CN113842457B

CN113842457B - Liquid formulation comprising anti-human interleukin-33 monoclonal antibody

Info

Publication number: CN113842457B
Application number: CN202111302350.XA
Authority: CN
Inventors: 薛刚; 朱华杰; 戴长松; 李帅; 许芹; 郭彩明; 陈卫; 吴亦亮
Original assignee: Jiangsu Quanxin Biomedical Co ltd
Current assignee: Jiangsu Quanxin Biomedical Co ltd
Priority date: 2021-11-04
Filing date: 2021-11-04
Publication date: 2022-05-20
Anticipated expiration: 2041-11-04
Also published as: CN113842457A

Abstract

The application discloses a liquid preparation of an anti-human interleukin-33 monoclonal antibody, which comprises the anti-human interleukin-33 monoclonal antibody with the protein concentration of 120-300 mg/mL and an amino acid additive with the amino acid concentration of 10-500 mM; the anti-human interleukin-33 monoclonal antibody comprises three heavy chain complementarity determining regions, CDR-H1, CDR-H2 and CDR-H3, and three light chain complementarity determining regions, CDR-L1, CDR-L2 and CDR-L3, wherein: CDR-H1 has the sequence shown in SEQ ID NO: 1; CDR-H2 has the sequence shown in SEQ ID NO: 2; CDR-H3 has the sequence shown in SEQ ID NO: 3; CDR-L1 has the sequence shown in SEQ ID NO: 4; CDR-L2 has the sequence shown in SEQ ID NO: 5; CDR-L3 has the sequence shown in SEQ ID NO: 6. The liquid preparation has low viscosity and high stability, can be injected by a syringe easily, and can be used as an injection, particularly a subcutaneous injection.

Description

Liquid formulation comprising anti-human interleukin-33 monoclonal antibody

Technical Field

The present application relates to the field of biotechnology. In particular, the present application relates to a liquid formulation comprising an anti-human interleukin-33 monoclonal antibody.

Background

The monoclonal antibody injection is used as a rapidly growing market and is mainly applied to the treatment of tumor treatment and autoimmune related diseases, more innovative treatment choices are brought to related patients due to higher targeting property and safety of the products, and more choices are provided for various monoclonal antibody injections for autoimmune related diseases which are difficult to treat by chemical small molecular drugs, such as asthma, atopic dermatitis, nasal polyp chronic sinusitis and the like. On the other hand, in order to reduce the clinical use cost of the biological preparation, the dosage form of the biological preparation is gradually changed from a freeze-dried dosage form to a water injection dosage form, and the administration route is also changed from an intravenous administration mode to a subcutaneous injection dosage form, so that the treatment time is greatly reduced, the self-injection at home can be realized, and the compliance of patients is improved. Because the administration dosage of the monoclonal antibody injection is usually in the range of 100-600 mg, and the volume of the subcutaneous injection liquid is generally limited to less than 2mL, in the case, a highly concentrated protein preparation must be prepared, and the protein content can reach 100mg/mL or more.

High concentrations of monoclonal antibody injections present many challenges for manufacturing processes, process scaling, and ultimately patient administration. The most important challenge is the ultra-high viscosity, and due to the biopolymer properties of monoclonal antibodies, the interaction force (such as hydrophobicity, charge action, etc.) between protein molecules is gradually enhanced as the protein concentration is increased, and the solution is expressed as a highly viscous solution. Even in some extreme cases, gel-like substances are formed, and the feed liquid with the property brings about not small challenges to the ultrafiltration membrane and the ultrafiltration equipment, such as reduction of tangential flow rate caused by rapid rise of pressure difference during final concentration, gradual runaway of concentration polarization until the phenomenon that protein precipitation blocks the membrane occurs, and thus necessarily causes reduction of recovery rate or process failure. On the other hand, even if the final high concentration protein solution is obtained by modifying the device or the film-coating type, it is difficult to put it into practical clinical use because it is necessary to take up it with a disposable sterile syringe or to use a final packaging form of a prefilled needle at the time of subcutaneous administration, and excessively high viscosity results in the need of a higher upper limit of a bolus force than that of an adult at the time of injection, and application of self-subcutaneous injection cannot be achieved. Another difficulty in concentrating high concentration monoclonal antibody solutions by ultrafiltration is that protein samples tend to aggregate to form soluble aggregates during high concentration, further aggregating to form protein precipitates.

Accordingly, there is a need to develop a low-viscosity, high-concentration liquid formulation comprising an anti-human interleukin-33 monoclonal antibody that is effective in reducing aggregation of the monoclonal antibody and improving its stability.

Disclosure of Invention

In order to solve the problems in the prior art, the application provides a liquid preparation containing an anti-human interleukin-33 monoclonal antibody, wherein the liquid preparation contains the anti-human interleukin-33 monoclonal antibody with the protein concentration of 120-300 mg/mL and an amino acid additive with the amino acid concentration of 10-500 mM, and the liquid preparation has the advantages of low viscosity, high stability and less aggregation of the anti-human interleukin-33 monoclonal antibody, and can be used for injections, particularly subcutaneous injections.

QX007N is a recombinant humanized monoclonal antibody which is developed by oneself and targets interleukin-33, interleukin-33 (interleukin 33, IL-33) is a protein coded by human gene IL-33, which is a member of interleukin 1 family and can effectively drive the production of accessory factor Th 2. IL-33 is one of the ligands of ST 2. In the inactive state, IL-33 is bound in the nucleus in its precursor form (270 amino acid residues in length) in a cellular homeostasis by binding to chromatin binding motifs. When cells are injured, IL-33 can be released extracellularly as an endogenous danger signal, and the signal is mainly transmitted through a binding receptor ST2, so that the IL-33 plays roles in immune regulation and the like as a cytokine. QX007N can specifically bind to IL-33, and prevent immune cells from releasing proinflammatory cytokines, so that asthma exacerbation is prevented, asthma control is improved, and the like.

Currently, no targeted human IL-33 monoclonal antibody medicine on the market exists at home and abroad, wherein the best progress of Etokimab of the companies REGN3500 and AnapotyBio developed by the cooperation of the regenerant and the Xenoriflu is currently carried out in 2/3-stage clinical research, and the intended administration mode is intravenous injection or subcutaneous injection. From the perspective of reducing clinical use cost of biological preparations and improving patient compliance, the preferable dosage form of the anti-human IL-33 monoclonal antibody medicament is a high-protein water needle which is used for subcutaneous injection, and if QX007N is prepared into subcutaneous injection, the protein content of the subcutaneous injection is as high as 100-150 mg/mL.

