NO. 17 and SEQ ID NO. 15, and/or LCDR1, LCDR2 and LCDR3 with amino acid sequences shown as SEQ ID NO. 19, SEQ ID NO. 16 and SEQ ID NO. 20 respectively;

(4) The novel coronavirus neutralizing antibody or antigen binding fragment thereof is a mixture of HUR33 variant and CB6 variant, wherein the HUR33 variant 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/or LCDR1, LCDR2 and LCDR3 with amino acid sequences shown as SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6 respectively; the CB6 variant comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences shown as SEQ ID NO. 18, SEQ ID NO. 17 and SEQ ID NO. 15 respectively, and/or LCDR1, LCDR2 and LCDR3 with amino acid sequences shown as SEQ ID NO. 21, SEQ ID NO. 16 and SEQ ID NO. 22 respectively;

(5) The novel coronavirus neutralizing antibody or antigen binding fragment thereof is a mixture of HUR33 variant and CB6 variant, wherein the HUR33 variant 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/or LCDR1, LCDR2 and LCDR3 with amino acid sequences shown as SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6 respectively; the CB6 variant comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences shown as SEQ ID NO. 18, SEQ ID NO. 17 and SEQ ID NO. 15, and/or LCDR1, LCDR2 and LCDR3 with amino acid sequences shown as SEQ ID NO. 19, SEQ ID NO. 16 and SEQ ID NO. 20 respectively;

(6) The novel coronavirus neutralizing antibody or antigen binding fragment thereof is a mixture of different CB6 variants, wherein the first CB6 variant comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences shown as SEQ ID NO. 18, SEQ ID NO. 17 and SEQ ID NO. 15 respectively and/or LCDR1, LCDR2 and LCDR3 with amino acid sequences shown as SEQ ID NO. 21, SEQ ID NO. 16 and SEQ ID NO. 22 respectively; the second CB6 variant comprises HCDR1, HCDR2 and HCDR3 having amino acid sequences shown in SEQ ID NO. 18, SEQ ID NO. 17 and SEQ ID NO. 15, and/or LCDR1, LCDR2 and LCDR3 having amino acid sequences shown in SEQ ID NO. 19, SEQ ID NO. 16 and SEQ ID NO. 20, respectively.

In some embodiments of the present application, the HUR33 variants used in the pharmaceutical compositions of the present application comprise a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO. 8 or SEQ ID NO. 9, and/or a light chain variable region having an amino acid sequence as set forth in SEQ ID NO. 7; the CB6 variant comprises a heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 23 and/or a light chain variable region with an amino acid sequence shown as SEQ ID NO. 25 or SEQ ID NO. 24.

HUR33 variant comprises a heavy chain variable region:

QVQLVQSGSELKKPGASVKVSCKASGYTFTDYGMNWVRQAPGQGLEWMGWINTYSGEPTYAD DFRGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARGGNWDWYFDVWGQGTLVTVSS (SEQ ID NO: 8); or (b)

QVQLVQSGSELKKPGASVKVSCKASGYTFTDYGMNWVRQAPGQGLEWMGWINTYSGEPTYAD DFRGRFVFSLDTSVSTAYLQINSLKAEDTAVYYCARGGNWDWYFDVWGQGTLVTVSS(SEQ ID NO:9)。

HUR33 variant comprises a light chain variable region:

DIQMTQSPSSLSASVGDRVTITCRASQSVSNFLHWYQQKPGKAPKLLIYYASQSISGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCQQSNTWPLTFGQGTKLEIK(SEQ ID NO:7)。

the CB6 variant comprises a heavy chain variable region:

EVQLVESGGGLVQPGGSLRLSCAASGFVVQANYMSWVRQAPGKGLEWVSVIYPGGSTFYADSVK GRFTISRDNSMNTLFLQMNSLRAEDTAVYYCARVLPMYGDYLDYWGQGTLVTVSS (SEQ ID NO: 23). The CB6 variant comprises a light chain variable region:

DIVMTQSPSSLSASVGDRVTITCRASQNIERYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCQQSASSTPEYTFGQGTKLEIK (SEQ ID NO: 25); or (b)

DIVMTQSPSSLSASVGDRVTITCRASQNILPYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCQQSSSAVPEYTFGQGTKLEIK(SEQ ID NO:24)。

In some embodiments of the present application, the HUR33 variants used in the pharmaceutical compositions of the present application further comprise a heavy chain constant region having the amino acid sequence shown in SEQ ID NO. 13 and a light chain constant region having the amino acid sequence shown in SEQ ID NO. 14.

HUR33 variants comprise a heavy chain constant region (IgG 1 heavy chain constant region):

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:13)。

HUR33 variants comprise a light chain constant region (IgG 1 light chain constant region):

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECS(SEQ ID NO:14)。

in some embodiments of the present application, the HUR33 variants used in the pharmaceutical compositions of the present application comprise a heavy chain having an amino acid sequence as shown in SEQ ID NO. 10 or SEQ ID NO. 11, and/or a light chain having an amino acid sequence as shown in SEQ ID NO. 12; the CB6 variant comprises a heavy chain with an amino acid sequence shown as SEQ ID NO. 26 and/or a light chain with an amino acid sequence shown as SEQ ID NO. 27 or SEQ ID NO. 28.

HUR33 variant comprises a heavy chain:

QVQLVQSGSELKKPGASVKVSCKASGYTFTDYGMNWVRQAPGQGLEWMGWINTYSGEPTYADDFRGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARGGNWDWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 10); or (b)

QVQLVQSGSELKKPGASVKVSCKASGYTFTDYGMNWVRQAPGQGLEWMGWINTYSGEPTYADDFRGRFVFSLDTSVSTAYLQINSLKAEDTAVYYCARGGNWDWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:11)。

HUR33 variant comprises a light chain:

DIQMTQSPSSLSASVGDRVTITCRASQSVSNFLHWYQQKPGKAPKLLIYYASQSISGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNTWPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:12)。

the CB6 variant comprises a heavy chain:

EVQLVESGGGLVQPGGSLRLSCAASGFVVQANYMSWVRQAPGKGLEWVSVIYPGGSTFYADSVKGRFTISRDNSMNTLFLQMNSLRAEDTAVYYCARVLPMYGDYLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:26)。

The CB6 variant comprises a light chain:

DIVMTQSPSSLSASVGDRVTITCRASQNIERYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSASSTPEYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 27); or (b)

DIVMTQSPSSLSASVGDRVTITCRASQNILPYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSSSAVPEYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:28)。

In some embodiments of the present application, the mass ratio of the HUR33 variant and the CB6 variant used in the pharmaceutical composition of the present application is (1-10): (10-1), about 1: 1. about 1: 2. about 1: 3. about 1: 4. about 1: 5. about 2: 1. about 2: 3. about 3: 1. about 3: 2. about 4:1 or about 5:1. the mass ratio of the HUR33 variant and the CB6 variant may be in a range formed by any two of the ratios described above as endpoints.

