CN116271016A

CN116271016A - Stable preparation of SARS-CoV-2neutralizing antibody

Info

Publication number: CN116271016A
Application number: CN202211293228.5A
Authority: CN
Inventors: 谢良志; 胡萍; 刘艳
Original assignee: Sinocelltech Ltd
Current assignee: Sinocelltech Ltd
Priority date: 2021-10-25
Filing date: 2022-10-21
Publication date: 2023-06-23

Abstract

The invention relates to the field of biological medicine preparations, and discloses a stable preparation of a SARS-CoV-2neutralizing antibody. Consists of SARS-CoV-2neutralizing antibody, buffer solution, osmotic pressure regulator and surfactant. The preparation can enhance antibody stability and prolong its effective period.

Description

Stable preparation of SARS-CoV-2neutralizing antibody

Technical Field

The invention relates to the field of biological medicine preparations, in particular to a stable SARS-CoV-2neutralizing antibody preparation.

Background

The preventive and therapeutic drugs developed at present for the COVID-19 mainly comprise vaccines, antibodies, small molecule drugs and the like. The antibody has the advantages of high safety, strong specificity, obvious curative effect, low adverse reaction and the like, and is considered as the drug with the most potential for treating the COVID-19. Meanwhile, antibodies have good application precedents in the prevention and treatment of sudden infectious diseases. Monoclonal antibodies against the SARS-CoV-2 receptor binding domain (Receptor binding domain, RBD) are useful as specific antibody agents for the prevention and treatment of acute respiratory infections caused by the SARS-CoV-2 virus.

However, prior to administration, the antibody formulation may undergo storage and transport processes during which the antibody may undergo physical and chemical degradation, which instabilities may reduce the efficacy and/or increase the immunogenicity of the antibody, and thus a stable formulation is required to ensure that the antibody still has the activity and safety required for treatment prior to administration.

Disclosure of Invention

The invention aims to provide a stable SARS-CoV-2neutralizing antibody preparation, which has good stability in the prescription of the preparation.

A first aspect of the invention relates to a stable formulation,

contains 1-150mg/mL of SARS-CoV-2neutralizing antibody, preferably 10-50mg/mL of SARS-CoV-2neutralizing antibody;

20-150mM buffer, preferably 40-100mM buffer;

50-200mM of an osmolality adjusting agent, preferably 100-120mM of an osmolality adjusting agent;

0.01 to 0.05wt% of a surfactant, preferably 0.02 to 0.03wt% of a surfactant;

the pH of the solution is 5.0-7.0, preferably 6.0-6.5.

In one embodiment, the buffer is selected from one or more of phosphate buffer, glycine buffer, or histidine buffer;

the osmotic pressure regulator is sodium chloride;

the surfactant is polysorbate 80.

In one embodiment, the pH of the formulation solution is 6.0.

In one embodiment, the formulation contains 25mg/mL SARS-CoV-2neutralizing antibody, 40mM histidine buffer, 120mM sodium chloride, and 0.03wt% polysorbate 80.

In one embodiment, the SARS-CoV-2neutralizing antibody comprises a light chain variable region and/or a heavy chain variable region, wherein the light chain variable region comprises a light chain CDR1 having the amino acid sequence of SEQ ID NO. 1, a light chain CDR2 having the amino acid sequence of SEQ ID NO. 2 and a light chain CDR3 having the amino acid sequence of SEQ ID NO. 3; the heavy chain variable region comprises a heavy chain CDR1 of amino acid sequence SEQ ID NO. 4, a heavy chain CDR2 of amino acid sequence SEQ ID NO. 5 and a heavy chain CDR3 of amino acid sequence SEQ ID NO. 6.

In one embodiment, the SARS-CoV-2neutralizing antibody comprises an amino acid sequence having at least 90%,92%,95%,98% or 100% sequence identity to the SARS-CoV-2neutralizing antibody light chain variable region sequence SEQ ID NO. 8 and/or an amino acid sequence having at least 90%,92%,95%,98% or 100% sequence identity to the SARS-CoV-2neutralizing antibody heavy chain variable region sequence SEQ ID NO. 7.

In one embodiment, the antibody further comprises a light chain constant region and a heavy chain constant region, preferably the light chain constant region is an amino acid sequence having at least 90%,92%,95%,98% or 100% sequence identity to the light chain constant region of SEQ ID NO. 10, and/or the heavy chain constant region is an amino acid sequence having at least 90%,92%,95%,98% or 100% sequence identity to the heavy chain constant region of amino acid sequence SEQ ID NO. 9.

In one embodiment, the antibody is an IgG antibody, preferably an IgG1 antibody.

In one embodiment, the antibody is a monoclonal antibody.

In one embodiment, the antibody,

its binding affinity KD with SARS-CoV-2RBD has an average value of 11.7E-11 to 1.3E-11M, preferably 5.9E-11 to 2.6E-11M, more preferably 3.9E-11M; and/or

The average KD of its binding affinity with SARS-CoV S1 is 11.1E-10 to 1.2E-10M, preferably 5.6E-10 to 2.5E-10M, more preferably 3.7E-10M.

In one embodiment, the formulation is in the form of an aqueous formulation or a lyophilized form.

In one embodiment, the formulation is stable for at least 12 months at 2-8 ℃ and at least 3 months at 25 ℃.