The specific technical scheme of the application is as follows:

the application provides a liquid preparation containing an anti-human interleukin-33 monoclonal antibody, which is characterized in that,

the liquid preparation comprises an anti-human interleukin-33 monoclonal antibody and an amino acid additive;

wherein the protein concentration of the human interleukin-33 resisting monoclonal antibody is 120-300 mg/mL, preferably 120-200 mg/mL, and more preferably 120-160 mg/mL; the amino acid concentration of the amino acid additive is 10-500 mM;

the anti-human interleukin-33 monoclonal antibody comprises three heavy chain complementarity determining regions, CDR-H1, CDR-H2 and CDR-H3, and three light chain complementarity determining regions, CDR-L1, CDR-L2 and CDR-L3, wherein:

CDR-H1 has the sequence shown in SEQ ID NO: 1;

CDR-H2 has the sequence shown in SEQ ID NO: 2;

CDR-H3 has the sequence shown in SEQ ID NO: 3;

CDR-L1 has the sequence shown in SEQ ID NO: 4;

CDR-L2 has the sequence shown in SEQ ID NO: 5;

CDR-L3 has the sequence shown in SEQ ID NO: 6.

In the present application, the anti-human interleukin-33 monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein,

the heavy chain variable region has the sequence shown in SEQ ID NO: 7;

the light chain variable region has the sequence shown in SEQ ID NO: 8.

In the present application, the amino acid additive is selected from one or two or more of arginine hydrochloride, histidine, lysine, methionine, proline.

In the present application, the amino acid additive includes one or two or three of arginine hydrochloride, histidine and methionine;

preferably, the amino acid additive comprises arginine hydrochloride, and the concentration of the arginine hydrochloride in the liquid preparation is 100-200 mM;

preferably, the amino acid additive comprises histidine, and the concentration of histidine in the liquid preparation is 10-50 mM;

preferably, the amino acid additive comprises methionine, and the concentration of methionine in the liquid formulation is 2-50 mM.

In the application, the liquid preparation further comprises a surfactant, and the content of the surfactant is 0.01-5 mg/mL, preferably 0.1-2 mg/mL, and more preferably 0.2-1 mg/mL;

preferably, the surfactant is polysorbate 80.

In the present application, when the concentration of the anti-human interleukin-33 monoclonal antibody in the liquid preparation is 180mg/mL or more, the viscosity of the liquid preparation is 15cP or less;

optionally, the viscosity of the liquid formulation is below 10cP when the concentration of the anti-human interleukin-33 monoclonal antibody in the liquid formulation is above 150 mg/mL.

In the application, the liquid preparation further comprises sucrose, and the concentration of the sucrose is 20-140 mg/mL, preferably 50-90 mg/mL.

In the application, the liquid preparation further comprises sorbitol, and the concentration of the sorbitol is 10-200 mg/mL, preferably 20-100 mg/mL, and more preferably 50-80 mg/mL.

In the application, the liquid preparation further comprises sodium chloride, and the concentration of the sodium chloride is 20-300 mg/mL, preferably 100-200 mg/mL.

In the application, the pH value of the liquid preparation is 5.5-6.5.

ADVANTAGEOUS EFFECTS OF INVENTION

The liquid formulation of the present application, which contains an anti-human IL-33 monoclonal antibody, contains an anti-human interleukin-33 monoclonal antibody, and has a low viscosity, can be easily injected by a syringe, and thus can be used as an injection, particularly a subcutaneous injection.

The monoclonal antibody against human IL-33 of the present application has an affinity for binding to human interleukin-33 comparable to that of the conventional monoclonal antibody against human IL-33 (Etokimab/ANB020), and has a neutralizing activity at a cellular level comparable to that of Etokimab/ANB 020.

The monoclonal antibody drug (Itepekimab/REGN 3500) which is developed by the company Sonofuran and targets interleukin-33 is intended to be used for treating atopic dermatitis diseases such as chronic obstructive pulmonary disease (clinical stage III), asthma (clinical stage II) and the like. Etokimab/ANB020 developed by AnaptysBio Inc. was used for chronic rhinosinusitis (phase II clinical).

The monoclonal antibody of the application shows a neutralizing activity equivalent to that of Etokimab/ANB020 (prepared according to the expression of a patent published sequence) at a cellular level, and is expected to show a good clinical effect in the aspects of preventing and treating related diseases.

Drawings

FIG. 1 is a diagram showing the result of nucleic acid electrophoresis for constructing a transient expression plasmid of QX007N (HZD 78-70). Wherein, M: marker; strip 1: PCR product 78VH-Hu 25; strip 2: pQX2.1, HindIII/NheI; the strip 3: PCR product 78VK-Hu 3-CK; the strip 4: pQX1, HindIII/BamHI.

Fig. 2 is a transient expression flow diagram.

FIG. 3 is an electrophoretically detected image of QX007N (HZD 78-70).

FIG. 4 shows that QX007N (HZD78-70) and Etokimab/ANB020 neutralize recombinant human interleukin-33 induced HEK Blue^TMNF-. kappa.B/AP-1 Signaling Activity Profile in IL-33 cells.

FIG. 5 is a graph showing that QX007N (HZD78-70) and Etokimab/ANB020 neutralize natural human interleukin-33-induced HEK Blue^TMNF-. kappa.B/AP-1 Signaling Activity Profile in IL-33 cells.

FIG. 6 is a graph showing the activity of QX007N (HZD78-70) and Etokimab/ANB020 in neutralizing recombinant human interleukin-33 in inducing IL-5 release from KU812 cells.

FIG. 7 is a graph showing the activity of QX007N (HZD78-70) and Etokimab/ANB020 in neutralizing recombinant human interleukin-33 in inducing IFN-. gamma.release from human whole blood.

Detailed Description

The following description of the exemplary embodiments of the present application, including various details of the embodiments of the present application to assist in understanding, should be taken as exemplary only. Accordingly, those of ordinary skill in the art will recognize that various changes and modifications of the embodiments described herein can be made without departing from the scope and spirit of the present application. Also, descriptions of well-known functions and constructions are omitted in the following description for clarity and conciseness.