In some embodiments of the present application, the novel coronavirus neutralizing antibody or antigen-binding fragment thereof has a concentration of about 1 to 300mg/mL, 10 to 200mg/mL, 20 to 150mg/mL, 30 to 120mg/mL, 30 to 100mg/mL, 20 to 100mg/mL, 5 to 90mg/mL, 5 to 85mg/mL, 10 to 90mg/mL, 10 to 85mg/mL, 20 to 90mg/mL, 20 to 85mg/mL, 30 to 90mg/mL, 30 to 85mg/mL, 40 to 90mg/mL, 40 to 85mg/mL, 50 to 90mg/mL, 50 to 85mg/mL, 60 to 90mg/mL, 10 to 80mg/mL, 20 to 80mg/mL, 30 to 80mg/mL, 40 to 80mg/mL, 50 to 80mg/mL, 10 to 60mg/mL, 20 to 60mg/mL, 30 to 60mg/mL, 10 to 50mg/mL, about 40 to about 40mg/mL, about 40 mg/mL. The concentration of the novel coronavirus-neutralizing antibody or antigen-binding fragment thereof may be about 1mg/mL, 5mg/mL, 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, 150mg/mL, 160mg/mL, 180mg/mL, 200mg/mL, 220mg/mL, 250mg/mL, 300mg/mL, or a range formed by any two of the foregoing as endpoints.

In some embodiments of the present application, the pharmaceutical composition further comprises a buffer selected from at least one of an acetate buffer, a citrate buffer, and a histidine buffer; preferably, the histidine buffer is histidine-histidine hydrochloride buffer; preferably, the concentration of the buffer is 1 to 100mM, 5 to 50mM, 10 to 40mM, 10 to 30mM, 15 to 25mM or about 20mM; preferably, the buffer is selected from histidine-histidine hydrochloride buffer at a concentration of about 20 mM. The concentration of the buffer may be about 1mM, 5mM, 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM or any two of the above ranges. The solvent used in the buffers described herein is water unless otherwise specified. The concentration of the buffer as described herein refers to the concentration of the buffer substance in the pharmaceutical composition, unless otherwise specified.

In some embodiments of the present application, the pH of the buffer is from 5.0 to 6.5, from 5.5 to 6.0, about 5.0, about 5.5, about 6.0, or about 6.5. The pH of the buffer may be about 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 endpoints.

In some embodiments of the present application, 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 of the present application, 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 of the present application, the citric acid buffer is a citric acid-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 of the present application, the pharmaceutical composition further comprises a stabilizer selected from at least one of arginine, arginine salt, proline, glycine, sodium chloride, mannitol, sorbitol, sucrose, maltose, xylitol, and trehalose; preferably, the concentration of the stabilizing agent in the pharmaceutical composition is 10 to 400mM, 50 to 300mM, 150 to 300mM, 200 to 300mM, 160 to 300mM, 180 to 280mM or about 230mM. The concentration of the above-mentioned stabilizer may be about 10mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 110mM, 120mM, 130mM, 140mM, 150mM, 160mM, 170mM, 180mM, 190mM, 200mM, 210mM, 220mM, 230mM, 240mM, 250mM, 260mM, 270mM, 280mM, 290mM, 300mM, 310mM, 320mM, 330mM, 340mM, 350mM, 360mM, 370mM, 380mM, 390mM, 400mM or a range formed by any two of the above-mentioned values as the end points. The concentration of the stabilizer as described herein refers to the concentration of the stabilizer in the pharmaceutical composition, unless otherwise specified.

In some embodiments of the present application, the stabilizer is sodium chloride; in the pharmaceutical composition, the concentration of the sodium chloride is 50-200 mM. The concentration of sodium chloride may be about 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 110mM, 120mM, 130mM, 140mM, 150mM, 160mM, 170mM, 180mM, 190mM, 200mM or any two of the above ranges.

In some embodiments of the present application, the stabilizer is mannitol; in the pharmaceutical composition, the concentration of mannitol is 100-300 mM or 200-300 mM. The concentration of mannitol may be about 100mM, 110mM, 120mM, 130mM, 140mM, 150mM, 160mM, 170mM, 180mM, 190mM, 200mM, 210mM, 220mM, 230mM, 240mM, 250mM, 260mM, 270mM, 280mM, 290mM, 300mM or any two of the above ranges.

In some embodiments of the present application, the stabilizing agent is sucrose; in the pharmaceutical composition, the concentration of sucrose is about 100 to 300mM, 150 to 300mM, 200 to 300mM or about 230mM. The concentration of sucrose may be about 100mM, 110mM, 120mM, 130mM, 140mM, 150mM, 160mM, 170mM, 180mM, 190mM, 200mM, 210mM, 220mM, 230mM, 240mM, 250mM, 260mM, 270mM, 280mM, 290mM, 300mM or any two of the above ranges.

In some embodiments of the present application, the stabilizing agent is trehalose; in the pharmaceutical composition, the trehalose is at a concentration of about 100 to 300mM, 150 to 300mM, 200 to 300mM, 160 to 300mM, 180 to 280mM, or about 230mM. The concentration of trehalose may be about 100mM, 110mM, 120mM, 130mM, 140mM, 150mM, 160mM, 170mM, 180mM, 190mM, 200mM, 210mM, 220mM, 230mM, 240mM, 250mM, 260mM, 270mM, 280mM, 290mM, 300mM or any two of the above ranges.

In some embodiments of the present application, the stabilizer is arginine or an arginine salt; preferably, the arginine salt is arginine hydrochloride. The concentration of arginine or arginine salt is 100-300 mM, 120-280 mM or 120-160 mM. The concentration of the arginine or arginine salt may be about 100mM, 110mM, 120mM, 130mM, 140mM, 150mM, 160mM, 170mM, 180mM, 190mM, 200mM, 210mM, 220mM, 230mM, 240mM, 250mM, 260mM, 270mM, 280mM, 290mM, 300mM.

In some embodiments of the present application, the stabilizer is sorbitol. In some embodiments, the concentration of sorbitol is 100 to 300mM, 200 to 300mM, or 220 to 250mM. The concentration of sorbitol may be 100mM, 110mM, 120mM, 130mM, 140mM, 150mM, 160mM, 170mM, 180mM, 190mM, 200mM, 210mM, 220mM, 230mM, 240mM, 250mM, 260mM, 270mM, 280mM, 290mM, 300mM or any two of the above ranges.

In some embodiments of the present application, the stabilizer is a combination of sodium chloride and mannitol. In some embodiments, the above-described stabilizer is a combination of 20-200 mM sodium chloride and 20-200 mM mannitol, or a combination of 30-100 mM sodium chloride and 100-180 mM mannitol, or a combination of 30-70 mM sodium chloride and 120-160 mM mannitol, or a combination of about 50mM sodium chloride and about 140mM, 145mM, or 150mM mannitol.

In some embodiments of the present application, the stabilizer is arginine hydrochloride in combination with sucrose. In some embodiments, the stabilizer is a combination of 20-200 mM arginine hydrochloride and 20-200 mM sucrose, or a combination of 20-80 mM arginine hydrochloride and 100-180 mM sucrose, or a combination of 30-70 mM arginine hydrochloride and 100-160 mM sucrose, or a combination of about 50mM arginine hydrochloride and about 120mM, 125mM, 130mM, or 135mM sucrose.

In some embodiments of the present application, the stabilizer is a combination of arginine hydrochloride and glycine. In some embodiments, the stabilizer is a combination of 20-200 mM arginine hydrochloride and 20-200 mM glycine, or a combination of 20-80 mM arginine hydrochloride and 100-180 mM glycine, or a combination of 30-70 mM arginine hydrochloride and 100-120 mM glycine, or a combination of about 50mM arginine hydrochloride and about 100mM, 105mM, 110mM, or 115mM glycine.

