CN113797333A

CN113797333A - Novel pharmaceutical composition of coronavirus antibody and application thereof

Info

Publication number: CN113797333A
Application number: CN202010535907.3A
Authority: CN
Inventors: 刘洪川; 刘沛想; 吴纯; 李园园; 潘隽; 张静; 李理; 周岳华
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: 2020-06-12
Filing date: 2020-06-12
Publication date: 2021-12-17
Also published as: WO2021249548A1; CN115666649A

Abstract

The invention provides a novel pharmaceutical composition of a coronavirus (COVID-19) humanized monoclonal antibody and application thereof in medicines. The pharmaceutical composition comprises an antibody or antigen-binding fragment thereof that specifically binds to COVID-19 RBD, a buffer, and optionally at least one stabilizer, and optionally a surfactant.

Description

Novel pharmaceutical composition of coronavirus antibody and application thereof

Technical Field

The present invention relates to the field of therapeutic pharmaceutical compositions. In particular, the invention relates to the field of pharmaceutical formulations comprising a humanized antibody that specifically binds to COVID-19 RBD.

Background

By 6, 9 and 2020, over 700 million globally diagnosed cases and over 40 million accumulated death cases of the novel coronavirus COVID-19(2019-nCoV) cause serious threats to the lives and health of the public.

However, no specific drug is currently available for this virus.

The culprit of the epidemic is that COVID-19 belongs to coronavirus. Severe acute respiratory syndrome coronavirus (SARS-CoV) and middle east respiratory syndrome coronavirus (MERS-CoV) of the same genus coronavirus have also caused epidemic in 2002-. According to the World Health Organization (WHO), it was counted that SARS-CoV co-caused 8000 human infections and 794 human deaths (https:// www.who.int /). The MERS-CoV infection virus cases are continuously increasing from 2012 to date, and 2499 infection cases and 861 death cases are diagnosed globally by the end of 2019. The world health organization formally named the novel coronavirus "2019 novel coronavirus (2019-nCoV)" at 12.1.2020, and later announced by the International Committee on Taxomy of viroses (ICTV) at 11.12.2.2020, the formal classification of the novel coronavirus was named Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the World Health Organization (WHO) announced on the same day as the International respiratory syndrome coronavirus 2 and the Innovation Forum, and formally named "COVID-19" for the disease caused by this virus.

To infect a cell, the virus first needs to bind to the host's receptor via the envelope protein. Antibodies, particularly neutralizing antibodies, block viral infection by binding to envelope proteins, blocking binding of the virus to cellular receptors. At the same time, the antibody binds to the envelope protein, thereby labeling the free virus or infected cells, recruiting immune cells and immune molecules such as macrophages or complement through the Fc region of the antibody, and removing the free virus and infected cells. Thus, antibodies that target the Receptor Binding Domain (RBD) not only have the ability to neutralize viral infection, but also act through the Fc region to facilitate viral and infected cell clearance.

Based on studies of other coronaviruses, particularly SARS-CoV and MERS-CoV, the important envelope protein for receptor binding is spike protein (S). S can be further divided into two parts, S1 and S2. The role of S2 is to mediate membrane fusion. Both the N-terminal (NTD) and C-terminal (CTD) of S1 may be RBDs. Through the research on COVID-19, the invention discovers that the CTD is the RBD of COVID-19, and the CTD binds to the receptor ACE 2. Antibodies that target RBD, and block S binding to ACE2, may therefore be neutralizing antibodies that inhibit viral infection. The invention aims to provide a specific humanized neutralizing antibody with a protective effect against COVID-19.

Disclosure of Invention

The pharmaceutical composition is a high-stability pharmaceutical composition containing a humanized monoclonal antibody specifically bound with COVID-19. In particular, the present inventors have found that humanized monoclonal antibodies that specifically bind to COVID-19 have the unexpected feature of high stability in a combination of a histidine buffer system and mannitol, sucrose or trehalose.

The present invention provides a pharmaceutical composition comprising: (1) a buffer solution; (2) a humanized monoclonal antibody or an antigen-binding fragment thereof, wherein the humanized monoclonal antibody specifically binds to COVID-19 RBD.

In some embodiments, the concentration of the humanized monoclonal antibody or antigen-binding fragment thereof in the pharmaceutical composition is from about 1 mg/mL to about 300mg/mL, preferably from about 10mg/mL to about 200mg/mL, more preferably from about 20mg/mL to about 150mg/mL, more preferably from about 40mg/mL to about 120 mg/mL; more preferably about 40-100 mg/mL; more preferably, the humanized monoclonal antibody or antigen-binding fragment thereof described above has a concentration of about 5mg/mL, 10mg/mL, 15mg/mL, 20mg/mL, 25mg/mL, 30mg/mL, 35mg/mL, 40mg/mL, 45mg/mL, 50mg/mL, 60mg/mL, 70mg/mL, 80mg/mL, 90mg/mL, 100mg/mL, 110mg/mL, 120mg/mL, 130mg/mL, 140mg/mL, 150mg/mL, 160mg/mL, 170mg/mL, 180mg/mL or 200mg/mL, preferably about 40mg/mL, 60mg/mL, 70mg/mL, 80mg/mL, 90mg/mL, 100mg/mL, 120 mg/mL.

In some embodiments, the buffer is selected from one or more of an acetate buffer, a citrate buffer, and a histidine buffer.

In some embodiments, the buffer is a histidine buffer, preferably the histidine buffer is selected from a histidine-hydrochloride buffer or a histidine-acetate buffer, preferably a histidine-hydrochloride buffer.

In some embodiments, the histidine-hydrochloride buffer is prepared from histidine and histidine hydrochloride, preferably L-histidine and L-histidine monohydrochloride. In some embodiments, the histidine buffer is made up of 1-30mM L-histidine and 1-30mM L-histidine monohydrochloride. In some embodiments, the histidine buffer is made up of histidine and histidine hydrochloride in a molar ratio of 1:1 to 1: 4. In some embodiments, the histidine buffer is made up of histidine and histidine hydrochloride in a molar ratio of 1: 1. In some embodiments, the histidine buffer is made up of histidine and histidine hydrochloride in a molar ratio of 1: 3. In some embodiments, the histidine preparation is: histidine buffer pH5.5 was prepared from 4.5mM L-histidine and 15.5mM L-histidine monohydrochloride. In some embodiments, the histidine preparation is: histidine buffer pH5.5 was prepared from 7.5mM L-histidine and 22.5mM L-histidine monohydrochloride. In some embodiments, the histidine preparation is: histidine buffer pH6.0 was prepared from 10mM histidine and 10mM histidine hydrochloride. In some embodiments, the histidine preparation is: histidine buffer pH6.0 was prepared from 15mM histidine and 15mM histidine hydrochloride.