CDR-H1 has the sequence shown in SEQ ID NO:1 (SYHMI);

CDR-H2 has the sequence shown in SEQ ID NO:2 (VIYPNSNIYYATWAKG);

CDR-H3 has the sequence shown in SEQ ID NO:3 (TIYVHVYSALSI);

CDR-L1 has the sequence shown in SEQ ID NO:4 (QASESVLNEVS);

CDR-L2 has the sequence shown in SEQ ID NO:5 (FASKLAS);

CDR-L3 has the sequence shown in SEQ ID NO:6 (QQDWSMDNIDNA).

In this application, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 respectively represent heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR 3.

Specifically, the protein concentration of the anti-human interleukin-33 monoclonal antibody may be, for example, 120mg/mL, 130mg/mL, 140mg/mL, 150mg/mL, 160mg/mL, 170mg/mL, 180mg/mL, 190mg/mL, 200mg/mL, 210mg/mL, 220mg/mL, 230mg/mL, 240mg/mL, 250mg/mL, 260mg/mL, 270mg/mL, 280mg/mL, 290mg/mL, 300mg/mL, or the like.

The amino acid concentration of the amino acid additive may be, for example, 10mM, 30mM, 50mM, 70mM, 90mM, 110mM, 130mM, 150mM, 170mM, 190mM, 210mM, 230mM, 250mM, 270mM, 290mM, 310mM, 330mM, 350mM, 370mM, 390mM, 410mM, 430mM, 450mM, 470mM, 490mM, 500mM, or the like.

In the present application, "monoclonal antibody" means an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, with the exception of possible variant antibodies (e.g., containing naturally occurring mutations or produced during the production of monoclonal antibody preparations), such variants typically being present in minute amounts. Unlike 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 single determinant on the antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention can be prepared by a variety of techniques including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods using transgenic animals comprising all or part of a human immunoglobulin locus, such methods and other exemplary methods of preparing monoclonal antibodies being described herein.

In the present application, a "monoclonal antibody" is generally a human antibody, which can be prepared using techniques well known to those skilled in the art, for example, human antibodies are generally described in van Dijk, m.a. and van de Winkel, j.g., curr. opin. pharmacol.5: 368-.

Antibodies can be prepared by administering an immunogen to transgenic animals that have been modified to stimulate the production of fully human antibodies or fully antibodies with human variable regions against an antigen challenge, these animals typically containing a portion or all of the human immunoglobulin locus that replaces the endogenous immunoglobulin locus, or that is present extrachromosomally or randomly integrated into the animal. In such transgenic mice, the endogenous immunoglobulin locus has generally been inactivated, and for a review of the methods of obtaining human antibodies from transgenic animals, see Lonberg, N., Nat. Biotech. (Nature Biotechnology) 23:1117-1125 (2005). See also, for example, XENOMOUSE described in U.S. Pat. Nos. 6,075,181 and 6,150,584^TMA technique; U.S. Pat. No.5,770,429

A technique; U.S. Pat. No.7,041,870

Techniques, and as described in U.S. patent application publication No. US 2007/0061900

Provided is a technique. May for example be passed through withThe human variable regions from the whole antibodies generated by such animals are further modified in combination with human constant regions.

Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human hybrid myeloma cells for use in the Production of human Monoclonal antibodies have been described (see, e.g., Kozbor, D., J.Immunol.133:3001-3005 (1984); Brodeur, B.R.et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York (1987), pp.51-63; and Borner, P.et al., J.Immunol.147:86-95 (1991)). Human antibodies produced via human B-cell hybridoma technology are also described in Li, j.etal, proc.natl.acad.sci.usas 103:3557-3562 (2006). Other methods include those described in, for example, U.S. Pat. No.7,189,826 (which describes the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue,26 (4); 265-268 (which describes a human-human hybridoma). The human hybridoma technique (Trioma technique) is also described in Vollmers, H.P.and Brandlein, S., Historagy and Histopathology 20: 927-.

Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from phage display libraries derived from humans, and then such variable domain sequences can be combined with the desired human constant domains.

Human antibodies can also be selected based on antibody libraries, i.e., human antibodies can be isolated by screening combinatorial libraries for antibodies having a desired activity or activities. For example, various methods for producing phage display libraries and screening such libraries for antibodies possessing desired binding characteristics are known in the art. Such Methods are reviewed, for example, in Hoogenboom, H.R.et al, Methods in Molecular Biology178:1-37(2001), and are further described, for example, in McCafferty, J.et al, Nature348:552-554 (1990); clackson, T.et al, Nature 352: 624-; marks, J.D.et al, J.mol.biol.222:581-597 (1992); marks, J.D.and Bradbury, A., Methods in Molecular Biology 248:161-175 (2003); sidhu, S.S.et al, J.mol.biol.338:299-310 (2004); lee, C.V.et al, J.mol.biol.340:1073-1093 (2004); fellouse, F.A., Proc.Natl.Acad.Sci.USA 101: 12467-; and Lee, C.V.et al, J.Immunol.methods 284:119-132 (2004).

In some phage display methods, repertoires of VH and VL genes are separately cloned by Polymerase Chain Reaction (PCR) and randomly recombined in a phage library, which is then screened for antigen-binding phages, as described in Winter, G.et al, Ann.Rev.Immunol.12:433-455 (1994). Phage typically display antibody fragments either as single chain fv (scfv) fragments or as Fab fragments. Libraries from immunized sources provide high affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, the non-immune repertoire can be cloned (e.g., from humans) to provide a single source of antibodies to a large panel of non-self and also self antigens in the absence of any immunization, as described by Griffiths, A.D.et al, EMBO J,12:725 (1993). Finally, the generation of an unimmunized library can also be synthesized by cloning unrearranged V gene segments from stem cells and encoding the highly variable CDR3 regions using PCR primers containing random sequences and effecting rearrangement in vitro, as described by Hoogenboom, H.R.and Winter, G., J.Mol.biol.227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example: U.S. Pat. No.5,750,373 and U.S. patent publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936 and 2009/0002360.