In some embodiments of the present application, the stabilizer is a combination of sodium chloride and sucrose. In some embodiments, the stabilizer is a combination of 20-200 mM sodium chloride and 20-200 mM sucrose, or a combination of 20-100 mM sodium chloride and 100-180 mM sucrose, or a combination of 30-70 mM sodium chloride and 120-160 mM sucrose, or a combination of about 50mM sodium chloride and about 120mM, 125mM, 130mM, or 135mM sucrose.

In some embodiments of the present application, the stabilizing agent is a combination of sodium chloride and trehalose. In some embodiments, the stabilizer is a combination of 20-200 mM sodium chloride and 20-200 mM trehalose, or a combination of 20-80 mM sodium chloride and 100-180 mM trehalose, or a combination of 30-70 mM sodium chloride and 120-160 mM trehalose, or a combination of about 50mM sodium chloride and about 120mM, 130mM, or 140mM trehalose.

In some embodiments of the present application, the pharmaceutical composition further comprises a surfactant selected from at least one of polysorbate 80, polysorbate 20, and poloxamer 188; preferably, in the pharmaceutical composition, the concentration of the surfactant is 0.01% to 0.1%, 0.01% to 0.08%, 0.01% to 0.06%, 0.01% to 0.05%, 0.01% to 0.04%, 0.02% to 0.08%, 0.02% to 0.06%, 0.02% to 0.05%, 0.02% to 0.04%, about 0.02%, about 0.03%, about 0.04%, or about 0.05% in w/v; preferably, the surfactant is selected from polysorbate 80, and the concentration of polysorbate 80 in the pharmaceutical composition is 0.02% or 0.04% in w/v. The concentration of the surfactant may be about 0.01%, 0.02%, 0.04%, 0.06%, 0.08%, 0.1% or any two of the above values as the endpoints. The concentration of surfactant as described herein refers to the concentration of surfactant in the pharmaceutical composition, unless otherwise specified.

In some embodiments of the present application, the pharmaceutical composition of the present application comprises the components shown below:

still more preferably, (a) 15mg/mL of said HUR33 variant and 15mg/mL of said CB6 variant; (b) 20mM histidine buffer, pH 5.5; (c) 230mM trehalose; and (d) polysorbate 80 at a concentration of 0.04% w/v; or (b)

(a) About 40mg/mL of the HUR33 variant and about 40mg/mL of the CB6 variant; (b) about 20mM histidine buffer, pH about 5.5; (c) 2 about 30mM trehalose; and (d) polysorbate 80 at a concentration of about 0.04% w/v; or (b)

The method of preparing the pharmaceutical composition is not particularly limited as long as the components in the pharmaceutical composition to be prepared are within the ranges described herein. For example, using Millipore for HUR33 variant stock samples and/or CB6 variant stock samples 0.11m ² Ultrafiltration concentration (UF/DF) is performed to a concentration of 1-2 times the preset final concentration of the HUR33 variant and/or CB6 variant, followed by dialysis into a buffer containing a stabilizer. Measurement after dialysis is completedAnd (3) determining the concentration of the HUR33 variant and/or the CB6 variant, adding a buffer solution containing a stabilizing agent and a surfactant according to the determined concentration, adjusting the final concentration of the HUR33 variant and/or the CB6 variant to a preset final concentration, and controlling the concentrations of other components to be corresponding preset final concentrations to obtain the pharmaceutical composition.

In yet another aspect, the present application provides a liquid formulation comprising the pharmaceutical composition described herein; preferably, the liquid formulation is an injection.

In some embodiments of the present application, the liquid formulation contains a dextrose solution or sodium chloride solution, and the pharmaceutical composition described herein; 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 neutralizing antibody or antigen binding fragment thereof 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 as used herein refers to the concentration of sodium chloride in the sodium chloride solution and the concentration of the glucose solution refers to the concentration of glucose in the glucose solution unless otherwise specified.

Medical uses and methods

In yet another aspect, the present application provides the use of a pharmaceutical composition or liquid formulation described herein in a medicament for preventing, treating, detecting or diagnosing a disease associated with a novel coronavirus (SARS-CoV-2) infection.

In yet another aspect, the present application provides the use of a pharmaceutical composition or liquid formulation described herein to prevent or treat a novel coronavirus infection by eliminating, inhibiting or reducing the activity of the novel coronavirus.

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

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

In yet another aspect, the present application also provides a combination therapy comprising administering to a subject a therapeutically effective amount of a pharmaceutical composition, lyophilized formulation or liquid formulation of any of the embodiments of the present application in combination with one or more other therapeutic agents to treat a disease caused by a novel coronavirus 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 present application also provides a method of reducing systemic or local viral load in a subject infected with a novel coronavirus comprising administering to the subject an effective amount of a pharmaceutical composition or liquid formulation of any of the embodiments of the present application.

In a further aspect, any of the pharmaceutical compositions or liquid formulations described herein before may also be used for the preparation of a medicament or formulation for the prevention, treatment, detection or diagnosis of a disease associated with a novel coronavirus.

The novel coronaviruses described herein, including novel coronavirus wild-type and/or mutant strains, comprise at least one of an Alpha (Alpha) mutant, a Beta (Beta) mutant, a Gamma (Gamma) mutant, a Delta (Delta) mutant, an Epsilon mutant, a Zeta mutant, an Eta mutant, a Theta mutant, an Iota mutant, a Kappa (Kappa) mutant, a Mu (Mu) mutant, and an ommicon (Omicron) mutant, preferably at least one of an Alpha mutant, a Beta mutant, a Gamma mutant, a Kappa mutant, an Epsilon mutant, and a Delta mutant.

Examples

The present 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 present application. Any modifications, equivalent substitutions, improvements, etc. which will be apparent to those skilled in the art with respect to the present embodiments without departing from the spirit and scope of the present application, are intended to be included within the scope of the present application. The experimental method without specific conditions being noted in the examples of the present application is generally carried out according to conventional conditions, such as an antibody technical laboratory manual, a molecular cloning manual, etc. of cold spring harbor; or according to the conditions recommended by the manufacturer of the raw materials or goods. The reagents of specific origin are not noted and are commercially available conventional reagents.

The amino acid sequence of the heavy chain of the HUR33 variant used in the examples below is shown in SEQ ID NO. 10, and the amino acid sequence of the light chain is shown in SEQ ID NO. 12; the heavy chain of the CB6 variant has an amino acid sequence shown in SEQ ID NO. 26, and the light chain has an amino acid sequence shown in SEQ ID NO. 27.

Preparation of HUR33 variants and CB6 variants: constructing pCAGGS vectors according to the amino acid sequences of the light chain and the heavy chain of the HUR33 variant and the CB6 variant after complete gene synthesis to obtain complete light chain expression plasmids and heavy chain expression plasmids of the HUR33 variant and the CB6 variant respectively; then, the plasmid is transfected into cells and then cultured to obtain target protein; specifically: the heavy and light chain expression plasmids of the HUR33 variant were expressed as 2:3, co-transfecting 293T cells in proportion, and after 4-6 hours of transfection, washing the cells for 2 times by PBS, and culturing by adopting a serum-free DMEM culture medium; cell supernatants were collected on days 3 and 7 after transfection, centrifuged to remove cell debris, and the antibody supernatants obtained in the two steps were mixed and purified to obtain the desired HUR33 variant and CB6 variant, respectively.

In examples 4 to 5 below, pharmaceutical composition FS5-1 was used as a test subject, and the pharmaceutical composition was diluted with the diluents corresponding to each example to bring the concentrations of HUR33 variant and CB6 variant to the corresponding values before the test.