In some embodiments, the histidine buffer is a histidine-acetate buffer, preferably in a molar ratio of 1:1 to 1.5:1, preferably such a buffer has a pH of 5.5 ± 0.3, preferably about 5.5, preferably such a buffer contains 15-20mM histidine and 12-15mM acetic acid.

In some embodiments, the buffer is an acetate buffer, preferably, 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 buffer is a citrate buffer, and preferably, the citrate buffer is a citrate-sodium citrate buffer.

In some embodiments, the buffer is a succinic acid buffer, and preferably, the succinic acid buffer is a succinic acid-sodium succinate buffer.

In some embodiments, the buffer has a concentration of about 1 mM to about 200mM, preferably about 1 mM to about 100mM, preferably about 5mM to about 50mM, preferably about 10mM to about 30 mM; preferably about 10-20 mM; preferably about 20 to about 30 mM; the above-mentioned buffer concentration is, for non-limiting examples, about 5mM, 10mM, 15mM, 20mM, 25mM, 30mM, 40mM, 45mM, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 105mM, 110mM, 115mM, 120mM, 130mM, 140mM, 150mM, 160mM, 170mM, or 180mM, or a range wherein any two of these ranges are defined as endpoints, and preferably about 10mM, 15mM, 20mM, 25mM, or 30 mM.

In some embodiments, the pH of the buffer is about 5.0 to about 6.5, preferably about 5.0 to about 6.0, preferably about 5.5 to about 6.5, preferably about 5.0 to about 5.5, preferably about 5.5 to about 6.0, preferably about 6.0 to about 6.5, and non-limiting examples of the pH of the buffer are 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, preferably about 5.5 or 6.0.

In some embodiments, the pharmaceutical composition further comprises a stabilizer.

In some embodiments, the stabilizing agent is selected from one or more of arginine hydrochloride, proline, glycine, sodium chloride, mannitol, sorbitol, sucrose, maltose, xylitol, and trehalose.

In some embodiments, the stabilizer is selected from one or more of mannitol, sucrose and trehalose.

In some embodiments, the concentration of the stabilizer is about 10mM to 400mM, preferably 50mM to 300mM, more preferably 100mM to 300mM, and still more preferably 200mM to 300 mM.

In some embodiments, the stabilizing agent is sodium chloride at a concentration of about 30 to 200 mM; or the stabilizer is mannitol at a concentration of about 100 and 300 mM; or the stabilizer is sorbitol at a concentration of about 100 and 300 mM; or the stabilizer is sucrose at a concentration of about 100 and 300 mM; or the stabilizer is trehalose at a concentration of about 100 and 300 mM; or the stabilizer is arginine hydrochloride at a concentration of about 30-200 mM; or the stabilizer is proline at a concentration of about 100 and 300 mM; or the stabilizer is glycine at a concentration of about 100 and 300 mM.

In some embodiments, the stabilizer is sodium chloride. In some embodiments, the stabilizer is sodium chloride at a concentration of about 30-200mM, preferably at a concentration of about 50-190mM, preferably about 100-180mM, preferably about 120-170mM, preferably about 130-150mM, non-limiting examples of the concentration of sodium chloride are about 100mM, 110mM, 120mM, 125mM, 130mM, 135mM, 140mM, 145mM, 150mM, 155mM, 160mM, 170mM, 180mM, 190mM, 200mM, preferably 135mM or 140 mM.

In some embodiments, the stabilizing agent is mannitol. In some embodiments, the stabilizer is mannitol at a concentration of about 100-300mM, preferably about 150-300mM, preferably about 180-280mM, preferably about 200-260mM, and non-limiting examples of the concentration of mannitol are about 200mM, 210mM, 220mM, 225mM, 230mM, 235mM, 240mM, 245mM, 250mM, 260mM, 270mM, 280mM, preferably 235 mM.

In some embodiments, the stabilizer is sorbitol. In some embodiments, the stabilizer is sorbitol at a concentration of about 100-300mM, preferably about 150-300mM, preferably about 180-280mM, preferably about 200-260mM, and non-limiting examples of the sorbitol concentration are about 200mM, 210mM, 220mM, 230mM, 235mM, 240mM, 250mM, 260mM, 270mM, 280mM, preferably 235 mM.

In some embodiments, the stabilizer is sucrose. In some embodiments, the stabilizer is sucrose at a concentration of about 100-300mM, preferably about 150-300mM, preferably about 180-280mM, preferably about 200-260mM, and non-limiting examples of the sucrose concentration are about 200mM, 210mM, 220mM, 230mM, 235mM, 240mM, 250mM, 260mM, 270mM, 280mM, preferably 235 mM.

In some embodiments, the stabilizing agent is trehalose. In some embodiments, the stabilizer is trehalose at a concentration of about 100-300mM, preferably about 150-300mM, preferably about 180-280mM, preferably about 200-260mM, non-limiting examples of trehalose concentrations are about 180mM, 200mM, 210mM, 220mM, 230mM, 235mM, 240mM, 250mM, 260mM, 270mM, 280mM, preferably 235 mM.

In some embodiments, the stabilizing agent is arginine hydrochloride. In some embodiments, the stabilizer is arginine hydrochloride at a concentration of about 30-200mM, preferably about 50-190mM, preferably about 100-180mM, preferably about 120-170mM, preferably about 130-150mM, and non-limiting examples of the arginine hydrochloride concentration are about 100mM, 110mM, 120mM, 125mM, 130mM, 135mM, 140mM, 145mM, 150mM, 155mM, 160mM, 170mM, 180mM, 190mM, 200mM, preferably 135mM or 140 mM.

In some embodiments, the stabilizing agent is proline. In some embodiments, the stabilizer is proline at a concentration of about 100-300mM, preferably about 150-300mM, preferably about 200-280mM, and non-limiting examples of the proline concentration are about 180mM, 200mM, 210mM, 220mM, 230mM, 240mM, 250mM, 260mM, 270mM, 280mM, preferably 240 mM.

In some embodiments, the stabilizer is glycine. In some embodiments, the stabilizer is glycine at a concentration of about 100-300mM, preferably about 150-300mM, preferably about 200-280mM, non-limiting examples of glycine concentrations are about 180mM, 200mM, 210mM, 220mM, 230mM, 240mM, 250mM, 260mM, 270mM, 280mM, preferably 260 mM.

In some embodiments, the above pharmaceutical composition further comprises a surfactant selected from polysorbate 80, polysorbate 20, or poloxamer 188.

In some embodiments, the surfactant is selected from polysorbate 80.

In some embodiments, the surfactant is selected from polysorbate 20.

In some embodiments, the surfactant concentration is from about 0.001% to about 0.1%, preferably from about 0.01% to about 0.1%, preferably from about 0.02% to about 0.08%, calculated as w/v; by way of non-limiting example, the concentration of the above surfactant is about 0.02%, 0.04%, or 0.08%.