In a second aspect, the invention relates to the use of a formulation according to the invention for the preparation of a medicament for the prevention and treatment of diseases caused by SARS-CoV-2 infection.

A third aspect of the invention relates to the use of the formulation of the invention for the prevention and treatment of diseases caused by SARS-CoV-2 infection.

Drawings

FIG. 1 is a graph showing the purity profile of each formulation sample in example 1 under freeze thawing conditions.

FIG. 2 is a graph showing the in vitro relative efficacy trend of each formulation sample of example 1 under freeze thawing conditions.

FIG. 3 is a graph showing the purity profile of each formulation sample of example 1 at 45 ℃.

FIG. 4 is a graph showing the main peak change trend at 45℃in each formulation sample of example 1.

FIG. 5 is a graph showing the in vitro relative efficacy trend of each formulation sample of example 1 at 45 ℃.

FIG. 6 is a graph showing the purity profile of each formulation sample in example 2 under freeze thawing conditions.

FIG. 7 is a graph showing the in vitro relative efficacy trend of each formulation sample of example 2 under freeze thawing conditions.

FIG. 8 is a graph showing the purity profile of each formulation sample of example 2 at 45 ℃.

FIG. 9 is a graph showing the main peak change trend at 45℃in each formulation sample in example 2.

FIG. 10 is a graph showing the in vitro relative efficacy trend of each formulation sample of example 2 at 45 ℃.

FIG. 11 is a graph showing the purity profile of each formulation sample in example 3 under freeze thawing conditions.

FIG. 12 is a graph showing the in vitro relative efficacy trend of each formulation sample of example 3 under freeze thawing conditions.

FIG. 13 is a graph showing the purity profile of each formulation sample in example 3 at 37 ℃.

FIG. 14 is a plot of the trend of the main peak change at 37℃for each of the formulation samples in example 3.

FIG. 15 is a graph showing the in vitro relative efficacy trend of each formulation sample of example 3 at 37 ℃.

Detailed Description

The present invention provides a stable preparation of SARS-CoV-2neutralizing antibody, and solves the problem of antibody stability in the course of storage and transportation. Ensuring that the antibody still has the activity and safety required for treatment before administration to a patient.

The term "formulation" refers to a composition that is effective in maintaining the biological activity of the active ingredient and that is free of other ingredients that are unacceptably toxic to the subject. Such formulations are sterile. "sterile" means free of living bacteria or free or substantially free of all living microorganisms and spores thereof.

As used herein, a "stable" formulation refers to a formulation in which the active ingredient substantially retains its physical and/or chemical stability and/or biological activity upon storage. Preferably, the formulation substantially retains its physical and chemical stability after storage, as well as its biological activity.

The terms "patient" or "subject" are used interchangeably to refer to any mammal suffering from a condition or disease according to the present invention. Preferably a human.

The stable formulation of the present invention contains SARS-CoV-2neutralizing antibody, buffer, osmotic pressure regulator and surfactant.

The terms "comprising" and "containing" mean that additional ingredients may be included in addition to the ingredients mentioned.

As used herein and in the appended claims, the singular forms "a," "an," "the," and "the" include plural referents unless the context clearly dictates otherwise.

As used herein, "buffer" refers to a buffer solution that resists changes in pH by the action of its acid-base complexing components. In an embodiment of the invention, histidine buffer is selected, preferably at a pH of about 5 to about 7, preferably about 6.

As used herein, "surfactant" refers to a surfactant, in one embodiment, polysorbate 80.

The term "osmolality adjusting agent" means a pharmaceutically acceptable osmolality adjusting agent. Suitable osmolality adjusting agents include, but are not limited to, salts, in one embodiment of the invention sodium chloride (NaCl), at a concentration of about 120mM.

The "stability" of a protein after storage at a selected temperature for a selected period of time can be assessed qualitatively and/or quantitatively in a number of different ways, including assessing aggregate formation (e.g., using size exclusion chromatography, by measuring turbidity, and/or by visual inspection); charge heterogeneity was assessed using cation exchange chromatography, image capillary isoelectric focusing (icIEF) or capillary zone electrophoresis; amino-terminal or carboxy-terminal sequence analysis; mass spectrometry; SDS-PAGE analysis to compare reduced and intact antibodies; peptide map (e.g., trypsin or LYS-C) analysis; the biological activity or antigen binding function of the antibody is evaluated, and the like.

In the embodiment of the invention, the purity of the sample after being stored for a selected time period is detected by adopting a molecular exclusion high performance liquid chromatography, and the sample is separated and quantified according to different molecular sizes, so that the content information of the monomer, aggregate and fragment of the sample is obtained; detecting the electric charge isomer by adopting an imaging capillary isoelectric focusing electrophoresis (IEF), and separating and quantifying according to the difference of isoelectric points of proteins so as to obtain the content information of acidic and alkaline proteins of a sample; separating and quantifying according to different protein charges by adopting a cation exchange high performance liquid chromatography method, so as to obtain sample charge heterogeneity information; measuring the molecular size variant of the SARS-COV-2neutralizing antibody according to the molecular weight by capillary gel electrophoresis under reducing and non-reducing conditions; calculating the in vitro relative titer of the sample and the EC50 percent of the control sample by detecting the EC50 of the sample, the working control sample and the SARS-CoV-2 spike protein; determining the biological activity of a sample by adopting a reporter gene method, wherein the detection principle is that SARS-CoV-2 pseudovirus with SARS-CoV-2 Spike protein (S) and Luciferase reporter gene is used for expressing a Luciferase gene (Luciferase) carried by the pseudovirus in cells after infecting cells expressing ACE2 and mediating bioluminescence; the SARS-CoV-2neutralizing antibody can block SARS-CoV-2 pseudovirus infection of high ACE2 expressing cell and inhibit expression of luciferase gene. Accordingly, the biological activity of the SARS-CoV-2neutralizing antibody was detected by the reporter gene method.