The antibody may also be a multispecific antibody, e.g., a bispecific antibody. Bispecific antibodies are monoclonal antibodies having binding specificity for at least two different sites. Techniques for generating multispecific antibodies include, but are not limited to, recombinant co-expression of two pairs of immunoglobulin heavy and light chains with different specificities (see Milstein, C.and Cuello, A.C., Nature305:537-540 (1983); WO 93/08829; and Traunecker, A.et al., EMBO J.10:3655-3659(1991)), and "node-in-hole" engineering (see, e.g., U.S. Pat. No.5,731,168). Effects can also be manipulated electrostatically by engineering the molecules for the generation of antibody Fc-heterodimers (WO 2009/089004); crosslinking two or more antibodies or fragments (see, e.g., U.S. Pat. No.4,676,980 and Brennan, M.et al, Science 229:81-83 (1985)); the use of leucine zippers to generate bispecific antibodies (see, e.g., Kostelny, S.A.et al, J.Immunol.148:1547-1553 (1992)); the "diabody" technique used to generate bispecific antibody fragments was used (see, e.g., Holliger, P.et al., Proc. Natl. Acad. Sci. USA 90: 6444-; and the use of single chain fv (scFv) dimers (see, e.g., Gruber, M.et al., J.Immunol.152:5368-5374 (1994)); and making a trispecific antibody (as described, for example, in Tutt, a.et al, j.immunol.147:60-69 (1991)) to generate a multispecific antibody.

Monoclonal antibodies described herein also include engineered antibodies having three or more functional antigen binding sites, including "octopus antibodies" (see, e.g., US 2006/0025576).

Antibodies herein also include multispecific antibodies described in WO 2009/080251, WO 2009/080252, WO2009/080253, WO 2009/080254, WO 2010/112193, WO 2010/115589, WO2010/136172, WO 2010/145792, and WO 2010/145793, WO 2011/117330, WO 2012/025525, WO 2012/025530, WO 2013/026835, WO2013/026831, WO 2013/164325, or WO 2013/174873.

The monoclonal antibodies described herein may also be antibody variants, for example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of an antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into, and/or substitutions of, residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, so long as the final construct possesses the desired characteristics, e.g., antigen binding. Thus, in certain embodiments, antibody variants are provided having one or more amino acid substitutions in which the site of interest for the substitution mutation comprises an HVR and an FR, e.g., amino acid substitutions can be introduced into an antibody of interest and screened for products having a desired activity, e.g., retained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

In the present application, the mammalian cells used for the in vitro fermentation production of the monoclonal antibody include, but are not limited to, various hybridoma cells, chinese hamster ovary Cells (CHO), and preferably CHO cells, which are currently used.

In this application, "affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" as used in this specification refers to an intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be determined by the equilibrium dissociation constant (K)_D) And (4) showing. Affinity can be measured by common methods known in the art.

In the present application, "ultrafiltration" refers to Tangential Flow Filtration (TFF), which is a Filtration mode in which the Flow direction of a liquid is Tangential (parallel) to the surface of a filter membrane, and compared with a conventional Filtration mode (NFF) in which the Flow direction is perpendicular to the filter membrane, the Tangential Flow filter membrane has less particle accumulation on the surface of the filter membrane, has stable Filtration speed, and is suitable for separation of large-volume samples. Wherein, the molecules larger than the membrane aperture are intercepted and gradually concentrated, and the substances smaller than the membrane aperture permeate the membrane and are separated from the macromolecular solution, thereby realizing the separation of macromolecules and micromolecules. Tangential flow ultrafiltration is thus commonly used for concentration of biologicals, dialysis, displacement of buffer solutions, separation of molecules of different sizes, etc.

In this application, human interleukin-33 means that human interleukin-33 located in the nucleus is hydrolyzed by protease to form mature human interleukin-33, which is secreted to the outside of the cell to exert the biological activity of human interleukin-33, and has the amino acid sequence shown in SEQ ID NO: 9, or a pharmaceutically acceptable salt thereof.

SEQ ID NO：9：

SITGISPITEYLASLSTYNDQSITFALEDESYEIYVEDLKKDEKKDKVLLSYYESQHPSNESGDGVDGKMLMVTLSPTKDFWLHANNKEHSVELHKCEKPLPDQAFFVLHNMHSNCVSFECKTDPGVFIGVKDNHLALIKVDSSENLCTENILFKLSET

In the present application, the "anti-human interleukin-33 monoclonal antibody" means a monoclonal antibody that: which is capable of binding human interleukin-33 with sufficient affinity such that the monoclonal antibody is useful as a diagnostic and/or therapeutic agent targeting human interleukin-33.

In the present application, the anti-human interleukin-33 monoclonal antibody does not bind to a target-independent protein. Here, "irrelevant protein" means a protein other than human interleukin-33 as a target; here, "not to bind" means: the binding ability of the anti-human IL-33 monoclonal antibody of the present invention to the unrelated protein is less than 10%, for example, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0, assuming that the binding ability of the anti-human IL-33 monoclonal antibody of the present invention to human interleukin-33 as its target is 100%.

The anti-human IL-33 monoclonal antibody of the present application may bind to interleukin-33 of human or cynomolgus monkey, and may not bind to interleukin-33 of other animal species. Here, "other animal species" means animal species other than human and cynomolgus monkey, such as pig, dog, rabbit, rat, mouse, guinea pig, and the like; here, in determining the species specificity of the anti-human IL-33 monoclonal antibody of the present application, "not binding" means: when the binding ability of the anti-human IL-33 of the present invention to human interleukin-33 as its target is defined as 100%, the binding ability of the anti-human IL-33 monoclonal antibody of the present invention to interleukin-33 of another animal species is less than 5%, for example, 4%, 3%, 2%, 1%, or 0%.

The anti-human IL-33 monoclonal antibodies of the present application have an equilibrium dissociation constant (K) of 1 μ M or less, 100nM or less, 50nM or less, 40nM or less_D)。

The experimental results show that the anti-human IL-33 monoclonal antibody of the application can specifically bind to human interleukin-33.

The anti-human IL-33 monoclonal antibody of the application is equivalent to or superior to the monoclonal antibody products of the same type on the market in terms of various biological activities. The biological activities include, for example, the activity of neutralizing recombinant/natural human interleukin-33 to induce NF-. kappa.B/AP-1 signal transduction in cells, the activity of neutralizing interleukin-33 to induce IL-5 release from KU812 cells, the activity of neutralizing interleukin-33 to induce IFN-. gamma.release from human whole blood, etc.