In some embodiments, the humanized monoclonal antibody or antigen-binding fragment thereof has HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO 1, SEQ ID NO 2, and SEQ ID NO 3, respectively, and LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO 4, SEQ ID NO 5, and SEQ ID NO 6, respectively.

In some embodiments, the humanized monoclonal antibody has a heavy chain variable region as shown in SEQ ID NO. 7 and a light chain variable region as shown in SEQ ID NO. 8.

In some embodiments, the humanized monoclonal antibody has a heavy chain amino acid sequence as set forth in SEQ ID NO. 9 and a light chain amino acid sequence as set forth in SEQ ID NO. 10.

In some embodiments, the above pharmaceutical composition comprises a component according to any one of (1) to (14), wherein the humanized monoclonal antibody or antigen binding fragment thereof is according to any one of the embodiments of the present invention:

(1) (ii) (a) about 20mg/mL to 150mg/mL of a humanized monoclonal antibody or antigen-binding fragment thereof; (b) about 5-50mM histidine buffer, pH about 5.0-6.5; (c) about 50-200mM sodium chloride; (d) and about 0.01% -0.1% polysorbate 80; or

(2) (ii) (a) about 20mg/mL to 150mg/mL of a humanized monoclonal antibody or antigen-binding fragment thereof; (b) about 5-50mM histidine buffer, pH about 5.0-6.5; (c) about 100mM mannitol; (d) and about 0.01% -0.1% polysorbate 80; or

(3) (ii) (a) about 20mg/mL to 150mg/mL of a humanized monoclonal antibody or antigen-binding fragment thereof; (b) about 5-50mM histidine buffer, pH about 5.0-6.5; (c) about 100mM sucrose; (d) and about 0.01% -0.1% polysorbate 80; or

(4) (ii) (a) about 20mg/mL to 150mg/mL of a humanized monoclonal antibody or antigen-binding fragment thereof; (b) about 5-50mM histidine buffer, pH about 5.0-6.5; (c) about 100mM trehalose; (d) and about 0.01% -0.1% polysorbate 80; or

(5) (ii) (a) about 20mg/mL to 150mg/mL of a humanized monoclonal antibody or antigen-binding fragment thereof; (b) about 10-30mM acetate buffer, pH about 5.5-6.0; (c) about 100mM mannitol; (d) and about 0.01% -0.1% polysorbate 80; or

(6) (ii) (a) about 20mg/mL to 150mg/mL of a humanized monoclonal antibody or antigen-binding fragment thereof; (b) about 10 to about 30mM citrate buffer, pH about 5.5 to about 6.0; (c) about 100mM mannitol; (d) and about 0.01% -0.1% polysorbate 80;

preferably, the pharmaceutical composition comprises:

(7) (ii) (a) about 40mg/mL to 120mg/mL of a humanized monoclonal antibody or antigen-binding fragment thereof; (b) about 10-30mM histidine buffer, pH about 5.5-6.0; (c) about 200mM mannitol; (d) and about 0.02% to 0.08% polysorbate 80; or

(8) (ii) (a) about 40mg/mL to 120mg/mL of a humanized monoclonal antibody or antigen-binding fragment thereof; (b) about 10-30mM histidine buffer, pH about 5.5-6.0; (c) about 200 and 300mM sucrose; (d) and about 0.02% to 0.08% polysorbate 80; or

(9) (ii) (a) about 40mg/mL to 120mg/mL of a humanized monoclonal antibody or antigen-binding fragment thereof; (b) about 10-30mM histidine buffer, pH about 5.5-6.0; (c) about 200mM trehalose; (d) and about 0.02% to 0.08% polysorbate 80;

more preferably, the pharmaceutical composition comprises:

(10) (a) about 40mg/mL of a humanized monoclonal antibody or antigen-binding fragment thereof; (b) about 20mM histidine buffer, pH about 6.0; (c) about 235mM of mannitol; (d) and about 0.02% polysorbate 80; or

(11) (a) about 40mg/mL of a humanized monoclonal antibody or antigen-binding fragment thereof; (b) about 20mM histidine buffer, pH about 6.0; (c) about 235mM of sucrose; (d) and about 0.02% polysorbate 80; or

(12) (a) about 40mg/mL of a humanized monoclonal antibody or antigen-binding fragment thereof; (b) about 20mM histidine buffer, pH about 6.0; (c) about 240mM trehalose; (d) and about 0.02% polysorbate 80; or

(13) (a) about 80mg/mL of a humanized monoclonal antibody or antigen-binding fragment thereof; (b) about 20mM histidine buffer, pH about 6.0; (c) about 235mM of sucrose; (d) and about 0.08% polysorbate 80; or

(14) (a) about 80mg/mL of a humanized monoclonal antibody or antigen-binding fragment thereof; (b) about 20mM histidine buffer, pH about 6.0; (c) about 240mM trehalose; (d) and about 0.08% polysorbate 80.

In some embodiments, the pharmaceutical composition is a liquid formulation or a lyophilized formulation.

In some embodiments, the pharmaceutical composition is a liquid formulation.

In some embodiments, the liquid formulation or lyophilized formulation described above is stable at 2-8 ℃ for at least 3 months, at least 6 months, at least 12 months, at least 18 months, or at least 24 months.

In some embodiments, the aqueous solution or lyophilized formulation is stable at 40 ℃ for at least 7 days, at least 14 days, or at least 28 days.

The invention also provides application of the pharmaceutical composition in preparing a medicament for treating or preventing COVID-19 infection.

The present invention also provides the use of a histidine buffer and one or more stabilizers selected from mannitol, sucrose and trehalose, and optionally a surfactant, preferably polysorbate 80, to improve the stability of a pharmaceutical formulation of a humanized monoclonal antibody that specifically binds to COVID-19, or to prepare a pharmaceutical formulation of a humanized monoclonal antibody that specifically binds to COVID-19 with improved stability. Preferably, the histidine buffer, stabilizer and surfactant and amounts thereof are as described in any embodiment herein; the stability is as described in any embodiment herein.

Drawings

FIG. 1: binding of humanized monoclonal antibody CB6 (batch No. 20200307) to COVID-19 RBD protein (Binding) ELISA assay.

FIG. 2: blocking (Blocking) ELISA assay of humanized monoclonal antibody CB6 (batch 20200307) with COVID-19 RBD protein.

FIG. 3: humanized monoclonal antibody CB6 (batch 20200306) blocked COVID-19 infection of Huh-7 cells.

FIG. 4: humanized monoclonal antibody CB6 (batch 20200306) blocked COVID-19 from infecting Vero E6 cells.

Detailed Description

Definitions and explanations

In order that the invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless otherwise defined herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It is to be understood that this invention is not limited to particular methods, reagents, compounds, compositions or biological systems, which can, 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.

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

The term "pharmaceutical composition" or "formulation" means a mixture containing one or more of the antibodies described herein and other components, such as physiologically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration to an organism, facilitate absorption of the active ingredient and 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 invention are stable upon storage and their stability is independent of lyophilization (or other state change methods, such as spray drying).