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

w represents a week;

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

HPLC means high performance liquid chromatography;

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;

NEM represents ethylmaleimide;

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

EC ₅₀ representing the half maximum effect concentration;

PBS represents phosphate buffer.

Example 1: stability test of pharmaceutical compositions comprising HUR33 variants

1.1 Experimental sample

The HUR33 variant sample (HUR 33-1) was subjected to Millipore0.11m ² Ultrafiltration concentration (UF/DF) was performed to a concentration of about 60mg/mL or 40mg/mL, respectively, followed by dialysis into the corresponding stabilizer-containing buffer. And respectively measuring the concentration of the HUR33 variants, respectively adding a corresponding buffer solution containing a stabilizing agent and a surfactant according to the measured concentrations, respectively adjusting the final concentration of the HUR33 variants to 50mg/mL, 30mg/mL, 20mg/mL and 10mg/mL, and respectively obtaining different pharmaceutical compositions to be tested according to the content of other components as shown in the table 1.

And filling the newly prepared different to-be-tested pharmaceutical compositions into 2R penicillin bottles in a sterile mode, wherein FS1-1 to FS1-5 are 0.5 mL/bottle, FS2-1 to FS2-4 are 2 mL/bottle, 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: 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 2W and 4W, hereinafter referred to simply as "long term (5.+ -. 3 ℃);

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

1.2.1 appearance test

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 line of sight, gently shaking or inverting to avoid 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.

Test results: the appearance test results are shown in table 2.

Table 2 appearance test results

As can be seen from the results of Table 2 above, the pharmaceutical compositions FS1-1 to FS1-5 were left for 4 weeks under high temperature conditions, and no apparent visible foreign matter was found, and no apparent opalescence was observed; after 4 weeks of standing under long term conditions, the appearance of opalescence was accentuated compared to T0, and FS1-3 and FS1-4 appeared more granular.

The pharmaceutical compositions FS2-1 to FS2-4 are placed for 4 weeks under the conditions of high temperature and long time, no obvious visible foreign matters are found in the FS2-1 and the FS2-2, no obvious opalescence is found in the FS2-3 and the FS2-4, the opalescence is aggravated after the pharmaceutical compositions are placed for 2 weeks, and the appearance is not obviously different from that of the pharmaceutical compositions after the pharmaceutical compositions are placed for 2 weeks.

1.2.2SEC-HPLC purity test

TestingThe method comprises the following steps: the column was used with a SEC column (Waters XBridge BEH,7.8 mm. Times.30 cm,3.5 μm,) The SEC purity was checked by HPLC (Waters e2695 instrument). Diluting each medicine composition (sample) to 4.0mg/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 consisted of a 50mM phosphate solution (containing 300mM sodium chloride) pH 6.8.+ -. 0.2. The relative percentages of main peak, polymer and fragment were calculated using area normalization. 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 3 below:

TABLE 3 SEC-HPLC purity test parameters

Test results: the SEC-HPLC purity test results are shown in Table 4.

Table 4 SEC-HPLC purity test results (%)

/>

Note that: "/" represents not measured

As can be seen from the results of Table 4 above, the pharmaceutical compositions FS1-1 to FS1-5 were left at high temperature for 4 weeks, and the SEC-HPLC purity was reduced, mainly the polymer was increased; wherein the purity of the FS1-1 is reduced most rapidly, and other pharmaceutical compositions have no obvious difference; after 4 weeks of standing under long term conditions, no significant change in SEC-HPLC purity occurred for all pharmaceutical compositions.

The pharmaceutical compositions FS2-1 to FS2-4 are placed for 4 weeks under the high temperature condition, the SEC-HPLC purity is slightly reduced, and no obvious difference exists between different pharmaceutical compositions; no significant change in SEC-HPLC purity occurred over 4 weeks of standing under long term conditions.

1.2.3CEX-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, uniformly mixing, adding into a lining pipe, and carrying out sample injection analysis; the mobile phase composition is phase a: 20mM MES solution (pH 6.20.+ -. 0.02); and B phase: 20mM MES+100mM NaCl solution (pH 6.20.+ -. 0.02). And calculating the percentages of the main peak, the acid peak and the alkaline peak by adopting a peak area normalization method. 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 5 below:

TABLE 5CEX-HPLC purity test parameters

Test results: the results of the CEX-HPLC purity test are shown in Table 6.

TABLE 6CEX-HPLC purity test results (%)

Note that: "/" represents not measured

As can be seen from the results of Table 6 above, the pharmaceutical compositions FS1-1 to FS1-5 were left at high temperature for 4 weeks, and the CEX-HPLC purity was significantly reduced, mainly due to the increase of the alkali peak; wherein the purity of the FS1-1 is reduced rapidly, and other pharmaceutical compositions have no obvious difference; after 4 weeks of long-term storage, all the pharmaceutical compositions were unchanged significantly.

The pharmaceutical compositions FS2-1 to FS2-4 are placed for 4 weeks under the condition of high temperature, the purity of CEX-HPLC is reduced, and no obvious difference exists between the pharmaceutical compositions; after 4 weeks of standing under long-term conditions, the CEX-HPLC purity of all the pharmaceutical compositions was not significantly changed.

1.2.4rCE-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 pretreatment method and the test method of the reference product are the same as those of the sample.

Test results: the results of rCE-SDS purity test are shown in Table 7.

TABLE 7 rCE-SDS purity test results (%)

Note that: "/" represents not measured

As can be seen from the results of Table 7, the pharmaceutical compositions FS1-1 to FS1-5 were left at high temperature for 4 weeks, and the rCE-SDS purity was decreased, and there was no significant difference between all the pharmaceutical compositions; after 4 weeks of long-term standing, no obvious change in rCE-SDS purity occurred.

The pharmaceutical compositions FS2-1 to FS2-4 are placed for 4 weeks under the high temperature condition, the purity of rCE-SDS is reduced, and no obvious difference exists among all the pharmaceutical compositions; after being placed for 4 weeks under long-term conditions, the purity is not obviously changed.

1.2.5nrCE-SDS purity test

The testing method comprises the following steps: the nrCE-SDS purity was detected using a capillary electrophoresis apparatus (Maurice apparatus). The amount of diluent and sample to be added when diluting each pharmaceutical composition (sample) to 1mg/mL was calculated as 100. Mu.L of the final volume, and then the diluted solution (1 wt% SDS+40mM phosphate buffer, pH 6.5), 5.0. Mu.L of 0.25M NEM, and the sample were added in this order to the 1.5mL EP tube and mixed well. 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. 75 mu L of the sample solution was taken out of the sample tube to a 96-well plate, and the sample solution was centrifuged at 1000g at room temperature for 10min to avoid air bubbles, and detected by a capillary electrophoresis apparatus (Maurice apparatus). 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 Table 8.

TABLE 8 nrCE-SDS purity test results (%)

Note that: "/" represents not measured

As can be seen from the results of Table 8, the purity of the nrCE-SDS was significantly reduced by allowing the pharmaceutical compositions FS1-1 to FS1-5 to stand at high temperature for 4 weeks, and there was no significant difference between all the pharmaceutical compositions; no significant change in nrCE-SDS purity occurred after 4 weeks of long-term storage.