In one embodiment, the amino acid sequence of the heavy chain of an anti-human IL-33 monoclonal antibody of the present application is as set forth in SEQ ID NO: 10 is shown in the figure; the amino acid sequence of the light chain is shown as SEQ ID NO: shown at 11.

SEQ ID NO：10：

EVQLVESGGGLVQPGGSLRLSCAASGFSLSSYHMIWVRQAPGKGLEWVGVIYPNSNIYYATWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTIYVHVYSALSIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO：11：

AFQMTQSPSSVSASVGDRVTITCQASESVLNEVSWYQQKPGKAPKLLIYFASKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQDWSMDNIDNAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Wherein, SEQ ID NO: 10 and 11 are both humanized sequences.

In one embodiment, the anti-human interleukin-33 monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein,

the heavy chain variable region has the amino acid sequence shown as SEQ ID NO: 7 (EVQLVESGGGLVQPGGSLRLSCAASGFSLSSYHMIWVRQAPGKGLEWVGVIYPNSNIYYATWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTIYVHVYSALSIWGQGTLVTVSS);

the light chain variable region has the sequence shown in SEQ ID NO: 8 (AFQMTQSPSSVSASVGDRVTITCQASESVLNEVSWYQQKPGKAPKLLIYFASKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQDWSMDNIDNAFGGGTKVEIK).

In one embodiment, the amino acid additive (amino acid protector) is selected from one or two or more of arginine hydrochloride, histidine, lysine, methionine and proline.

In one embodiment, the amino acid additive is selected from one or two or more of arginine hydrochloride, histidine, lysine, methionine, proline.

In one embodiment, the amino acid additive comprises one or two or three of arginine hydrochloride, histidine and methionine.

In one embodiment, the amino acid additive comprises arginine hydrochloride, and the concentration of arginine hydrochloride in the liquid formulation is 100-200 mM, and may be, for example, 100mM, 110mM, 120mM, 130mM, 140mM, 150mM, 160mM, 170mM, 180mM, 190mM, 200mM, and the like.

In one embodiment, the amino acid additive comprises histidine, and the concentration of histidine in the liquid formulation is 10 to 50mM, and may be, for example, 10mM, 12mM, 14mM, 16mM, 18mM, 20mM, 22mM, 24mM, 26mM, 28mM, 30mM, 32mM, 34mM, 36mM, 38mM, 40mM, 42mM, 44mM, 46mM, 48mM, 50mM, etc.

In one embodiment, the amino acid additive comprises methionine, and the concentration of methionine in the liquid formulation is 2 to 50mM, and may be, for example, 2mM, 4mM, 6mM, 8mM, 10mM, 12mM, 14mM, 16mM, 18mM, 20mM, 22mM, 24mM, 26mM, 28mM, 30mM, 32mM, 34mM, 36mM, 38mM, 40mM, 42mM, 44mM, 46mM, 48mM, 50mM, and the like.

In one embodiment, the amino acid additive comprises arginine hydrochloride and histidine, wherein the concentration of arginine hydrochloride is 100-200 mM and the concentration of histidine is 10-50 mM in the liquid formulation.

In one embodiment, the amino acid additive comprises arginine hydrochloride and methionine, and the concentration of arginine hydrochloride is 100-200 mM and the concentration of methionine is 2-50 mM in the liquid formulation.

In one embodiment, the amino acid additive comprises histidine and methionine, and the concentration of histidine is 10 to 50mM and the concentration of methionine is 2 to 50mM in the liquid formulation.

In one embodiment, the amino acid additive comprises arginine hydrochloride, histidine and methionine, wherein arginine hydrochloride is present in a concentration of 100 to 200mM, preferably 150mM, histidine is present in a concentration of 10 to 50mM, preferably 20mM, and methionine is present in a concentration of 2 to 50mM, preferably 10mM, in the liquid formulation.

In one embodiment, the amino acid additive consists of arginine hydrochloride, histidine and methionine, wherein arginine hydrochloride has a concentration of 100-200 mM, preferably 150mM, histidine has a concentration of 10-50 mM, preferably 20mM, and methionine has a concentration of 2-50 mM, preferably 10 mM.

In one embodiment, the liquid preparation further comprises a surfactant, wherein the surfactant is contained in an amount of 0.01 to 5mg/mL, preferably 0.1 to 2mg/mL, more preferably 0.2 to 1mg/mL, and may be, for example, 0.01mg/mL, 0.1mg/mL, 0.2mg/mL, 0.3mg/mL, 0.4mg/mL, 0.5mg/mL, 0.6mg/mL, 0.7mg/mL, 0.8mg/mL, 0.9mg/mL, 1mg/mL, 2mg/mL, 3mg/mL, 4mg/mL, 5mg/mL, or the like. The surfactant used in the present application is not limited in kind, and may be, for example, polysorbate 80.

In a specific embodiment, the viscosity of the liquid formulation is 15cP or less at a concentration of the anti-human interleukin-33 monoclonal antibody in the liquid formulation of 180mg/mL or more.

In a specific embodiment, the viscosity of the liquid formulation is 10cP or less at a concentration of the anti-human interleukin-33 monoclonal antibody in the liquid formulation of 150mg/mL or more.

In one embodiment, the liquid preparation further comprises sucrose, and the concentration of the sucrose is 20-140 mg/mL, preferably 50-90 mg/mL, and may be, for example, 20mg/mL, 30mg/mL, 40mg/mL, 50mg/mL, 60mg/mL, 70mg/mL, 80mg/mL, 90mg/mL, 100mg/mL, 120mg/mL, 140mg/mL, or the like.

In one embodiment, the liquid preparation further comprises sorbitol, wherein the concentration of sorbitol is 10-200 mg/mL, preferably 20-100 mg/mL, more preferably 50-80 mg/mL, and may be, for example, 10mg/mL, 20mg/mL, 30mg/mL, 40mg/mL, 50mg/mL, 60mg/mL, 70mg/mL, 80mg/mL, 90mg/mL, 100mg/mL, 120mg/mL, 140mg/mL, 160mg/mL, 180mg/mL, 200mg/mL, or the like.