The term "antibody" means an immunoglobulin molecule, meaning any form of antibody that exhibits the desired biological activity. Including but not limited to monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies), even including antibody fragments. Typically, full length antibody structures preferably comprise 4 polypeptide chains, 2 heavy (H) chains and 2 light (L) chains, typically interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region and a heavy chain constant region. Each light chain comprises a light chain variable region and a light chain constant region. In addition to the typical full length antibody structure, the structure includes other derived forms.

The term "variable region" refers to a domain in an antibody heavy or light chain that is involved in binding an antibody to an antigen. The variable regions of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures and can be further subdivided into regions of hypervariability (called Complementarity Determining Regions (CDRs)) interspersed with regions that are more conserved, called Framework Regions (FR).

The term "complementarity determining regions" (CDRs, e.g., CDR1, CDR2, and CDR 3) refers to amino acid residues of an antibody variable region whose presence is necessary for antigen binding. Each variable region typically has 3 CDR regions identified as CDR1, CDR2, and CDR3. Each complementarity determining region may comprise amino acid residues from a "complementarity determining region" as defined by Kabat (Kabat et al Sequences of Proteins of Immulological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,MD.1991)) and/or those residues from a "hypervariable loop" (Chothia and Lesk; j Mol Biol 196:901-917 (1987)).

Each heavy and light chain variable region typically comprises 3 CDRs and up to 4 FRs, arranged from amino-terminus to carboxy-terminus, for example, in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

The Complementarity Determining Regions (CDRs) and Framework Regions (FRs) of a given antibody may be identified using the Kabat system (Kabat et al, sequences of Proteins of Immunological Interest,5th edition, U.S. department of health and public service, PHS, NIH, NIH publication No. 91-3242, 1991).

The term "constant region" refers to such amino acid sequences on the light and heavy chains of an antibody that are not directly involved in binding of the antibody to an antigen, but exhibit multiple effector functions, such as antibody-dependent cellular cytotoxicity.

An "antigen-binding fragment of an antibody" comprises a portion of an intact antibody molecule that retains at least some of the binding specificity of the parent antibody, typically including at least a portion of the antigen-binding or variable regions (e.g., one or more CDRs) of the parent antibody. Examples of antigen binding fragments include, but are not limited to Fv, fab, fab ', fab' -SH, F (ab ') 2, fd fragment, fd' fragment, single chain antibody molecules (e.g., scFv, di-scFv or tri-scFv, diabody or scFab), single domain antibodies.

An "antibody fragment" is a non-intact antibody molecule that retains at least some of the biological properties of the parent antibody, examples of which include, but are not limited to, fc fragments in addition to those recited above for the "antigen binding fragment".

The term "chimeric" antibody refers to an antibody in which a portion of the heavy and/or light chains are derived from a particular source or species, while the remainder are derived from a different source or species. A "humanized antibody" is a subset of a "chimeric antibody".

The term "humanized antibody" or "humanized antigen-binding fragment" is defined herein as an antibody or antibody fragment that: (i) Antibodies derived from non-human sources (e.g., transgenic mice carrying a heterologous immune system) and based on human germline sequences; or (ii) a chimeric antibody in which the variable region is of non-human origin and the constant region is of human origin; or (iii) CDR grafting, wherein the CDRs of the variable region are from a non-human source, and one or more framework regions of the variable region are of human origin, and the constant region (if any) is of human origin. The purpose of "humanization" is to eliminate the immunogenicity of antibodies of non-human origin in humans while at the same time retaining the affinity as much as possible. It may be advantageous to select human framework sequences most similar to those of antibodies of non-human origin as templates for humanization engineering. In some cases, it may be desirable to replace one or more amino acids in a human framework sequence with corresponding residues in a non-human framework to avoid loss of affinity.

"monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the population comprising a single antibody is identical except for possible mutations (e.g., natural mutations) that may be present in minute amounts. Thus, the term "monoclonal" indicates the nature of the antibody, i.e., not a mixture of unrelated antibodies. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a separate one of the determinants on the antigen. In addition to their specificity, monoclonal antibody preparations have the advantage that they are generally not contaminated with other antibodies. The term "monoclonal" is not to be construed as requiring production of the antibody by any particular method. The term monoclonal antibody specifically includes chimeric, humanized and human antibodies.

Antibodies "specifically bind" to an antigen of interest, such as a virus-associated antigen protein (herein, spike protein S), i.e., bind the antigen with sufficient affinity such that the antibody is useful as a therapeutic, targets cells or tissues expressing the antigen, and does not significantly cross-react with other proteins or with proteins other than the homologs and variants (e.g., mutant forms, splice variants, or proteolytically truncated forms) of the antigen targets mentioned above.