The purity of the nrCE-SDS is reduced after the pharmaceutical compositions FS2-1 to FS2-4 are placed for 4 weeks under the high temperature condition, and no obvious difference exists among all pharmaceutical composition groups; no significant change in nrCE-SDS purity occurred after 4 weeks of standing under long term conditions.

1.2.6 binding Activity test

The testing method comprises the following steps: the plates were wrapped with His-tagged RBD protein solution (self-made by the monarch, suzhou) at a concentration of 3.0 μg/mL and incubated at 37℃for 90min; plates were washed and blocked with PBS containing 2% (w/v) skimmed milk; HUR33 variants were added in a gradient of PBS containing 2% (w/v) skim milk (HUR 33 variants were diluted to 40. Mu.g/mL with PBS containing 2% (w/v) skim milk, then diluted in a 4-fold gradient to a final concentration of 0.009537 ng/mL) and incubated at 37℃for 60min. Detection was performed with 5000-fold dilution of goat anti-human IgG antibody (Fc-specific, coupled peroxidase) (manufacturer: sigma: A0170) as detection antibody, incubation at 37℃for 60min, followed by development with 0.1mg/mL TMB (manufacturer: sigma: T2885), termination of the reaction with 2M HCl after 15min, reading of the plate at 450nm/620nm, followed by four-parameter logistic regression (4 PL) model fitting curve using GraphPad Prism software, giving the EC of the experimental group ₅₀ (half maximum effect concentration) value. Binding activity (relative activity,%) =ec of reference substance ₅₀ EC of pharmaceutical composition ₅₀ ×100％。

Test results: the binding activity test results are shown in Table 9.

Table 9 binding Activity test results (%)

Note that: "/" represents not measured

From the results of Table 9 above, it is clear that the binding activity of the pharmaceutical compositions FS1-1 to FS1-5 did not change significantly when left for 4 weeks under high temperature conditions and long term conditions.

The pharmaceutical compositions FS2-1 to FS2-4 were left for 4 weeks under high temperature conditions and long term conditions without significant changes in the binding activity of all the pharmaceutical compositions.

1.2.7 sub-visible particle test

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 10 below.

Table 10 sub-visible particle test parameters

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

Test results: the sub-visible particle test results are shown in table 11. 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 11 sub-visible particle test results (Unit: particles/mL)

From the results of Table 11 above, it is clear that the pharmaceutical compositions FS2-1 to FS2-4 were left for 4 weeks under high temperature conditions and long term conditions, and that no significant changes in the sub-visible particles of all the pharmaceutical compositions were observed.

From the above, it is clear from the results of FS1-1 to FS1-5 that there is no significant difference between all the pharmaceutical compositions in terms of appearance, rCE-SDS, nCE-SDS and cell activity at high temperature; the results of SEC-HPLC purity and CEX main peak purity show that the FS1-1 purity is relatively fast to drop, and other pharmaceutical compositions have no obvious difference; under long-term conditions, FS1-1, FS1-2 and FS1-5 are relatively superior in appearance, and other pharmaceutical compositions have no obvious difference, and all other pharmaceutical compositions are detected to have no abnormality. Overall, the pharmaceutical compositions FS1-2 and FS1-5 are more stable than other pharmaceutical compositions.

As can be seen from the results of FS2-1 to FS2-4, no significant differences were observed between all the pharmaceutical compositions from the results of SEC-HPLC, CEX-HPLC, rCE-SDS, nrCE-SDS, binding activity and sub-visible particles, and from the appearance, no further enhancement of the opalescence was observed in the 4-week samples at long or high temperatures at concentrations of 30mg/mL for the HUR33 variants in FS2-3 and FS2-4, and no significant differences were observed in the two groups. Overall, the stability of the pharmaceutical composition FS2-3 is better than other pharmaceutical compositions.

Example 2: stability test of pharmaceutical compositions comprising CB6 variants

2.1 Experimental sample

Variant CB6Sample (CB 638) using Millipore0.11m ² Ultrafiltration concentration (UF/DF) was performed to a concentration of about 60mg/mL or 40mg/mL, respectively, followed by dialysis into the corresponding stabilizer-containing buffer. And respectively measuring the concentration of the HUR33 variants, respectively adding a corresponding buffer solution containing a stabilizing agent and a surfactant according to the measured concentrations, respectively adjusting the final concentration of the HUR33 variants to 50mg/mL, 30mg/mL, 20mg/mL and 10mg/mL, and respectively obtaining different pharmaceutical compositions to be tested according to the content of other components as shown in the table 12.

And filling the newly prepared different to-be-tested pharmaceutical compositions into 2R penicillin bottles in a sterile mode, wherein FS3-1 to FS3-4 are 0.5 mL/bottle, FS4-1 to FS4-4 are 2 mL/bottle, and then carrying out stability lofting and detection according to stability investigation conditions. The components of the different pharmaceutical compositions tested are shown in table 12, wherein: histidine buffer refers to L-histidine-L-histidine hydrochloride buffer.

TABLE 12 different compositions of the test pharmaceutical compositions

2.2 Experimental methods and results

The stability investigation conditions included:

2.2.1 appearance test

The testing method comprises the following steps: and the same as 1.2.1 appearance test.

Test results: the appearance test results are shown in table 13.

TABLE 13 appearance test results

As can be seen from the results of Table 13, the pharmaceutical compositions FS3-1 to FS3-4 were left for 4 weeks under high temperature conditions, and no apparent visible foreign matters and no apparent opalescence were found; after 4 weeks of standing under long term conditions, all pharmaceutical compositions appeared to have more particles than T0, and FS3-3 appeared to have a large number of fine sand-like particles.

The pharmaceutical compositions FS4-1 to FS4-4 were left for 4 weeks under high temperature and long term conditions without visible foreign matter and opalescence.

2.2.2SEC-HPLC purity test

The testing method comprises the following steps: and 1.2.2SEC-HPLC purity.

Test results: the SEC-HPLC purity test results are shown in Table 14.

Table 14 SEC-HPLC purity test results (%)

Note that: "/" represents not measured

As can be seen from the results in Table 14, the pharmaceutical compositions FS3-1 to FS3-4 were left at high temperature for 4 weeks, and the SEC-HPLC purity was reduced, mainly due to the increase of the fragment peaks, without significant differences between all the pharmaceutical compositions; after 4 weeks of standing under long term conditions, no significant change in SEC-HPLC purity occurred for all pharmaceutical compositions.

The purity of the SEC-HPLC is reduced after the pharmaceutical compositions FS4-1 to FS4-4 are placed for 4 weeks under the high temperature condition, and no obvious difference exists between different pharmaceutical compositions; no significant change in SEC-HPLC purity occurred over 4 weeks of standing under long term conditions.

2.2.3CEX-HPLC purity test

The testing method comprises the following steps: by using a column equipped with a cation chromatography column (ProPac ^TM CEX-HPLC purity detection was performed by HPLC (Waters e2695 instrument) of WCX-10, 4X 250 mm. Diluting each pharmaceutical composition (sample) to 1.0mg/mL by using a mobile phase A, uniformly mixing, adding into a lining pipe, and carrying out sample injection analysis;the mobile phase composition is phase a: 20mM MES solution (pH 6.50.+ -. 0.02); and B phase: 20mM MES+150mM NaCl solution (pH 6.50.+ -. 0.02). And calculating the percentages of the main peak, the acid peak and the alkaline peak by adopting a peak area normalization method. 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 15 below:

TABLE 15 CEX-HPLC purity test parameters

Test results: the results of the CEX-HPLC purity test are shown in Table 16.