In one embodiment, the liquid formulation further comprises sodium chloride, wherein the concentration of the sodium chloride is 20-300 mg/mL, preferably 100-200 mg/mL, and may be, for example, 20mg/mL, 30mg/mL, 40mg/mL, 50mg/mL, 60mg/mL, 70mg/mL, 80mg/mL, 90mg/mL, 100mg/mL, 120mg/mL, 140mg/mL, 160mg/mL, 180mg/mL, 200mg/mL, 220mg/mL, 240mg/mL, 260mg/mL, 280mg/mL, 300mg/mL, or the like.

In a specific embodiment, the pH of the liquid formulation is 5.5 to 6.5, and may be, for example, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, and the like.

Examples

Hereinafter, the present application will be described more specifically with reference to examples. It should be understood that the present application is not limited to these embodiments.

EXAMPLE 1 preparation of anti-human Interleukin-33 monoclonal antibody QX007N

Human interleukin-33 is purchased from Shanghai near-shore science and technology Limited, and is used for immunizing New Zealand rabbits, and a B cell cloning technology is used for obtaining an antigen binding specific antibody clone, so that a monoclonal antibody which is bound with the human interleukin-33 and has the human interleukin-33 inhibitory activity is screened. Firstly, detecting cell supernatant by Binding ELISA, and selecting a clone combined with human interleukin-33; reuse of HEK Blue^TMIL-33 reporter gene cell assay was performed to select clones with human interleukin-33 inhibitory activity. The immunization and screening process is entrusted to a commercial company for completion.

12 clones were selected in sequence for recombinant expression and sequencing. 78# was determined to have optimal cell neutralizing activity and was humanized engineered to 78 #. Carrying out homology alignment of human IgG embryonic line sequences (Germine) by using NCBI Igblast, selecting IGHV3-66 a 01 as a heavy chain CDR grafting template, and grafting CDR regions (namely CDR-H1(SEQ ID No:1), CDR-H2(SEQ ID No:2) and CDR-H3(SEQ ID No:3)) of the heavy chain of clone No. 78 into a framework region of IGHV3-66 a 01; selecting IGKV1-12 x 01 as light chain CDR grafting template, grafting CDR regions (namely CDR-L1(SEQ ID No:4), CDR-L2(SEQ ID No:5) and CDR-L3(SEQ ID No:6)) of 78# clone light chain into framework region of IGKV1-12 x 01; the framework region was back-mutated at a specific site to obtain the variable region of monoclonal antibody QX007N of the present application. Finally, the humanized heavy chain variable region has the sequence as shown in SEQ ID NO: 7; the humanized light chain variable region has the amino acid sequence shown as SEQ ID NO: 8.

The gene of the heavy chain variable region (SEQ ID NO: 7) and the gene of the light chain full length (SEQ ID NO: 11) were obtained by PCR amplification. The heavy chain expression plasmid pQX2.1 is cut by HindIII and NheI enzyme; the HindIII and BamHI are used for double enzyme digestion of the transient expression plasmid pQX 1; the PCR amplified genes were inserted into the corresponding expression plasmids using the Infusion recombinase to construct the heavy chain expression plasmid pQX2.1-78VH-Hu25 and the light chain expression plasmid pQX2.2-78VK-Hu3, respectively. Wherein, pQX2.2 refers to pQX1 plasmid expressing light chain.

The results of double restriction by electrophoresis of nucleic acids are shown in FIG. 1. As can be seen from the results in FIG. 1, the PCR amplification results of the heavy chain variable region and the light chain of the antibody are full length, and the results of double digestion of the heavy chain and light chain expression plasmids are obtained, wherein the sizes of the heavy chain and light chain plasmids are about 5000bp, the light chain full length is about 781bp, and the heavy chain variable region is about 480 bp.

The correct sequence of the heavy chain expression plasmid pQX2.1-78VH-Hu25 and the light chain expression plasmid pQX2.2-78VK-Hu3 were co-transfected into ExpicHO-S cells. One day before transfection, ExpCHO-S cells were diluted to 3X 10⁶Individual cells/ml were passaged before transfection. On the day of transfection, cell density was diluted to 6X 10⁶Individual cells/ml, 125ml shake flasks with 25ml cells, waiting for transfection. The transfection and expression process is shown in FIG. 2.

On day 5 post-transfection, culture supernatants were harvested and purified in one step with ProteinA. The purified antibody was detected by SDS-PAGE and designated as QX007N (HZD78-70), and the results of detection of the antibody by protein electrophoresis are shown in FIG. 3. The protein electrophoresis was performed using a denaturing reduced gel, and the results in FIG. 3 show two bands, approximately 50kDa and 25kDa in size, respectively, consistent with the theoretical molecular weights of the heavy (49.3kDa) and light (23.4kDa) chains.

Example 2 equilibrium dissociation constant (K)_D) Measurement of (2)

BiacoreT200 was used to test the affinity of QX007N (HZD78-70) for human interleukin-33, all at 25 ℃. A commercial Protein A chip is adopted, and a proper amount of antibody is fixed by a capture method, so that Rmax is about 50RU, and the capture flow rate is 10 mul/min. The antigen is subjected to gradient dilution, the flow rate of the instrument is switched to 30 mul/min, the antigen sequentially flows through a reference channel and a channel for fixing the antibody according to the sequence of the concentration from low to high, and the antigen flows through a buffer solution to be used as a negative control. After each binding and dissociation, the chip was regenerated with glycine of pH 1.5. Selecting a 1:1 binding model in Kinetics options by using self-contained analysis software of an instrument for fitting, and calculating a binding rate constant k of the antibody_aDissociation rate constant k_dAnd equilibrium dissociation constant K_DThe value is obtained.

In addition, when QX007N (HZD78-70) was compared with the affinity of Etokimab/ANB020, a human interleukin-33 monoclonal antibody developed by AnatypBio, the method of detection of the known antibody was the same as that of QX007N, and the results are shown in Table 1. Wherein the Etokimab/ANB020 constructs an expression plasmid according to an APE4909 sequence provided by a patent WO2015106080A2, and is obtained by transforming an ExpiCHO-S cell in self.

TABLE 1

Example 3 neutralization of human Interleukin-33 induced HEK Blue^TMActivity detection of NF- κ B/AP-1 Signal transduction in IL-33 cells

HEK Blue^TMIL-33 cells were generated by stable transfection of human embryonic kidney cells HEK 293 with the human IL1RL1 gene, and TNF-. alpha.and IL-1. beta. responses were blocked, thus HEK-Blue^TMIL-33 cells respond specifically to IL-33. Interleukin-33 binds to cell surface IL-1RL1/IL-1RAcP triggering a signaling cascade, resulting in NF-. kappa.B/AP-1 signaling and production of secreted alkaline phosphatase (SEAP), thereby detecting the biological activity of interleukin-33 or performing antibody screening.