The term "binding affinity" refers to the strength of the sum of the non-covalent interactions between a single binding site of a molecule and its binding partners. As used herein, "binding affinity" refers to an inherent binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen), unless otherwise indicated. "KD", "binding rate constant kon" and "dissociation rate constant koff" are generally used to describe the affinity between a molecule (e.g., an antibody) and its binding partner (e.g., an antigen), i.e., how tightly a ligand binds to a particular protein. Binding affinity is affected by non-covalent intermolecular interactions such as hydrogen bonding, electrostatic interactions, hydrophobic and van der Waals forces between two molecules. In addition, the binding affinity between a ligand and its target molecule may be affected by the presence of other molecules. Affinity can be analyzed by conventional methods known in the art, including ELISA as described herein.

An "isolated" antibody is an antibody that has been identified and isolated from cells that naturally express the antibody. Isolated antibodies include in situ antibodies within recombinant cells and antibodies that are typically prepared by at least one purification step.

"sequence identity" between two polypeptide or nucleic acid sequences means the number of identical residues between the sequences as a percentage of the total number of residues. In calculating the percent identity, sequences being compared are aligned in a manner that produces a maximum match between the sequences, with gaps in the alignment (if any) being resolved by a particular algorithm. Preferred computer program methods for determining identity between two sequences include, but are not limited to, GCG program package, including GAP, BLASTP, BLASTN and FASTA (Altschul et al, 1990, J.mol. Biol. 215:403-410). The above procedure is publicly available from the international biotechnology information center (NCBI) and other sources. The well-known Smith Waterman algorithm can also be used to determine identity.

In one embodiment of the invention, SARS-CoV-2neutralizing antibody, described in patent application PCT/CN2021/089748, filed on 26, 4, 2021, is used, and in a particularly preferred embodiment, is a CoV2-HB27-Fd6-IgG1 antibody having reduced Fc function. PCT/CN2021/089748 is incorporated into the specification and claims by reference.

In a particularly preferred embodiment of the invention, the formulation contains 25mg/mL CoV2-HB27-Fd6-IgG1 antibody, 40mM histidine buffer, 120mM sodium chloride, and 0.03wt% polysorbate 80. The preparation has good stability, and can be stably stored at 2-8deg.C for at least 12 months and at 25 deg.C for at least 3 months.

The formulations of the present invention may be provided in liquid form or may be provided in lyophilized form. The lyophilized formulation can be reconstituted prior to administration.

The preparation of the invention is used for treating SARS-CoV-2 infection.

Examples

The invention will be more fully understood by reference to the following examples. However, they should not be construed as limiting the scope of the invention. All documents, patents and patent applications are incorporated herein by reference.

In the following examples, the SARS-CoV-2neutralizing antibody used is the CoV2-HB27-Fd6-IgG1 antibody, and its preparation, characterization and characterization is described in PCT/CN2021/089748 filed on month 26 of 2021.

The light chain CDR1-CDR3 amino acid sequences of the antibodies CoV2-HB27-Fd6-IgG1 are respectively SEQ ID NO. 1-3, the heavy chain CDR1-CDR3 amino acid sequences are respectively SEQ ID NO. 4-6, the heavy chain variable region amino acid sequence is SEQ ID NO. 7, the light chain variable region amino acid sequence is SEQ ID NO. 8, the heavy chain constant region amino acid sequence is SEQ ID NO. 9, and the light chain constant region sequence is SEQ ID NO. 10.

In the following examples, the detection methods used are as follows:

1) Size exclusion high performance liquid chromatography (SEC-HPLC)

Using a chromatographic column: TSKgel G3000SWX gel filtration chromatographic column (7.8X105 mM,5 μm) (code 0008541, manufacturer Tosoh) mobile phase SEC mobile phase (200 mM disodium hydrogen phosphate, 100mM arginine, pH6.50,1% isopropyl alcohol), UV detection wavelength 280nm, column temperature 25 ℃, 80 μg sample was injected into liquid chromatograph, and sample purity was calculated by area normalization method (rule 0514 of Chinese pharmacopoeia (2020 edition four parts)).

2) Imaging capillary isoelectric focusing electrophoresis (IEF)

The imaging capillary isoelectric focusing electrophoresis apparatus iCE3 is adopted for measurement, and the relevant parameters of iCIEF are as follows: the sample is evenly mixed with Pharmalyte3-10for IEF,1%methyl cellulose (1% MC), pI marker with pI of 5.12 and 9.33 and water, so as to prepare a sample solution for analysis, wherein the final concentration of protein in the final solution is 0.25mg/mL, the final concentration of ampholyte is 4%, and the final concentration of MC is 0.35%. in the iCE3 analysis, the focusing conditions were: 1500v,1 min; 3000V,6 minutes. After applying a voltage across the capillary, the sample will be focused at its pI point. The detection wavelength is 280nm, the UV absorption peak is photographed, the focused spectrum can be obtained, and the content information of the protein of the acid peak, the main peak and the alkaline peak of the sample is obtained through calculation (the general rule 0542 capillary electrophoresis method of Chinese pharmacopoeia (four parts of 2020 edition)).

3) Cation exchange high performance liquid chromatography (CEX-HPLC)

CoV2-HB27-Fd6-IgG1 antibody was eluted gradient using a chromatography column weak cation exchange column WCX-10 (4X 250 mm) (Product No.:054993, celastday) with phase A (10 mM PB, pH=7.0), phase B (10mM PB,200mM NaCl,pH =7.0) as the mobile phase, UV detection wavelength 280nm, column temperature 35 ℃. 80 mug of the sample is injected into a liquid chromatograph, and the purity of the sample is calculated according to an area normalization method (general rules 0512 and 0513 in Chinese pharmacopoeia (four parts of 2020).