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

The term "excipient" refers to an agent that can be added to a formulation to provide a desired characteristic (e.g., consistency, improved stability) and/or to adjust osmotic pressure. Examples of commonly used excipients include, but are not limited to, sugars, polyols, amino acids, surfactants, and polymers.

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

The term "buffer pH of about 5.0-6.5" refers to an agent that, through the action of its acid/base conjugate components, renders a solution containing the agent resistant to pH changes. The buffer used in the formulation of the present invention 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 6.5, or a pH in the range of about 5.0 to about 6.0.

Examples of "buffers" that control the pH within this range herein include acetates (e.g., sodium acetate), succinates (e.g., sodium succinate), gluconic acid, histidine hydrochloride, methionine, citrates, phosphates, citrate/phosphates, imidazole, acetic acid, acetates, citrates, combinations thereof, and other organic acid buffers.

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

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

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

A "succinate buffer" is a buffer that includes succinate ions. Examples of the succinate buffer include sodium succinate-succinate, potassium succinate-succinate, calcium succinate-succinate, magnesium succinate-succinate, and the like. The preferred succinate buffer is sodium succinate-succinate buffer.

The term "stabilizer" means 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 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), octopine (opines), alanine, octopine (strombine), and the N-oxide of Trimethylamine (TMAO), human serum albumin (hsa), albumin (bsa), α -bovine serum albumin, globulin, α -lactalbumin, GABA, lysozyme, myoglobin, ovalbumin, and rnasea. Some stabilizers, such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, sucrose, etc., may also act to control osmotic pressure. The stabilizer used in the present invention is one or more selected from the group consisting of a polyol, an amino acid, a salt and a sugar. Preferred sugars are sucrose and trehalose, and the preferred polyol is mannitol. Preferred amino acids are arginine or its salts (e.g. arginine hydrochloride), glycine, proline. 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, more preferably arginine hydrochloride, sodium chloride-sucrose, arginine hydrochloride-mannitol, arginine hydrochloride-sucrose, more preferably arginine hydrochloride-sucrose. In a particularly preferred embodiment, the stabilizer used in the present invention is selected from one or more of mannitol, sucrose and trehalose.

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 particulate matter in the formulation. Exemplary surfactants include, but are not limited to, nonionic surfactants such as polyoxyethylene sorbitan fatty acid esters (e.g., polysorbate 20 and polysorbate 80), polyethylene-polypropylene copolymers, polyethylene-polypropylene glycols, polyoxyethylene-stearates, polyoxyethylene alkyl ethers, such as polyoxyethylene monolauryl ether, alkylphenylpolyoxyethylene ether (Triton-X), polyoxyethylene-polyoxypropylene copolymers (poloxamers, pluronics), Sodium Dodecyl Sulfate (SDS). In a particularly preferred embodiment, the surfactant used in the present invention is polysorbate 80.

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

The term "stable" formulation is one in which the antibody substantially retains its physical and/or chemical stability and/or biological activity during the manufacturing process and/or upon storage. The pharmaceutical preparation may be stable even if the contained antibody fails to maintain 100% of its chemical structure or biological function after storage over a certain period of time. In certain instances, an antibody structure or function that maintains about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% after storage over a period of time may also be considered "stable". Various analytical techniques for measuring Protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery 247-: 29-90 (both incorporated by reference).

After storage of the formulation at a temperature and for a period of time, its stability can be measured by determining the percentage of native antibody remaining therein (among other methods). The percentage of native antibody can be measured by size exclusion chromatography (e.g., size exclusion high performance liquid chromatography [ SEC-HPLC ]), among other methods, "native" referring to unaggregated and undegraded. In some embodiments, the stability of a protein is determined as a 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 is stable for storage at room temperature, about 25-30 ℃, or 40 ℃ 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.

Stability can be measured by determining (among other methods) the percentage of antibody that migrates ("acidic form") in the fraction where this main fraction of antibody ("predominantly charged form") is acidic during ion exchange, where stability is inversely proportional to the percentage of acidic form antibody. The percentage of "acidified" antibody can be measured by, among other methods, ion exchange chromatography (e.g., cation exchange high performance liquid chromatography [ CEX-HPLC ]). In some embodiments, an acceptable degree of stability means that no more than about 49%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the antibody in acidic form can be detected in the formulation after storage of the formulation at a temperature and for a time. The certain time period of storage prior to measuring stability can 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, a temperature that allows for storage of the pharmaceutical formulation can be any temperature in the range of about-80 ℃ to about 45 ℃, e.g., storage 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 shows substantially no signs of, for example, aggregation, precipitation and/or denaturation when visually inspected for color and/or clarity or measured by UV light scattering or by pore size 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 extent that a visible precipitate is formed.

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

The level of turbidity which varies with, for example, the concentration of one or more components in the solution (e.g., protein and/or salt concentration) is also referred to as the "opacification" or "opacified appearance" of the formulation. The turbidity level can be calculated with reference to a standard curve generated using suspensions of known turbidity. Reference standards for determining the turbidity level of a pharmaceutical composition may be based on the "European Pharmacopoeia" standards (European Pharmacopoeia), fourth edition, "European commission for Quality of Medicine instructions" (EDQM), Strasbourg, France). According to the european pharmacopoeia standard, a clear solution is defined as a solution having a turbidity lower than or equal to that 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 association or non-ideal effects, which typically varies linearly with concentration. Other methods for assessing physical stability are well 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 chemically altered forms of the antibody. Chemical changes may include size changes (e.g., clipping), which may be assessed using, for example, pore size 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" if it is biologically active for its intended purpose. For example, a formulation of the invention may be considered stable if, after storage of the formulation for a certain period of time (e.g., 1 to 12 months) at a temperature, e.g., 5 ℃, 25 ℃, 45 ℃, etc., the formulation contains a humanized monoclonal antibody that binds to COVID-19 with an affinity that is at least 90%, 95% or more of the binding affinity of the antibody prior to said storage. Binding affinity can also be determined using, for example, ELISA or plasmon resonance techniques.

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

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

The terms "administering," "administering," and "treating" refer to introducing a composition comprising a therapeutic agent into a subject using any of a variety of methods or delivery systems known to those of skill in the art. Routes of administration of anti-PD-1 antibodies include intravenous, intramuscular, subcutaneous, peritoneal, spinal or other parenteral routes of administration, such as injection or infusion. "parenteral administration" refers to modes of administration other than enteral or topical administration, typically by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraframe, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, and via in vivo electroporation.

Antibodies

The term "antibody" as used herein is to be understood as including whole antibody molecules and 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 COVID-19(2019, a novel coronavirus) or an epitope thereof.