Using HEK Blue^TMIL-33 cell assay QX007N (HZD 7)8-70) neutralizing activity against human interleukin-33. HEK Blue^TMIL-33 cells at 4X 10 per well⁴Individual cells were plated into 96 wells at 37 ℃ and 5% CO₂Incubated overnight under conditions. Diluting the antibody to a concentration range of 0-500 ng/ml, mixing the diluted solution with 2ng/ml recombinant human interleukin-33, incubating for 1h, adding into cells at 37 deg.C and 5% CO₂Culturing for 24 hr, collecting cell culture supernatant, adding QUANTI-Blue at a ratio of 1:10^TMIn a detection reagent (InvivoGen, rep-qbs2), and reacted at 37 ℃ for 1 hour, and OD was detected using a Varioskan LUX multifunctional microplate reader_630nmValues, data were analyzed using softMaxPro software using four-parameter curve fitting (fig. 4) to analyze antibody antagonistic activity.

The result shows that QX007N (HZD78-70) can inhibit HEK Blue induced by recombinant human interleukin-33^TMNF- κ B/AP-1 Signal transduction in IL-33 cells, IC thereof₅₀It was 6.67 ng/ml.

Example 4 neutralization of native human Interleukin-33 induced HEK Blue^TMActivity detection of NF- κ B/AP-1 Signal transduction in IL-33 cells

Natural human interleukin-33 was prepared, and the neutralizing activity of QX007N (HZD78-70) against natural human interleukin-33 was verified. Culturing HFL-1 cells, inducing with 200ng/ml TNF-alpha for 24 hr, collecting cells, lysing the cells by repeated freeze thawing, collecting cell lysate supernatant containing human interleukin-33, and processing with HEK Blue^TMIL-33 cells demonstrated activity.

HEK Blue^TMIL-33 cells at 4X 10 per well⁴Individual cells were plated into 96 wells at 37 ℃ and 5% CO₂Culturing overnight under the condition, diluting the antibody to the concentration of 0-1000 ng/ml, adding the diluent and natural human interleukin-33, mixing, adding into cells at 37 deg.C and 5% CO₂Culturing for 24 hr, collecting cell culture supernatant, adding QUANTI-Blue at a ratio of 1:10^TMIn the detection reagent, and reacted at 37 ℃ for 1 hour, and OD was detected using a Varioskan LUX multifunctional microplate reader_630nmValues, analysis of the antibodies using 4-parameter curve fitting analysis of the data using SoftMax Pro software (FIG. 5)The activity was neutralized.

The results in FIG. 5 show that QX007N (HZD78-70) was able to inhibit natural human interleukin-33 from inducing HEK Blue^TMNF- κ B/AP-1 Signal transduction in IL-33 cells, IC thereof₅₀It was 3.91 ng/ml.

EXAMPLE 5 Activity assay for neutralizing IL-5 Release from human Interleukin-33 induced KU812 (human peripheral blood basophilic leukemia cells)

The activity of QX007N (HZD78-70) in neutralizing human interleukin-33 was evaluated using as an index that human interleukin-33 induces the release of IL-5 from KU812 (human peripheral blood basophilic leukemia cells). KU812 cells (2X 10) were seeded in 96-well plates⁵Cells/well), followed by addition of antibody and recombinant human interleukin-33 (final concentration 4ng/ml) at 37 ℃ and 5% CO₂Cultured for 24 hours under the conditions, and the cell culture supernatant was collected by means of Human IL-5DuoSet ELISA (R)&D, DY205) and detecting the expression level of IL-5 in the supernatant, and OD was detected using a Varioskan LUX multifunctional microplate reader_450nmData were analyzed using 4-parameter curve fitting using SoftMax Pro software (figure 6) to further analyze the neutralizing activity of the antibodies.

The results in FIG. 6 show that QX007N (HZD78-70) was able to neutralize IL-5 releasing activity of human interleukin-33-induced KU812 (human peripheral blood basophilic leukemia cells), the IC thereof₅₀5.87 ng/ml.

Example 6 Activity assay for neutralizing human Interleukin-33 inducing human Whole blood to release IFN- γ

The neutralization activity of QX007N (HZD78-70) was further characterized by taking monocytes in human whole blood as the basis of the assay and IFN-gamma as the index of the assay. Whole blood from healthy volunteers was plated (100. mu.L/well) followed by addition of antibody and recombinant human interleukin-33 (final concentration 4ng/ml) at 37 ℃ and 5% CO₂Incubated for 24 hours under the conditions, and OD was detected using a Varioskan LUX multifunctional microplate reader_450nmValues, the data were analyzed using 4-parameter curve fitting using SoftMax Pro software (figure 7) to further analyze the antagonistic activity of the antibodies.

The results in FIG. 7 show that QX007N (HZD78-70) was able to neutralize the activity of human interleukin-33 to induce IFN-. gamma.release from human whole blood with an IC50 of 16 ng/ml.

Example 7 high concentration Ultrafiltration concentration of anti-human IL-33 monoclonal antibody

CHO cells are used as host cells and are fermented on a bioreactor with the scale of 2L to produce the antibody QX007N obtained in example 1, clarified fermentation liquor is obtained by a centrifugal machine or a deep membrane filtration, Protein A chromatography is adopted to capture target Protein, and low pH value virus inactivation and anion and cation chromatography are carried out to remove impurities, thus obtaining an intermediate sample to be ultrafiltered.

Adopting the intermediate to carry out ultrafiltration, wherein the ultrafiltration equipment adopts a Labscale small-sized ultrafiltration instrument (clamping two 50cm pieces) of Mercury²Pellicon XL membrane package, cutoff 30 kDa). At 120 to 300L/m²H, concentrating the intermediate sample to about 30-50 mg/mL on the basis of maintaining TMP at 0.6-1.5 bar; then, performing replacement by using equal volume of buffer solution, wherein the replacement buffer solution is 20mM histidine-hydrochloric acid buffer solution, the pH value is 6.0, and when the volume of the histidine-hydrochloric acid buffer solution is 7 times of the weight of the concentrated sample, the replacement is finished to obtain ultrafiltration replacement concentrated solution; sampling and measuring the concentration of the protein in the ultrafiltration displacement concentrated solution, calculating the theoretical volume of the ultrafiltration displacement concentrated solution at the moment, adding 1/9 amino acid additive mother liquor of the theoretical volume of the ultrafiltration displacement concentrated solution, uniformly mixing to obtain a buffer system shown in table 2, and concentrating by using a merck millipore 30kDa ultrafiltration centrifugal tube until the concentration of the protein is the concentration of the table 2 to obtain a concentrated solution of the anti-human IL-33 monoclonal antibody.