Time (min)	Phase A (%)	Phase B (%)	Flow rate (mL/min)	Protective pressure (bar)
					0.00	100	0	0.5	200
40.00	88	12	0.5	200
					50.00	0	100	0.5	200
50.01	100	0	0.5	200
					70.00	100	0	0.5	200

4) Capillary gel electrophoresis (CE-SDS)

The sample Purity was calculated by an area normalization method (see general rule 3127 and general rule 0542 of Chinese pharmacopoeia (2020 edition three division), european pharmacopoeia 2.2.47, united states Pharmacopeia <1053 >) using a Capillary electrophoresis system (model: PA 800 plus) from Beckman Coulter, the Capillary tube being Capilliary (50. Mu.mI. D. Bore-fused Silica), and using an IgG Purity/Heterogeneity Assay Kit kit provided from Beckman Coulter, the detection wavelength being 220nm, the temperature of the kit being 25 ℃ C. And subjecting the sample to Capillary electrophoresis analysis.

5) In vitro relative efficacy

Combining SARS-CoV-2 spike protein (RBD) (source: coV2-HB27-Fd6-IgG1 working control (source: peking GmbH, biotechnology Co., ltd.) with a solid phase carrier to form a solid phase antigen, washing to remove unbound antigen and impurities, adding 100.00ng/mL, 50.00ng/mL, 25.00ng/mL, 12.50ng/mL, 6.25ng/mL, 3.13ng/mL, 1.56ng/mL, 0.78ng/mL, 0.26ng/mL, 0.09ng/mL, 0.03ng/mL of CoV2-HB27-Fd6-IgG1 working control (source: peking GmbH, biotechnology Co., ltd., sequence with antibody sequence in the sample to be tested) and blank, and combining the sample to be tested with the solid phase antigen to form a solid phase antigen antibody complex; then adding goat anti-human IgG (Fc) marked by horseradish peroxidase, combining the antibody on the solid-phase immune complex with the enzyme-labeled antibody, adding a substrate for color development, catalyzing a substrate by enzyme on the solid phase to become a colored product, reading the absorbance at 450nm by an enzyme-labeled instrument after stopping the reaction, fitting a four-parameter equation, and calculating EC ₅₀ The binding activity of CoV2-HB27-Fd6-IgG1 was examined (Du L, et al (2009) The spike protein of SARS-CoV-a target for vaccine and therapeutic development. Nat. Rev Microbiol.7 (3): 226-36.).

6) Biological Activity (pseudo-virus neutralization Activity method)

The samples and the CoV2-HB27-Fd6-IgG1 control were subjected to gradient dilution with DMEM complete medium to make it workConcentration (sample concentration at neutralization with pseudovirus) of 500.000, 166.667, 55.556, 18.519, 6.137, 2.058, 0.686, 0.229, 0.076ng/mL,50 μl/well was added to 96-well plates; pseudovirus PSV-Luc-Spike (M) -B13 from China cell engineering Co., ltd was diluted to 1.3X10 with DMEM complete medium ⁴ TCID50/mL, 50. Mu.L/well was added to a 96-well plate containing the existing antibody, incubated at 37℃for 1h, log-phase Huh-7 cells were obtained, 20K/100. Mu.L/well was added to a 96-well plate incubated for 1h, at 37℃and 5% CO ₂ Incubating for 20-28 hours. Negative control groups were set up respectively: adding cells, not adding samples, and not adding pseudotoxin; sample group: adding cells, adding a sample, and adding pseudotoxin; positive control group: adding cells, not adding samples, and not adding pseudotoxin. After the incubation, the supernatant was aspirated, 1 Xlysate was added for 10min, and 40. Mu.L/well of cell lysate was taken to 96 wells Bai Deban, placed on a microplate chemiluminescent detector, and 40. Mu.L/well of Luciferase Assay System was added for bioluminescence detection. Calculating the average bioluminescence intensity (Relative Light Units, RLU) between the sample and each control compound hole, and calculating the neutralization inhibition rate corresponding to each concentration point according to a formula: neutralization inhibition ratio (%) = (mean of luminescence intensity of positive control group-mean of luminescence intensity of sample group)/(mean of luminescence intensity of positive control group-mean of luminescence intensity of negative control group) ×100%. Calculating a standard curve by adopting an automatic analysis function of statistical software GraphPad Prism, wherein the abscissa is the concentration of an internal reference substance or a sample to be detected, the ordinate is the neutralization inhibition rate, an S-shaped curve is obtained, the regression equation is a four-parameter equation, and half-value Effective Concentrations (EC) of the sample and the internal reference substance are calculated respectively ₅₀ ). Calculating sample and working reference EC ₅₀ And using the average of the two independent results as the detection result of Jianhui Nie, qianqian Li, jiajin Wu et al, quantification of SARS-CoV-2neutralizingantibody by a pseudotyped virus-based assay.NATURE PROTOCOLS,2020,15 (11): 3699-3715.