The term "full length antibody" or "whole antibody molecule" as used herein refers to an immunoglobulin molecule comprising four peptide chains, two heavy (H) chains (about 50-70kDa in length) and two light (L) chains (about 25kDa in length) 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 herein as VL) and a light chain constant region. The light chain constant region consists of one domain CL. The VH and VL regions can be further subdivided into Complementarity Determining Regions (CDRs) with high variability and regions that are spaced apart to be more conserved, called Framework Regions (FRs). Each VH or VL region is formed by, in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 are composed of 3 CDRs and 4 FRs arranged from amino terminus to carboxy terminus. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq).

As used herein, the term "CDR" refers to complementarity determining regions within an antibody variable sequence. There are 3 CDRs in each variable region of the heavy and light chains, 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.

The precise amino acid sequence boundaries of the variable region CDRs of the antibodies of the invention may be determined using any of a number of well-known protocols, including the Kabat protocol described by Kabat et al (1991), "Sequences of Proteins of Immunological Interest, 5 th edition Public Health Service, National Institutes of Health, Bethesda, MD (" Kabat "numbering scheme) and the IMGT protocol described by Lefranc M. -P.et al (1999Nucleic Acids Research,27, 209-212).

As used herein, an "antigen-binding fragment" includes a fragment of an antibody or derivative thereof, typically including at least one fragment of an antigen-binding region or variable region (e.g., one or more CDRs) of a 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 Fv fragments; a diabody; a linear antibody; single chain antibody molecules, such as sc-Fv; nanobodies (nanobodies) and multispecific antibodies formed from antibody fragments. When the binding activity of an antibody is expressed on a molar concentration basis, the binding fragment or derivative thereof typically retains at least 10% of the antigen binding activity of the parent antibody. Preferably, the binding fragment or derivative thereof retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the antigen binding affinity of the parent antibody. It is also contemplated that antigen-binding fragments of an antibody may include conservative or non-conservative amino acid substitutions (referred to as "conservative variants" or "functionally conservative variants" of the antibody) that do not significantly alter its biological activity.

When referring to ligand/receptor, antibody/antigen or other binding pairs, "specific" binding refers to determining the presence or absence of a protein in a heterogeneous population of proteins and/or other biological agents. For example, the monoclonal antibody of the present invention binds to COVID-19 RBD protein. Thus, under the conditions specified, a particular ligand/antigen binds to a particular receptor/antibody and does not bind in significant amounts to other proteins present in the sample.

The humanized monoclonal antibodies or antigen binding fragments thereof described herein include any of the humanized monoclonal antibodies described in application No. 202010114283.8, the disclosure of which is hereby incorporated by reference in its entirety. In some embodiments, the CDR sequences of the antibodies used in the methods and compositions of the invention comprise CDR sequences from antibody CB6 described in 202010114283.8.

A non-limiting, exemplary antibody used in the examples herein is selected from the humanized antibody CB6 described in CN201811515045.7, which is capable of specifically binding to human COVID-19 RBD. Wherein antibody CB6 has amino acid sequences of HCDR1, HCDR2 and HCDR3 shown as SEQ ID NO 1, SEQ ID NO 2 and SEQ ID NO 3, respectively, and LCDR1, LCDR2 and LCDR3 shown as SEQ ID NO 4, SEQ ID NO 5 and SEQ ID NO 6, respectively; preferably, antibody CB6 has the amino acid sequence shown in SEQ ID NO. 7 for the heavy chain variable region and SEQ ID NO. 8 for the light chain variable region; preferably, antibody CB6 has the heavy chain amino acid sequence shown as SEQ ID NO. 9 and the light chain amino acid sequence shown as SEQ ID NO. 10.

Pharmaceutical preparation

The pharmaceutical composition is a high-stability pharmaceutical composition containing a humanized antibody specifically bound with COVID-19. In particular, the present inventors have found that a combination of a histidine buffer system and mannitol, sucrose or trehalose has high stability.

The humanized monoclonal antibody in the pharmaceutical composition of the invention has the amino acid sequences of HCDR1, HCDR2 and HCDR3 shown as SEQ ID NO 1, SEQ ID NO 2 and SEQ ID NO 3, respectively, and LCDR1, LCDR2 and LCDR3 shown as SEQ ID NO 4, SEQ ID NO 5 and SEQ ID NO 6, respectively; preferably, the humanized monoclonal antibody in the pharmaceutical composition of the present invention has a heavy chain variable region having an amino acid sequence shown in SEQ ID NO. 7 and a light chain variable region shown in SEQ ID NO. 8; more preferably, the humanized monoclonal antibody in the pharmaceutical composition of the present invention has a heavy chain amino acid sequence as shown in SEQ ID NO. 9 and a light chain amino acid sequence as shown in SEQ ID NO. 10, respectively.

In the pharmaceutical compositions of the present invention, the humanized monoclonal antibody or antigen-binding fragment thereof is present in a concentration of about 1 to about 300mg/mL, preferably about 10 to about 200mg/mL, more preferably about 20 to about 150mg/mL, more preferably about 40 to about 120mg/mL, more preferably about 40 to about 100 mg/mL; more preferably, the humanized monoclonal antibody or antigen-binding fragment thereof described above has a concentration of about 5mg/mL, 10mg/mL, 15mg/mL, 20mg/mL, 25mg/mL, 30mg/mL, 35mg/mL, 40mg/mL, 45mg/mL, 50mg/mL, 60mg/mL, 70mg/mL, 80mg/mL, 90mg/mL, 100mg/mL, 110mg/mL, 120mg/mL, 130mg/mL, 140mg/mL, 150mg/mL, 160mg/mL, 170mg/mL, 180mg/mL or 200mg/mL, preferably about 40mg/mL, 60mg/mL, 70mg/mL, 80mg/mL, 90mg/mL, 100mg/mL, 120 mg/mL.

The buffer in the pharmaceutical composition of the present invention may be selected from the group consisting of an acetate buffer, a citrate buffer and a histidine buffer to provide a pH of about 5.0 to 6.5, preferably about 5.0 to 6.0, more preferably about 5.5-6.0, more preferably about 6.0, to the pharmaceutical composition of the present invention. In another aspect, the buffer used in the pharmaceutical compositions of the present invention has a pH of about 5.0 to about 6.5, preferably about 5.0 to about 6.0, more preferably about 5.5 to about 6.0, and more preferably about 6.0.