TABLE 16 CEX-HPLC purity test results (%)

Note that: "/" represents not measured

As can be seen from the results in Table 16, the pharmaceutical compositions FS3-1 to FS3-4 were left at high temperature for 4 weeks, and the CEX-HPLC purity was significantly reduced, mainly due to the increase of the acidic peak; wherein the purity of FS3-1 and FS3-4 decreases slowly; after 4 weeks of long-term storage, all the pharmaceutical compositions were unchanged significantly.

The pharmaceutical compositions FS4-1 to FS4-4 are placed for 4 weeks under the condition of high temperature, the purity of CEX-HPLC is reduced, and no obvious difference exists between the pharmaceutical compositions; after 4 weeks of standing under long-term conditions, the CEX-HPLC purity of all the pharmaceutical compositions was not significantly changed.

2.2.4rCE-SDS purity test

The testing method comprises the following steps: purity was measured as 1.2.4 rCE-SDS.

Test results: the results of rCE-SDS purity test are shown in Table 17.

TABLE 17 rCE-SDS purity test results (%)

Note that: "/" indicates not measured

As can be seen from the results of Table 17, the pharmaceutical compositions FS3-1 to FS3-4 were left at high temperature for 4 weeks, and the rCE-SDS purity was decreased, and there was no significant difference between all the pharmaceutical compositions; after 4 weeks of long-term standing, no obvious change in rCE-SDS purity occurred.

The pharmaceutical compositions FS4-1 to FS4-4 are placed for 4 weeks under the high temperature condition, the purity of rCE-SDS is reduced, and no obvious difference exists among all the pharmaceutical compositions; after being placed for 4 weeks under long-term conditions, the purity is not obviously changed.

2.2.5nrCE-SDS purity test

The testing method comprises the following steps: purity was measured as 1.2.5 nrCE-SDS.

Test results: the results of the nrCE-SDS purity test are shown in Table 18.

TABLE 18 nrCE-SDS purity test results (%)

Note that: "/" indicates not measured

As can be seen from the results in Table 18, the purity of the nrCE-SDS was significantly reduced by allowing the pharmaceutical compositions FS3-1 to FS3-4 to stand at high temperature for 4 weeks, and there was no significant difference between all the pharmaceutical compositions; no significant change in nrCE-SDS purity occurred after 4 weeks of long-term storage.

The purity of the pharmaceutical compositions FS4-1 to FS4-4 is reduced after the pharmaceutical compositions are placed for 4 weeks under the high temperature condition, and no obvious difference exists among all pharmaceutical composition groups; no significant change in nrCE-SDS purity occurred after 4 weeks of standing under long term conditions.

2.2.6 binding Activity test

The testing method comprises the following steps: the plates were wrapped with His-tagged RBD protein solution (Xujun Suzhou) at a concentration of 3.0 μg/mL and incubated at 37℃for 90min; plates were washed and blocked with PBS containing 2% (w/v) skimmed milk; adding a concentrated solution comprising CB6 variant (CB 6 variant) diluted in gradient with PBS solution containing 2% (w/v) skimmed milkThe solution was diluted to 40. Mu.g/mL with PBS containing 2% (w/v) skimmed milk, diluted 4-fold, and incubated at 37℃for 60min at a final concentration of 0.009537 ng/mL. Detection was performed with 5000-fold dilution of goat anti-human IgG antibody (Fc-specific, coupled peroxidase) (manufacturer: sigma: A0170) as detection antibody, incubation at 37℃for 60min, followed by development with 0.1mg/mL TMB (manufacturer: sigma: T2885), termination of the reaction with 2M HCl after 15min, reading of the plate at 450nm/620nm, followed by four-parameter logistic regression (4 PL) model fitting curve using GraphPad Prism software, giving the EC of the experimental group ₅₀ (half maximum effect concentration) value. Binding activity (relative activity,%) =ec of reference substance ₅₀ EC of pharmaceutical composition ₅₀ ×100％。

Test results: the binding activity test results are shown in Table 19.

Table 19 binding Activity test results (%)

Note that: "/" indicates not measured

From the results of Table 19 above, it is clear that the binding activity of the pharmaceutical compositions FS3-1 to FS3-4 did not change significantly when left for 4 weeks under high temperature conditions and long term conditions.

The pharmaceutical compositions FS4-1 to FS4-4 were left for 4 weeks under high temperature conditions and long term conditions without significant changes in the binding activity of all the pharmaceutical compositions.

2.2.7 sub-visible particle test

The testing method comprises the following steps: as in the 1.2.7 sub-visible particle test.

Test results: the sub-visible particle test results are shown in table 20. 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)

From the results in Table 20 above, it is clear that the pharmaceutical compositions FS4-1 to FS4-4 were left for 4 weeks under high temperature conditions and long term conditions, and that no significant changes in the sub-visible particles of all the pharmaceutical compositions were observed.

From the above, it is clear from the results of FS3-1 to FS3-4 that there is no significant difference between all the pharmaceutical compositions from the viewpoints of appearance, SEC-HPLC, rCE-SDS, nrCE-SDS and cell activity results at high temperature; from CEX-HPLC results, the purity of FS3-3 and FS3-4CEX-HPLC is reduced relatively slowly, and other pharmaceutical compositions have no obvious difference; under long term conditions, FS3-1, FS3-2 and FS3-4 are relatively better from the appearance, and all other tests have no abnormality. Overall, the pharmaceutical compositions FS3-1, FS3-2 and FS3-4 are more stable than other pharmaceutical compositions.

As is evident from the results of FS4-1 to FS4-4, no significant differences between all pharmaceutical compositions were seen from the appearance, SEC-HPLC, CEX-HPLC, rCE-SDS, nrCE-SDS, binding activity and sub-visible particle results, and long-term stability was good.

Example 3: stability test of pharmaceutical compositions comprising HUR33 variants and CB6 variants

3.1 Experimental sample

The sample of HUR33 variant (HUR 33-1) or CB6 variant (CB 638) was used as Millipore, respectively0.11m ² Ultrafiltration concentration (UF/DF) was performed to a concentration of about 100mg/mL, respectively, followed by dialysis into the corresponding stabilizer-containing buffer. And respectively measuring the concentration of the HUR33 variant and/or the CB6 variant, respectively adding a corresponding buffer solution containing a stabilizing agent and a corresponding surfactant according to the measured concentrations, respectively adjusting the final concentration of the HUR33 variant and/or the CB6 variant to 80mg/mL, 50mg/mL and 30mg/mL, respectively mixing the concentrations and the equal volumes of the HUR33 variant and the CB6 variant, respectively, and obtaining different pharmaceutical compositions to be tested according to the content of other components as shown in the table 21.

And filling the newly prepared different pharmaceutical compositions to be tested into 2R penicillin bottles in a sterile mode, 2 mL/bottle, and then carrying out stability lofting and detection according to stability investigation conditions. The components of the different pharmaceutical compositions tested are shown in table 21, wherein: histidine buffer refers to L-histidine-L-histidine hydrochloride buffer.

TABLE 21 different compositions of the test pharmaceutical compositions

3.2 Experimental methods and results

The stability investigation conditions included:

3.2.1 appearance test

Test results: the appearance test results are shown in table 22.