7) Protein content (UV method)

The ultraviolet-visible spectrophotometer is used, the sample is diluted to have absorbance of 0.3-0.7 by the corresponding preparation buffer solution of the test sample, a cuvette of 1cm is used, the corresponding preparation buffer solution is used as a blank control, and the absorbance of the diluted sample is measured at the wavelength of 280 nm. Calculating the protein content according to the formula C=A×dilution/(epsilon L), namely the actual protein content of the measured sample, wherein C is the protein content (mg/mL); a is absorbance; epsilon is the extinction coefficient; l is the thickness (cm) of the liquid layer (general rule 0401 in Chinese pharmacopoeia (three parts of 2020).

Example 1: screening study of buffer System

The formulation of the CoV2-HB27-Fd6-IgG1 preparation of this example is shown in the following Table:

table 1 formulations of different buffer systems

Preparation method of CoV2-HB27-Fd6-IgG1 preparation:

the antibodies were changed to the target buffer (all components except polysorbate 80 and antibody) by ultrafiltration, the required amount of polysorbate 80 was replenished, and the antibody concentration was adjusted to 50mg/mL, and after aseptic packaging, placed under the following conditions, respectively:

1) Freezing below-25 ℃, thawing at room temperature, and taking out for analysis and detection in 0 and 4 freeze thawing cycles respectively, wherein the detection items comprise SEC-HPLC and in vitro relative efficacy;

2) Analytical tests were performed in an incubator at 45℃and removed at week 0, day 5, day 10, and week 4, respectively, and the test items included SEC-HPLC, IEF, and in vitro relative potency.

The analysis and detection method comprises the following steps:

and (3) purity detection: size exclusion high performance liquid chromatography (SEC-HPLC); the detection principle is that separation and quantification are carried out according to different molecular sizes, so that content information of sample monomers, aggregates and fragments is obtained.

Charge isomers: imaging capillary isoelectric focusing electrophoresis (IEF); the detection principle is that the protein content information of the acid peak, the main peak and the alkaline peak of the sample is obtained by separating and quantifying according to the difference of isoelectric points of the protein, and the main peak content is more suitable for antibody products.

Relative potency in vitro: the detection principle of the method is that the EC50 of the sample and the working control substance and the EC50 of SARS-CoV-2 spike protein (RBD) are detected respectively, and the percentage of the EC50 of the sample and the control substance is calculated, wherein the higher the value is, the higher the in-vitro relative efficacy of the sample is, and the better the sample quality is.

The detection results are shown in the accompanying drawings 1-5 and the tables 2-6.

TABLE 2 purity of CoV2-HB27-Fd6-IgG1 formulation under freeze-thawing conditions

TABLE 3 relative potency in vitro under freeze-thawing conditions of CoV2-HB27-Fd6-IgG1 formulation

TABLE 4 purity of CoV2-HB27-Fd6-IgG1 preparation at 45 ℃

TABLE 5 main peak of CoV2-HB27-Fd6-IgG1 preparation at 45 ℃

TABLE 6 relative potency of CoV2-HB27-Fd6-IgG1 formulation in vitro at 45 ℃

The detection result shows that:

freeze thawing for 4 times, wherein the purity of the CoV2-HB27-Fd6-IgG1 in F1 and F2 is higher than that of F3, and the in vitro relative efficacy is higher than 70%;

the purity of CoV2-HB27-Fd6-IgG1 in F1, F2 and F3 is equivalent after the mixture is placed at 45 ℃ for 0, 5 days, 10 days and 4 weeks, the main peak content of F1 is higher than that of F2 and F3, and the in vitro relative efficacy is higher than 70 percent.

In conclusion, the stability of F1 is superior to other formulations.

Example 2: screening study of pH

TABLE 7 formulations of different pH

Formulation number

SARS-CoV-2neutralizing antibodies

Histidine buffer

Sodium chloride

Polysorbate

80

pH

F1

50mg/mL

40mM

120mM

0.03wt％

5.0

F2

50mg/mL

40mM

120mM

0.03wt％

6.0

F3

50mg/mL

40mM

120mM

0.03wt％

6.5

F4

50mg/mL

40mM

120mM

0.03wt％

7.0

Preparation method of CoV2-HB27-Fd6-IgG1 preparation:

The analysis and detection method comprises the following steps:

The detection results are shown in the accompanying figures 6-10 and the tables 8-12.

TABLE 8 purity of CoV2-HB27-Fd6-IgG1 formulations under freeze-thawing conditions

TABLE 9 relative potency of CoV2-HB27-Fd6-IgG1 formulations in vitro under freeze-thawing conditions

TABLE 10 purity of CoV2-HB27-Fd6-IgG1 preparation at 45 ℃

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TABLE 11 main peak of CoV2-HB27-Fd6-IgG1 preparation at 45 ℃

TABLE 12 relative potency of CoV2-HB27-Fd6-IgG1 formulations in vitro at 45 ℃

The detection result shows that:

freeze thawing for 4 times, wherein the purity of CoV2-HB27-Fd6-IgG1 in F1, F2, F3 and F4 is higher than 95%, and the in vitro relative efficacy is higher than 70%;

the purity of the CoV2-HB27-Fd6-IgG1 in F2 is higher than that of F1, F3 and F4 after being placed at 45 ℃ for 0, 5 days, 10 days and 4 weeks, the main peak contents of F2 and F3 are slightly higher than that of F1 and F4, and the in vitro relative efficacy is higher than 70 percent.