Particularly preferred buffers in the pharmaceutical composition of the invention are histidine buffers, including histidine-hydrochloride buffers or histidine-acetate buffers, preferably histidine-hydrochloride buffers. More preferably, the histidine-hydrochloride buffer is made of histidine and histidine hydrochloride, preferably L-histidine and L-histidine monohydrochloride. In some embodiments, the histidine buffer is made up of 1-20mM L-histidine and 1-20mM L-histidine monohydrochloride. In some embodiments, the histidine buffer is made up of histidine and histidine hydrochloride in a molar ratio of 1:1 to 1: 4. In some embodiments, the histidine buffer is made up of histidine and histidine hydrochloride in a molar ratio of 1: 1. In some embodiments, the histidine buffer is made up of histidine and histidine hydrochloride in a molar ratio of 1: 3. In some embodiments, the histidine buffer is: histidine buffer with a pH of about 5.5 was prepared from 4.5mM L-histidine and 15.5mM L-histidine monohydrochloride. In some embodiments, the histidine buffer is: histidine buffer with a pH of about 5.5 was prepared from 7.5mM L-histidine and 22.5mM L-histidine monohydrochloride. In some embodiments, the histidine buffer is: histidine buffer with a pH of about 6.0 was prepared from 15mM L-histidine and 15mM L-histidine monohydrochloride. In some embodiments, the histidine buffer is: histidine buffer with a pH of about 6.0 made from 10mM L-histidine and 10mM L-histidine monohydrochloride.

Accordingly, the pharmaceutical composition of the present invention may contain: a histidine-histidine hydrochloride buffer at a pH of 5.5 to 6.0, in a concentration of 10 to 30mM in the pharmaceutical composition; and 40-120mg/mL, preferably 40-100mg/mL, of a humanized monoclonal antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, in particular a CB6 antibody or antigen-binding fragment thereof as described herein.

In some embodiments, the pharmaceutical compositions of the present invention further comprise a stabilizer. Preferably, the stabilizer is selected from one or more of arginine hydrochloride, proline, glycine, sodium chloride, mannitol, sorbitol, sucrose, maltose, xylitol and trehalose. Preferably, the stabilizer in the pharmaceutical composition is selected from mannitol, sucrose and trehalose. The concentration of the stabilizer in the pharmaceutical composition of the present invention is about 10mM-400mM, preferably 50mM-300mM, more preferably 100mM-300 mM. In some embodiments, the stabilizing agent is sodium chloride at a concentration of about 30-200 mM; or the stabilizer is mannitol at a concentration of about 100-300mM, preferably 200-300 mM; or the stabilizer is sucrose at a concentration of about 100-300mM, preferably 200-300 mM; or the stabilizer is trehalose at a concentration of about 100-300mM, preferably 200-300 mM.

Thus, in some embodiments, the pharmaceutical compositions of the present invention comprise: a histidine-histidine hydrochloride buffer at a pH of 5.5 to 6.0, in a concentration of 10 to 30mM in the pharmaceutical composition; 40-120mg/mL, preferably 40-100mg/mL, of a humanized monoclonal antibody or antigen-binding fragment thereof according to any of the preceding embodiments, in particular a CB6 antibody or antigen-binding fragment thereof as described herein; and 100mM-300mM stabilizer, preferably, the stabilizer comprises one of mannitol, sodium chloride, sucrose and trehalose, preferably 100-300mM mannitol, 100-300mM sucrose and 100-300mM trehalose. In some embodiments, the stabilizer is 200-300mM sucrose. In some embodiments, the stabilizer is 200-300mM trehalose. In some embodiments, the stabilizer is 200-300mM mannitol.

In some embodiments, the pharmaceutical compositions of the present invention further comprise a surfactant. Preferred surfactants are selected from polysorbate 80, polysorbate 20 and poloxamer 188. The most preferred surfactant is polysorbate 80. The concentration of the surfactant in the pharmaceutical composition of the present invention is about 0.001% to about 0.1%, preferably about 0.02% to about 0.08%, in w/v. By way of non-limiting example, the concentration of surfactant in the pharmaceutical composition of the present invention is about 0.02%, 0.04%, or 0.08%.

Thus, in some embodiments, the pharmaceutical compositions of the present invention comprise: a histidine-histidine hydrochloride buffer at a pH of 5.5 to 6.0, in a concentration of 10 to 30mM in the pharmaceutical composition; 40-120mg/mL, preferably 40-100mg/mL, of a humanized monoclonal antibody or antigen-binding fragment thereof according to any of the preceding embodiments, in particular a CB6 antibody or antigen-binding fragment thereof as described herein; 100mM-300mM stabilizer, preferably, the stabilizer is 100-300mM sucrose, or 100-300mM mannitol, or 100-300mM trehalose; and 0.02% -0.08% by w/v of polysorbate 80.

The pharmaceutical composition of the present invention may be a liquid formulation, or a lyophilized formulation.

Medical use and method

The invention also provides a pharmaceutical composition according to any embodiment of the invention for treating or preventing a disease associated with infection with COVID-19, the use of a pharmaceutical composition according to any embodiment of the invention for preparing a medicament for treating or preventing a disease associated with infection with COVID-19, and a method for administering a therapeutically effective amount of a pharmaceutical composition according to any embodiment of the invention to a subject or patient in need thereof to treat or prevent a disease associated with infection with COVID-19.

In the present invention, the disease associated with COVID-19 infection refers to the disease that occurs and progresses due to COVID-19 infection.

The present invention will be illustrated below 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 invention. The methods and materials used in the examples are, unless otherwise indicated, conventional in the art.

Example 1: buffer solution system, pH, auxiliary material and protein concentration screening experiment

In the liquid pharmaceutical composition, the stability of the antibody is closely influenced by the buffer solution system, pH, the auxiliary materials and the protein concentration, and each antibody with unique physicochemical properties has the most suitable type, pH and auxiliary material conditions of the buffer solution. This example is directed to screening an optimal buffer system, pH and excipients to optimize the stability of the humanized antibodies disclosed herein for clinical use.

This example was performed with antibody CB6 at concentrations of about 20mg/mL and 40 mg/mL. The sample was prepared using Millipore Pellicon 30.11m²And (3) carrying out ultrafiltration concentration and liquid change on the membrane, placing the sample in a corresponding prescription after the liquid change, and placing the sample in a sealed centrifugal tube for buffer liquid screening. Buffer system acetic buffer, citric acid buffer and histidine buffer were screened at pH from 5.5 to 6.0 (as shown in table 1). Auxiliary materials sodium chloride, sucrose, trehalose or mannitol were screened for comparative testing. Namely, the different auxiliary materials are respectively added into the buffer solution containing the antibody CB6 with the concentration of about 20mg/mL or 40mg/mL, and the specific prescription information is shown in Table 1. The samples were placed at 40. + -. 2 ℃ and taken out at week 0, week 2 and week 4, respectively, for analysis.

The major pathways for protein degradation are the formation of aggregates, cleavage products and charged variants. The percentage of the native form (protein monomers) to the aggregated form was determined by size exclusion chromatography (SEC-HPLC) and the percentage of the acidic and basic forms of the antibody was determined by cation exchange chromatography (CEX-HPLC). The influence of different buffer systems, pH, excipients, protein concentration on the stability of antibody CB6 was examined by fitting a straight line with SEC-HPLC monomer content and CEX-HPLC main peak content for four weeks (4W) and calculating the slope of decline (%/week).