Table 22 appearance test results

As can be seen from the results of Table 22 above, the pharmaceutical compositions FS5-1 to FS5-3 were left for 4 weeks under high temperature conditions, the opalescence was enhanced, and more particles appeared in FS 5-3; after being placed for 2 weeks under long-term conditions, all prescriptions show the opalescence enhancement phenomenon, the opalescence enhancement degree has concentration dependency, and the opalescence phenomenon is more obvious along with the higher sample concentration.

3.2.2SEC-HPLC purity test

The testing method comprises the following steps: SEC purity was checked by HPLC (Waters e2695 instrument) fitted with a SEC column (TSKgel G3000SWXL,7.8 mm. Times.30 cm,5 μm). Diluting each medicine composition (sample) to 4.0mg/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 consisted of a 50mM phosphate solution (containing 300mM sodium chloride and 300mM L-arginine hydrochloride) at pH 6.8.+ -. 0.2. The relative percentages of main peak, polymer and fragment were calculated using area normalization. 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 23 below:

TABLE 23 SEC-HPLC purity test parameters

Chromatographic conditions	Chromatographic parameters
		Chromatographic column	TSKgel G3000SWXL，7.8mm×30cm，5μm
Detection wavelength	280nm
		Column temperature box	25±3℃
Sample tray temperature	5±3℃
		Flow rate	1.0mL/min
Sample injection amount	25 μL (sample concentration 4.0 mg/mL)
		Mobile phase	50mM phosphate, 300mM sodium chloride, 300mM L-arginine hydrochloride, pH 6.8.+ -. 0.2
Elution mode	Isocratic elution
		Elution time	20min

Test results: the SEC-HPLC purity test results are shown in Table 24.

Table 24 SEC-HPLC purity test results (%)

As can be seen from the results in Table 24, the pharmaceutical compositions FS5-1 to FS5-3 were left at high temperature for 4 weeks, the SEC-HPLC purity was slightly lowered, and there was no significant difference between all the pharmaceutical compositions; after 4 weeks of standing under long term conditions, no significant change in SEC-HPLC purity occurred for all pharmaceutical compositions.

3.2.3CEX-HPLC purity test

The testing method comprises the following steps: by using a column equipped with a cation chromatography column (ProPac ^TM CEX-HPLC purity detection was performed by HPLC (Waters e2695 instrument) with WCX-10, 4X 250 mm. Diluting each pharmaceutical composition (sample) to 1.0mg/mL by using a mobile phase A, uniformly mixing, adding into a lining pipe, and carrying out sample injection analysis; the mobile phase composition is phase a: 20mM MES solution (pH 6.50.+ -. 0.02); and B phase: 20mM MES+100mM NaCl solution (pH 6.50.+ -. 0.02). The percentages of CB6 variant (main peak 1), HUR33 variant (main peak 2) and components 1 to 3 were calculated separately using an area normalization method. 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 25 below:

TABLE 25 CEX-HPLC purity test parameters

Test results: the results of the CEX-HPLC purity test are shown in Table 26.

TABLE 26 CEX-HPLC purity test results (%)

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As is clear from the results in Table 26, the pharmaceutical compositions FS5-1 to FS5-3 were left for 4 weeks under high temperature conditions and long term conditions, and the CEX-HPLC purity was lowered, and no significant difference was observed between the pharmaceutical compositions.

3.2.4rCE-SDS purity test

rCE-SDS test method: each pharmaceutical composition (sample) was diluted to 0.8mg/mL with a diluent (1X Sample Buffer Maurice CE-SDS PLUS, manufacturer: proteinmiple, cat# 046-567), and then 95. Mu.L of the diluted sample was mixed with 5. Mu.L of 2-mercaptoethanol uniformly, centrifuged at 3000rpm at room temperature for 30s, and 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 pretreatment method and the test method of the reference product are the same as those of the sample.

The nrCE-SDS method was used to test the purity of 1.2.5 nrCE-SDS.

Test results: the results of the rCE-SDS and nCE-SDS purity tests are shown in Table 27.

TABLE 27 purity test results of rCE-SDS and nrCE-SDS (%)

As is clear from the results in Table 27, the pharmaceutical compositions FS5-1 to FS5-3 were left for 4 weeks under high temperature conditions and long term conditions, and the purity of rCE-SDS and nCE-SDS were reduced, and there was no significant difference between all the pharmaceutical compositions.

3.2.5 binding Activity assay

The testing method comprises the following steps: binding activity was tested with 1.2.6.

Test results: the binding activity test results are shown in table 28.

Table 28 binding Activity test results (%)

From the results of Table 28 above, it is clear that the binding activity of the pharmaceutical compositions FS5-1 to FS5-3 did not change significantly when left for 4 weeks under high temperature conditions and long term conditions.

From the above, the results of FS5-1 to FS5-3 show that no significant difference appears between all the pharmaceutical compositions from the results of SEC-HPLC and binding activity, and that the appearance of FS5-3 is relatively poor at high temperature from the appearance, and that opalescence enhancement appears between all the pharmaceutical compositions. Based on the concentration dependency of opalescence of the pharmaceutical composition under high temperature and long-term conditions, the FS5-1 stability is relatively good in combination with the test result and risk controllability of the pharmaceutical composition only containing HUR33 variant or CB6 variant.

Example 4: antigen affinity assay of pharmaceutical compositions

4.1 Experimental methods

The concentrations of the HUR33 and CB6 variants were diluted to 5. Mu.g/mL using a molecular interaction analyzer Octet RBD 96e (Sartorius Fortebio company) using PBST, which was composed of PBS (manufacturer Sigma, cat. No. P4417) and 0.05vol% P20 (manufacturer Cytiva, cat. No. BR 100054), pH 7.4, captured on the surface of Protein A (Protein A) probes (manufacturer Sartorius Fortebio, cat. No. 18-5010), respectively; immersing into RBD solution (Leun) of Spike protein of SARS-COV-2 wild strain with concentration of 15nM and 60nM respectively, and detecting to obtain binding signal; dissociation in PBST, detection to obtain dissociation signal. The data were analytically fitted using Octet analysis software to obtain a binding dissociation curve.

4.2 experimental results

The experimental results are shown in Table 29 and FIGS. 1 and 2, and the association rate constant (ka), dissociation rate constant (kd) and affinity (K) are shown in Table 29 _D ) Values.

TABLE 29 affinity of pharmaceutical composition FS5-1 for RBD of SARS-COV-2 wild-type strain Spike protein

The above results indicate that both the HUR33 variant and the CB6 variant of the pharmaceutical composition FS5-1 of the present application have binding activity to the wild-type strain RBD protein.

Example 5: binding Activity of pharmaceutical composition to SARS-COV-2 wild-type RBD

5.1 Experimental methods

The plates were wrapped with His-tagged RBD protein solution (Xujun Suzhou) at a concentration of 3.0 μg/mL and incubated at 37℃for 90min; plates were washed and blocked with PBS containing 2% (w/v) skimmed milk; the pharmaceutical compositions FS5-1 containing HUR33 variant and CB6 variant, diluted in gradient with PBS containing 2% (w/v) skimmed milk, were added respectively (the concentrations of HUR33 variant and CB6 variant were diluted to 40. Mu.g/mL with PBS containing 2% (w/v) skimmed milk, then diluted in gradient 4-fold, and the final concentration was 0.009537 ng/mL), and incubated at 37℃for 60min. Detection was performed with 5000-fold dilution of goat anti-human IgG antibody (Fc-specific, coupled peroxidase) (manufacturer: sigma: A0170) as detection antibody, incubation at 37℃for 60min, followed by development with 0.1mg/mL TMB (manufacturer: sigma: T2885), termination of the reaction with 2M HCl after 15min, reading of the plate at 450nm/620nm, followed by four-parameter logistic regression (4 PL) model fitting curve using GraphPad Prism software, giving the EC of the experimental group ₅₀ (half maximum effect concentration) value. The mixed liquid containing no HUR33 variant or CB6 variant and other pharmaceutical composition FS5-1 is used as a negative control group, and the sample treatment method is the same as that of the experimental group.