Overall, the stability of F2 is superior to other formulations.

Example 3: screening study of antibody concentration

TABLE 13 formulations of different antibody concentrations

Formulation number

SARS-CoV-2neutralizing antibodies

Histidine buffer

Sodium chloride

Polysorbate

80

pH

F1

25mg/mL

40mM

120mM

0.03wt％

6.0

F2

40mg/mL

40mM

120mM

0.03wt％

6.0

F3

50mg/mL

40mM

120mM

0.03wt％

6.0

F4

100mg/mL

40mM

120mM

0.03wt％

6.0

F5

150mg/mL

40mM

120mM

0.03wt％

6.0

Preparation method of CoV2-HB27-Fd6-IgG1 preparation:

the antibodies were changed to the target buffer (all components except polysorbate 80 and antibody) by ultrafiltration, the required amount of polysorbate 80 was replenished, and the antibody concentration was adjusted to 25mg/mL, 40mg/mL, 50mg/mL, 100mg/mL, 150mg/mL, and after aseptic packaging, each was placed in:

2) Analytical tests were performed in an incubator at 37℃and removed at weeks 0,1 and 4, respectively, and the test items included SEC-HPLC, IEF and in vitro relative efficacy.

The analysis and detection method comprises the following steps:

and (3) purity detection: size exclusion high performance liquid chromatography (SEC-HPLC).

Charge isomers: imaging capillary isoelectric focusing electrophoresis (IEF).

The results are shown in FIGS. 11-15, and tables 14-18.

TABLE 14 purity of CoV2-HB27-Fd6-IgG1 formulation under freeze-thawing conditions

TABLE 15 relative potency of CoV2-HB27-Fd6-IgG1 formulations in vitro under freeze-thawing conditions

TABLE 16 purity of CoV2-HB27-Fd6-IgG1 preparation at 37 ℃

TABLE 17 main peak of CoV2-HB27-Fd6-IgG1 preparation at 37 ℃

TABLE 18 relative potency of CoV2-HB27-Fd6-IgG1 formulations in vitro at 37 ℃

The detection result shows that:

freeze thawing for 4 times, the purity of CoV2-HB27-Fd6-IgG1 in F1, F2, F3, F4 and F5 is higher than 95%, and the in vitro relative efficacy is higher than 70%;

the purity of the CoV2-HB27-Fd6-IgG1 in F1, F2, F3, F4 and F5 is higher than 95 percent, the change trend is consistent, the in vitro relative efficacy is higher than 70 percent, and the main peak change trend is basically consistent after the CoV2-HB27-Fd6-IgG1 is placed for 0,1 and 4 weeks, which shows that the stability of the CoV2-HB27-Fd6-IgG1 in F1, F2, F3, F4 and F5 is equivalent.

Example 4: prescription verification experiment 1

The SARS-CoV-2neutralizing antibody preparation (preparation formula 25mg/mLCoV2-HB27-Fd6-IgG1 antibody +40mM histidine buffer +120mM sodium chloride +0.03wt% polysorbate 80, pH 6.0) was sterilized, filtered, filled into penicillin bottles, and then plugged, capped, labeled, prepared to obtain samples, and examined for light stability and transport stability:

1) Light stability: horizontally placing the sample under 4500+/-500 lx illumination intensity, 2-8 ℃, examining the stability of the sample on days 6 and 12, and packaging the sample with tinfoil paper to make test control under the same conditions. Protein content, SEC-HPLC, CE-SDS, in vitro relative titers and biological activity assays were performed on days 6, 12, respectively.

2) Transportation stability: samples were tested by cold chain (2-8 ℃) transport company by land transport, air transport, 1 and 2 round trip transport, and then sampled, the test items included protein content, SEC-HPLC, CE-SDS, in vitro relative titers, and biological activity.

The analysis and detection method comprises the following steps:

protein content: the detection principle is that the protein content information of the sample is obtained by detecting the absorbance at 280nm and combining with the lambert beer law.

And (3) purity detection: size exclusion high performance liquid chromatography (SEC-HPLC);

and (3) purity detection: capillary gel electrophoresis (CE-SDS), which adopts sodium dodecyl sulfate capillary electrophoresis, and under reducing and non-reducing conditions, determining molecular size variant of SARS-COV-2neutralizing antibody according to molecular weight;

relative potency in vitro: the detection principle of the method is that by respectively detecting the EC50 of a sample, a working reference substance and SARS-CoV-2 spike protein (RBD), the percentage of the EC50 of the sample and the reference substance is calculated, and the higher the value is, the higher the in-vitro relative efficacy of the sample is, and the better the sample quality is;

biological Activity: pseudo virus neutralization activity method; SARS-CoV-2 pseudovirus (SARS-CoV-2 PsV) has SARS-CoV-2 Spike protein (S) and Luciferase reporter gene, and when SARS-CoV-2PsV infects ACE 2-expressing cell, the Luciferase gene (Luciferase) carried by pseudovirus is expressed in the cell and mediates bioluminescence. The SARS-COV-2neutralizing antibody is recombinant anti-SARS-CoV-2 spike protein monoclonal antibody, which can block SARS-CoV-2PsV from infecting Huh-7 cell with high expression ACE2 and inhibit expression of luciferase gene. Accordingly, the biological activity of the SARS-COV-2neutralizing antibody was detected by the reporter gene method.

The results are shown in tables 19 to 20.