Stability was assessed by the following parameters: (1) visual appearance and visible foreign matter; (2) measuring the protein content by an ultraviolet spectrophotometry; (3) SEC-HPLC measurement of antibody monomer, dimer or fragment content; (4) CEX-HPLC measures antibody main charge, acidic charge or basic charge content; (5) detecting the molecular weight of the antibody by an NR-CE-SDS method; (6) detecting the molecular weight of the antibody by an R-CE-SDS method; (7) the ELISA method was used to detect the antibody binding activity.

Table 1: prescription information in buffer system, pH, adjuvant and protein concentration screening experiments

The results are shown in Table 2.

Recipe 1 and recipe 5 have heavy opalescence under the condition that the protein concentration is 40mg/ml, the concentration is 20mg/ml, and the protein content and the appearance of other recipe concentrations are 20mg/ml and 40mg/ml, which indicates that the sodium chloride and pH6.0 citric acid buffer system is not suitable for the high-concentration stability of the product.

In SEC-HPLC experimental detection, under the condition of 40 +/-2 ℃ acceleration, the monomer content of the prescription 5 is reduced at a high speed, the monomer content of the

prescriptions

2, 3 and 4 is reduced at a low speed, the average reduction rate is 0.4 percent per week, and the protein concentration has little influence on the purity under the conditions of 20mg/ml and 40 mg/ml.

The CEX-HPLC decreasing rate results show that the CEX main peak content of formula 5 and formula 6 decreases faster under the accelerated condition of 40 +/-2 ℃. The decrease rates of the CEX main peak contents of

formulas

1, 2, 3 and 4 were relatively low, and the protein concentrations at 20mg/ml and 40mg/ml had little effect on the purity.

The binding activity (Elisa method) and the purity of NR-CE-SDS were not abnormal, and the purity of R-CE-SDS was slightly decreased.

Table 2: buffer solution system, pH, auxiliary material and protein concentration screening experiment result

The detection data are integrated, and the comparison of the formula 1 to the formula 4 shows that the auxiliary materials mannitol, sucrose and trehalose are superior to sodium chloride, and the comparison of the formula 2 and the formula 5 shows that a histidine buffer system is superior to a citric acid buffer system, which is shown in that the latter has heavier opalescence; the SEC-HPLC purity and the CEX main peak of the acetic acid buffer system containing the auxiliary material mannitol (formula 6) are reduced rapidly at pH5.5, so that the histidine buffer system containing the auxiliary materials mannitol, sucrose and trehalose (formula 2/3/4) at pH6.0 is insensitive to protein concentration and has better stability.

According to the screening result, a histidine buffer system with pH6.0 is selected, and mannitol, sucrose and trehalose are used as auxiliary materials for subsequent research.

Example 2: prescription stability study of formulations

2.1 Shake stability

The antibody stock solution was selected and formulated as shown in Table 3, with an antibody concentration of 40 mg/mL. The product is filled into 2.0mL penicillin bottles by hand in an aseptic filling mode, after the product is continuously shaken at the temperature of 25 +/-2 ℃ at 80rpm or 150rpm, the content of antibody monomers, polymers or fragments is measured by appearance and SEC-HPLC for stability investigation, and the specific information is shown in Table 4.

Table 3: stability review prescription information

Table 4: examination of Shake stability

After continuous shaking at 80rpm or 150rpm at 25 + -2 deg.C, the three formulations showed no significant change in appearance and SEC-HPLC purity, showing better stability, see Table 5.

Table 5: prescription screening-shaking

2.2 dilution stability of physiological saline

The antibody stock solution was selected and formulated as shown in Table 3, with an antibody concentration of 40 mg/mL. The samples were diluted to different concentrations with physiological saline (0.9% NaCl), and left at 25. + -. 2 ℃ for 8 hours for testing, the stability of the samples was examined, the prescription conditions were confirmed, and the screening results are shown in Table 6.

Table 6: prescription stability screening results

The three prescription antibody preparations have good stability under the condition of dilution by normal saline and have good compatibility with infusion tubes and infusion bags.

The stability investigation experiment of shaking and physiological saline dilution is carried out on the sample under the condition of the prescription, so that the monomer content is not greatly influenced, and the good stability of the prescription is represented.

By investigating different buffer systems, different pH conditions, different antibody concentrations and different auxiliary material compositions, the stability of the humanized antibody CB6 is explored and researched, and the relatively optimal formula of the hydro-acupuncture preparation is determined. Antibody CB6 selects histidine and histidine hydrochloride buffer solution to adjust pH, mannitol, sucrose or trehalose to adjust osmotic pressure of the preparation, and adds polysorbate 80 to increase solubility of the preparation.

Example 3: binding specificity and high binding activity of monoclonal antibody preparation and COVID-19 virus S protein RBD

The recombinant SARS-CoV-2(COVID-19) S protein RBD (Novoprotein, cat # DRA32) was diluted to 3.0. mu.g/mL coating and shaken on a microplate shaker for 2 h. Plates were washed and blocked with 2% skim milk. Control antibody (isotype control antibody of IgG1 subtype) and CB6 antibody (4-fold gradient dilution from 40. mu.g/mL to 0.009537ng/mL, configured as per formula 2) were added at different concentrations, incubated for 1 hour and plates were washed. And then, mixing with the mixture of 1: goat anti-human IgG (Fc-specific) peroxidase antibody (Sigma, cat # a0170) diluted in 5000 was incubated for 1 hour, and then incubated with HRP substrate TMB (Sigma, cat # T2885) for 15 minutes to develop color, and binding signals of the antibody to RBD of COVID-19 virus S protein were detected. EC was fitted using a slope curve fit of logarithm (agonist) to response variable (GraphPad Prism)₅₀。

The results of the experiment are shown in FIG. 1. The CB6 antibody and the recombinant SARS-CoV-2S protein RBD have higher Binding specificity and Binding activity, EC through the Binding ELISA determination₅₀It was 21.7 ng/mL.

Example 4: the monoclonal antibody preparation can effectively block the combination of the RBD of the COVID-19 virus S protein and the receptor ACE2 thereof

Recombinant human ACE2(C-6His) (Novoprotein, cat # C419) was diluted to 3.0. mu.g/mL coated plates and incubated at 37 ℃ for 90 min. Plates were washed and blocked with 2% skim milk. Recombinant SARS-CoV-2S protein RBD (C-mFc) (Novoprotein, cat No. DRA32) was treated with 2% skim milk) Diluted to 1.0. mu.g/mL, followed by control antibody (a cognate control antibody of the IgG1 subtype) and CB6 antibody (diluted 2-fold gradient from 400. mu.g/mL to 0.2. mu.g/mL, as prescribed 2). The mixture was added to the plate and incubated for 1 hour and the plate washed. By reacting with 1: a5000 dilution of peroxidase-labeled goat anti-mouse Fc fragment secondary antibody (Sigma, cat # A2554) was incubated for 1 hour, followed by addition of TMB (Sigma, cat # T2885) and incubation for 20 minutes. IC of JS016 fitted using software GraphPad Prism₅₀。

The results of the experiment are shown in FIG. 2. The CB6 antibody can effectively block the combination of the RBD of the COVID-19 virus S protein and the receptor ACE2 thereof, IC, determined by Blocking ELISA (Blocking)₅₀22.8 μ g/mL, indicating that the CB6 antibody can inhibit the binding of COVID-19 RBD to the coated ACE2 receptor fusion protein.