5.2 experimental results

The experimental results are shown in table 30 and fig. 3.

Table 30 EC ₅₀ Results

	Experimental group	Negative control group
			EC ₅₀ Value (ng/mL)	7.7	NA

Note that: "NA" means no significant activity

As can be seen from Table 30 and FIG. 3, the binding curves of the pharmaceutical compositions FS5-1 and RBD his containing HUR33 variant and CB6 variant are "S" with specific binding activity, EC ₅₀ A value of 7.7ng/mL; while the negative control group had no specific binding activity to RBD his.

The foregoing description of the preferred embodiments of the present invention is not intended to limit the invention to the particular embodiments disclosed, but on the contrary, the intention is to cover all modifications, equivalents, alternatives, and alternatives falling within the spirit and scope of the invention.

Claims

1. A pharmaceutical composition comprising a novel coronavirus neutralizing antibody or antigen binding fragment thereof selected from at least one of a HUR33 variant and a CB6 variant;

the HUR33 variant 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/or LCDR1, LCDR2 and LCDR3 with amino acid sequences shown as SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6 respectively;

The CB6 variant comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences shown as SEQ ID NO. 18, SEQ ID NO. 17 and SEQ ID NO. 15 respectively, and/or LCDR1, LCDR2 and LCDR3 with amino acid sequences shown as SEQ ID NO. 21, SEQ ID NO. 16 and SEQ ID NO. 22 respectively or as SEQ ID NO. 19, SEQ ID NO. 16 and SEQ ID NO. 20 respectively.

2. The pharmaceutical composition of claim 1, wherein the HUR33 variant comprises a heavy chain variable region having an amino acid sequence as set forth in SEQ ID No. 8 or SEQ ID No. 9, and/or a light chain variable region having an amino acid sequence as set forth in SEQ ID No. 7;

the CB6 variant comprises a heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 23 and/or a light chain variable region with an amino acid sequence shown as SEQ ID NO. 25 or SEQ ID NO. 24.

3. The pharmaceutical composition of claim 1 or 2, wherein the HUR33 variant further comprises a heavy chain constant region having an amino acid sequence as set forth in SEQ ID No. 13 and a light chain constant region having an amino acid sequence as set forth in SEQ ID No. 14.

4. A pharmaceutical composition according to any one of claims 1 to 3 wherein the HUR33 variant comprises a heavy chain having the amino acid sequence shown in SEQ ID No. 10 or SEQ ID No. 11 and/or a light chain having the amino acid sequence shown in SEQ ID No. 12;

The CB6 variant comprises a heavy chain with an amino acid sequence shown as SEQ ID NO. 26 and/or a light chain with an amino acid sequence shown as SEQ ID NO. 27 or SEQ ID NO. 28.

5. The pharmaceutical composition of any one of claim 1 to 4, wherein,

the HUR33 variant comprises:

the amino acid sequences are shown as HCDR1, HCDR2 and HCDR3 shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3 respectively, and the amino acid sequences are shown as LCDR1, LCDR2 and LCDR3 shown as SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6 respectively; or (b)

A heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 8 and a light chain variable region with an amino acid sequence shown as SEQ ID NO. 7; or (b)

A heavy chain with an amino acid sequence shown as SEQ ID NO. 10 and a light chain with an amino acid sequence shown as SEQ ID NO. 12;

the CB6 variant comprises:

the amino acid sequences are shown as HCDR1, HCDR2 and HCDR3 shown as SEQ ID NO. 18, SEQ ID NO. 17 and SEQ ID NO. 15 respectively, and the amino acid sequences are shown as LCDR1, LCDR2 and LCDR3 shown as SEQ ID NO. 21, SEQ ID NO. 16 and SEQ ID NO. 22 respectively; or (b)

A heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 23 and a light chain variable region with an amino acid sequence shown as SEQ ID NO. 25; or (b)

The heavy chain with the amino acid sequence shown in SEQ ID NO. 26 and the light chain with the amino acid sequence shown in SEQ ID NO. 27.

6. The pharmaceutical composition of any one of claims 1-5, wherein the mass ratio of the HUR33 variant and the CB6 variant is (1-10): (10-1) or about 1:1.

7. the pharmaceutical composition of any one of claims 1-6, wherein the concentration of the novel coronavirus neutralizing antibody or antigen-binding fragment thereof is 5-90 mg/mL, 5-85 mg/mL, 20-40 mg/mL, or about 30mg/mL.

8. The pharmaceutical composition of any one of claims 1-7, wherein the pharmaceutical composition further comprises a buffer selected from at least one of an acetate buffer, a citrate buffer, and a histidine buffer; preferably, the histidine buffer is histidine-histidine hydrochloride buffer; preferably, the concentration of the buffer is 10 to 30mM, 15 to 25mM or about 20mM; preferably, the buffer is selected from histidine-histidine hydrochloride buffer at a concentration of about 20 mM.

9. The pharmaceutical composition of claim 8, wherein the pH of the buffer is 5.0-6.5 or 5.5-6.0.

10. The pharmaceutical composition of any one of claims 1-9, wherein the pharmaceutical composition further comprises a stabilizer selected from at least one of arginine, arginine salt, proline, glycine, sodium chloride, mannitol, sorbitol, sucrose, maltose, xylitol, and trehalose; preferably, the concentration of the stabilizing agent in the pharmaceutical composition is 180 to 280mM or about 230mM.

11. The pharmaceutical composition of claim 10, wherein the stabilizer is sodium chloride; in the pharmaceutical composition, the concentration of the sodium chloride is 50-200 mM; or (b)

The stabilizer is mannitol; in the pharmaceutical composition, the concentration of mannitol is 100-300 mM or 200-300 mM; or (b)

The stabilizer is sucrose; in the pharmaceutical composition, the concentration of sucrose is 180-280 mM or about 230mM; or (b)

The stabilizer is trehalose; in the pharmaceutical composition, the trehalose is at a concentration of 180-280 mM or about 230mM.

12. The pharmaceutical composition of any one of claims 1-11, wherein the pharmaceutical composition further comprises a surfactant selected from at least one of polysorbate 80, polysorbate 20, and poloxamer 188; preferably, in the pharmaceutical composition, the concentration of the surfactant is 0.01-0.05% or 0.02-0.04% in w/v; preferably, the surfactant is selected from polysorbate 80, and the concentration of polysorbate 80 in the pharmaceutical composition is 0.02% -0.04% in w/v.

13. The pharmaceutical composition of any one of claims 1-12, comprising:

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

15. The liquid formulation of claim 14, wherein the liquid formulation contains a dextrose solution or a sodium chloride solution and the pharmaceutical composition of any one of claims 1-13; 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 neutralizing antibody or antigen binding fragment thereof 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.

16. Use of a pharmaceutical composition according to any one of claims 1-13 or a liquid formulation according to claim 14 or 15 in the manufacture of a medicament for the prevention, treatment, detection or diagnosis of a disease associated with a novel coronavirus infection.