The detection result shows that:

1) After 6 days and 12 days of illumination, the protein content, the purity (SEC-HPLC and CE-SDS), the in vitro relative efficacy and the biological activity of the sample are not changed obviously basically;

2) After air transportation and land transportation, the protein content, the purity (SEC-HPLC), the in vitro relative efficacy and the biological activity of the sample are not changed obviously basically;

in summary, the CoV2-HB27-Fd6-IgG1 formulation has good light stability and transport stability.

Example 5: prescription verification experiment 2

Three batches of SARS-CoV-2neutralizing antibody preparation (formulation 25mg/mLCoV2-HB27-Fd6-IgG1 antibody+40 mM histidine buffer+120 mM sodium chloride+0.03 wt% polysorbate 80, pH 6.0) were sterile filtered and filled into penicillin bottles, then capped, labeled, and then subjected to stability studies at 2-8deg.C and 25+ -2deg.C, including protein content, SEC-HPLC, CEX-HPLC, CE-SDS, in vitro relative titers, and biological activity.

The analysis and detection method comprises the following steps:

charge isomers: cation exchange high performance liquid chromatography (CEX-HPLC); the detection principle is that separation and quantification are carried out according to different charges of proteins, so as to obtain sample charge heterogeneity information;

The results of the experiments are shown in tables 21 to 26.

Experimental results show that the CoV2-HB27-Fd6-IgG1 preparation disclosed by the invention has good stability, can be stably stored for at least 12 months at the temperature of 2-8 ℃ and can be stably stored for at least 3 months at the temperature of 25 ℃.

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Sequence listing

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Claims

1. A stable preparation, characterized in that,

20-150mM buffer, preferably 40-100mM buffer;

0.01 to 0.05wt% of a surfactant, preferably 0.02 to 0.03wt% of a surfactant;

the pH of the solution is 5.0-7.0, preferably 6.0-6.5.

2. A formulation according to claim 1, characterized in that,

the buffer solution is one or more selected from phosphate buffer solution, glycine buffer solution or histidine buffer solution;

the osmotic pressure regulator is sodium chloride;

the surfactant is polysorbate 80.

3. Formulation according to any one of claims 1-2, characterized in that the pH of the formulation solution is 6.0.

4. A preparation according to any one of claims 1-3, which contains 25mg/mL SARS-CoV-2neutralizing antibody, 40mM histidine buffer, 120mM sodium chloride and 0.03 wt.% polysorbate 80.

5. The preparation according to claim 1 to 4, wherein the SARS-CoV-2neutralizing antibody comprises a light chain variable region and/or a heavy chain variable region,

wherein the light chain variable region comprises a light chain CDR1 having an amino acid sequence of SEQ ID NO. 1, a light chain CDR2 having an amino acid sequence of SEQ ID NO. 2, and a light chain CDR3 having an amino acid sequence of SEQ ID NO. 3;

the heavy chain variable region comprises a heavy chain CDR1 having an amino acid sequence of SEQ ID NO. 4 and a heavy chain CDR1 having an amino acid sequence of SEQ ID NO. 4

The heavy chain CDR2 and the amino acid sequence of SEQ ID NO. 5 are the heavy chain CDR3 of SEQ ID NO. 6.

6. Preparation according to claim 5, characterized in that the SARS-CoV-2neutralizing antibody comprises an amino acid sequence having at least 90%,92%,95%,98% or 100% sequence identity to the SARS-CoV-2neutralizing antibody light chain variable region sequence SEQ ID NO. 8 and/or an amino acid sequence having at least 90%,92%,95%,98% or 100% sequence identity to the SARS-CoV-2neutralizing antibody heavy chain variable region sequence SEQ ID NO. 7.

7. The formulation according to claim 5 or 6, wherein the antibody further comprises a light chain constant region and a heavy chain constant region, preferably the light chain constant region is an amino acid sequence having at least 90%,92%,95%,98% or 100% sequence identity to the light chain constant region of SEQ ID No. 10 and/or the heavy chain constant region is an amino acid sequence having at least 90%,92%,95%,98% or 100% sequence identity to the heavy chain constant region of amino acid sequence SEQ ID No. 9.

8. The formulation according to claim 7, said antibody being an IgG antibody, preferably an IgG1 antibody.

9. The formulation according to claim 7 or 8, wherein the antibody is a monoclonal antibody.

10. The preparation according to any one of claim 7 to 9, wherein the antibody,

a) The average value of the binding affinity KD of the anti-SARS-CoV-2 RBD is 11.7E-11-1.3E-11M,

preferably 5.9E-11 to 2.6E-11M, more preferably 3.9E-11M; and/or

b) The average KD of its binding affinity with SARS-CoV S1 is 11.1E-10 to 1.2E-10M, preferably 5.6E-10 to 2.5E-10M, more preferably 3.7E-10M.

11. The formulation according to any one of claims 1-10, which is in the form of an aqueous formulation or in lyophilized form.

12. Use of a formulation according to any one of claims 1-11 for the preparation of a medicament for the prevention and treatment of diseases caused by SARS-CoV-2 infection.

13. Use of a formulation according to any one of claims 1-11 for the prevention and treatment of diseases caused by SARS-CoV-2 infection.

14. The formulation according to any one of claims 1-11, which is stable for at least 12 months at 2-8 ℃ and for at least 3 months at 25 ℃.