Example 5: the monoclonal antibody preparation can effectively block the infection of pseudovirus on target cells

The COVID-19 pseudovirus (with the final concentration of 10000 TCID) expressing the complete sequence COVID-19 spike protein and luciferase reporter gene₅₀/ml) was mixed with a control antibody (anti-KLH antibody, LALA) or a CB6 antibody after gradient dilution (from 10. mu.g/ml to 0.128ng/ml, 5-fold gradient dilution, formulated according to formula 2) 1:1 and incubated for 1 h.

Huh-7 cells were plated at 5X 10 per well⁴The individual cells were seeded in a 96-well white-walled background plate, and then 100. mu.l of a mixture of the antibody and pseudovirus was added to the cells, which were incubated in an incubator at 37 ℃ for 24 hours. After incubation, 70. mu.l of One-Glo was added to each well^TMFirefly luciferase substrate and fluorescence detection was performed using a microplate reader (Perkinelmer/Envision).

Vero E6 cells were plated at 1X 10 per well⁴Individual cells were seeded in white-walled, bottomed 96-well plates and then placed in an incubator at 37 ℃ for 3 hours until the cells were adherent. After the cells adhered to the wall, 100. mu.l of the mixture of the antibody and pseudovirus was added to the cells, and incubated in an incubator at 37 ℃ for 22 hours. After incubation, 70. mu.l of One-Glo was added to each well^TMFirefly luciferase substrate and fluorescence detection was performed using a microplate reader (Perkinelmer/Envision).

Inhibition rate ═ 1- (experiment group fluorescence intensity average)Mean-average of blank)/(average of fluorescence intensity of negative control-average of blank)]X 100%. Plotting and fitting IC using the software GraphPad Prism₅₀。

The CB6 antibody can effectively inhibit the COVID-19 pseudovirus from infecting Huh-7 and VeroE6 cells and IC₅₀0.1831nM (0.02747. mu.g/ml) and 0.07628nM (0.01144. mu.g/ml), respectively, and the detailed results are shown in FIGS. 3 and 4.

Sequence listing

<110> Shanghai Junshi biomedical science and technology Co., Ltd

SUZHOU JUNMENG BIOSCIENCES Co.,Ltd.

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1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Arg Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Pro

85 90 95

Glu Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 9

<211> 449

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Asn

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Val Ile Tyr Ser Gly Gly Ser Thr Phe Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Met Asn Thr Leu Phe Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Val Leu Pro Met Tyr Gly Asp Tyr Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 10

<211> 216

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Arg Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Pro

85 90 95

Glu Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val

100 105 110

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys

115 120 125

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

130 135 140

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

145 150 155 160

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

165 170 175

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

180 185 190

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

195 200 205

Lys Ser Phe Asn Arg Gly Glu Cys

210 215

Claims

1. A pharmaceutical composition comprising:

(1) a buffer solution; and

(2) a humanized monoclonal antibody or an antigen-binding fragment thereof, wherein the humanized monoclonal antibody specifically binds to COVID-19 RBD.

2. The pharmaceutical composition of claim 1, wherein the buffer is selected from the group consisting of an acetate buffer, a citrate buffer, and a histidine buffer; preferably, the buffer is a histidine buffer; preferably, the buffer is a histidine-hydrochloride buffer;

preferably, the buffer has a concentration of about 1 mM to about 100mM, preferably about 5mM to about 50mM, more preferably about 10mM to about 30 mM;

preferably, the pH of the buffer is about 5.0 to about 6.5, preferably about 5.5 to about 6.0.

3. The pharmaceutical composition of claim 1 or 2, wherein the pharmaceutical composition further comprises a stabilizer; preferably, the stabilizer is selected from one or more of arginine hydrochloride, proline, glycine, sodium chloride, mannitol, sorbitol, sucrose, maltose, xylitol and trehalose; more preferably, the stabilizer is one or more selected from mannitol, sucrose and trehalose.

4. The pharmaceutical composition according to claim 3, wherein the stabilizer is present in a concentration of 10mM to 400mM, preferably 50mM to 300 mM; preferably, the stabilizer is sodium chloride at a concentration of about 50-200 mM; or the stabilizer is mannitol at a concentration of about 100 and 300 mM; or the stabilizer is sucrose at a concentration of about 100 and 300 mM; or the stabilizer is trehalose at a concentration of about 100 and 300 mM; preferably, the stabilizer is mannitol at a concentration of about 200-300mM, or sucrose at a concentration of about 200-300mM, or trehalose at a concentration of about 200-300 mM.

5. The pharmaceutical composition of any one of claims 1-4, wherein the pharmaceutical composition further comprises a surfactant; preferably, the surfactant is selected from polysorbate 80, polysorbate 20 or poloxamer 188, preferably the surfactant is polysorbate 80; preferably, the surfactant concentration is from about 0.01% to about 0.1%, preferably from about 0.02% to about 0.08%, by w/v.

6. The pharmaceutical composition of any one of claims 1-5, wherein said humanized monoclonal antibody has HCDR1, HCDR2, and HCDR3 having amino acid sequences shown in SEQ ID NO 1, SEQ ID NO 2, and SEQ ID NO 3, respectively, and LCDR1, LCDR2, and LCDR3 having amino acid sequences shown in SEQ ID NO 4, SEQ ID NO 5, and SEQ ID NO 6, respectively.

7. The pharmaceutical composition of any one of claims 1-5, wherein the humanized monoclonal antibody has a heavy chain variable region having an amino acid sequence set forth in SEQ ID NO. 7 and a light chain variable region having an amino acid sequence set forth in SEQ ID NO. 8; preferably, the humanized monoclonal antibody has a heavy chain amino acid sequence as shown in SEQ ID NO. 9 and a light chain amino acid sequence as shown in SEQ ID NO. 10.

8. The pharmaceutical composition of any one of claims 1-7, wherein the humanized monoclonal antibody or antigen-binding fragment thereof is present at a concentration of about 1-300mg/mL, preferably about 10-200mg/mL, more preferably about 20-150mg/mL, more preferably about 40-120 mg/mL.

9. The pharmaceutical composition according to any one of claims 1 to 7, which comprises a component represented by any one of the following (1) to (14):

10. Use of a pharmaceutical composition according to any one of claims 1 to 9 in the manufacture of a medicament for the treatment or prevention of a COVID-19 infection.