The concentration of QX007N in the concentrated solution was measured by uv spectrophotometry using the following method:

1. the wavelength of the spectrophotometer was adjusted to 280nm and the calibration was performed using blank buffer or water as a control.

2. Diluting a sample with blank buffer solution or water to be detected, measuring the light absorption value of the sample at 280nm (the light absorption value is ensured to be between 0.5 and 1.5), and calculating the concentration of the sample according to the following formula (the extinction coefficient of QX007N is 1.513).

The viscosity of the concentrated solution was measured using a μ VISC viscometer from Sharp, and the procedure was as follows:

1. taking a disposable special syringe, extracting 200-400 microliters of a sample to be detected, exhausting bubbles in the syringe, and lightly wiping residual liquid with dust-free paper;

2. placing the injector into a pipe groove for fixing, clicking an instrument host interface, setting a shearing rate, clicking 'Run' to detect after setting is finished, recording a viscosity value, and generally measuring for multiple times to obtain an average value;

3. for the detection of a plurality of samples, after the previous sample is detected, the injector is taken out, the liquid in the tube is emptied, and after the tube opening is lightly wiped by the dust-free paper, the next sample can be sucked and measured.

After filtering the samples with a 0.2 μm filter, the samples were mixed with different buffers or additive stocks of Table 2 so that the protein concentration is shown in Table 2, and then the viscosity values were determined as described above, and the results are shown in Table 2.

TABLE 2

As can be seen from the results in Table 2, the viscosity of the anti-human IL-33 antibody increased exponentially with the increase in protein concentration, and the viscosity of the monoclonal antibody reached 31.1cP at a concentration of 200mg/mL without the addition of additives, which is a high value at which concentration could not be achieved using conventional ultrafiltration membranes. The viscosity value of the antibody under the same protein concentration condition can be reduced to below 50% of the original value and is less than 15cP by adding 100-200 mM of amino acid or a combination of the amino acid and the amino acid. It is generally accepted that liquid formulations having a viscosity of less than 10cP are suitable for use as subcutaneous injections. The viscosity value of the sample liquid medicine can be obviously reduced by adding the liquid preparation of sodium chloride and an amino acid protective agent (arginine hydrochloride, lysine, proline or a combination of several amino acids), and the amino acid protective agent has an additional anti-aggregation effect, so that the amino acid protective agent is preferably used as a viscosity regulator, and the subcutaneous injection administration concentration of the QX007N monoclonal antibody injection can reach at least 150 mg/mL.

Example 8 preparation of a Low viscosity liquid formulation comprising a high concentration of QX007N

By performing affinity chromatography, low pH inactivation, anion exchange chromatography, cation exchange chromatography, hydrophobic chromatography, tangential flow ultrafiltration concentration, etc. on CHO cell fermentation broth expressing anti-human IL-33 monoclonal antibody, adding mother liquor of amino acid protectant before the second step of ultrafiltration concentration, viscosity value during concentration can be greatly reduced, and ultrafiltration concentrate (containing 20mM buffer salt such as histidine salt, citrate or other buffer salt, 150mM arginine hydrochloride, pH 6.0) can be obtained. Then the mixture is evenly mixed with mother liquor of polysorbate 80 as a surfactant, and the mixture is filtered by a filter membrane of 0.2 mu m to obtain stock solution. The SEC-HPLC analysis confirmed that the sample contained the active ingredient QX007N monomer of more than 95%, the polymer of less than 2%, and substantially no host-foreign protein. The concentration of QX007N in the ultrafiltration concentrate was approximately 150mg/mL as determined by UV spectrophotometry. The viscosity of this feed liquid was measured to be 6.1cP using a μ VISC viscometer of Sharey.

The concentration of the antibody in the obtained liquid preparation was measured by the above-mentioned UV spectrophotometry.

The obtained liquid preparation is subjected to purity detection by an SEC-HPLC method, and the operation steps are as follows:

1. high performance liquid chromatography (Agilent 1260 or equivalent instrument), chromatography column (Waters,

specification: 3.5 μm, 7.8 × 300 mm; or an equivalent chromatographic column), wherein the mobile phase is PBS buffer solution;

2. unscrewing a quaternary pump pipeline, flushing the pipeline for 3 minutes by 100% mobile phase at the flow rate of 5mL/min, screwing a quaternary pump switch, connecting a chromatographic column, and flushing the chromatographic column by 100% mobile phase at the flow rate of 1.0mL/min for 30 minutes;

3. sample analysis method settings: the flow rate is 1.0mL/min, the analysis time is 15min, and the sample amount is 50 mg; the detection wavelength is 280 nm; before sample injection, the system adaptability is checked (continuous sample injection is carried out by using a reference substance, the sample injection frequency is not less than 5 times, and the result is calculated after 5 times), then 1-needle blank sample is analyzed, and then the sample is analyzed.

4. The chromatographic peaks of each sample are sequentially integrated according to the sequence of polymers, main peaks, degradation products and the like, all chromatographic peaks which are inconsistent with the retention time of a blank sample are integrated, the ratio of each peak is calculated by an area normalization method, and the measurement result is shown in table 3, wherein the QX007N monomer in the liquid preparation is more than 95 percent, the polymer is less than 2 percent, and the liquid preparation is basically free of host impurity proteins.

TABLE 3

From the results in Table 3, it is clear that the viscosity of the second concentration step of the ultrafiltration process was reduced from the original 31.5cP to 10.5cP after arginine addition, and that arginine hydrochloride also acts as a protectant to some extent to inhibit polymer formation. After the high-concentration anti-human IL-33 monoclonal antibody is added with arginine hydrochloride, the viscosity value is obviously reduced, excessive back pressure is not caused, the control of the flow rate and the membrane pressure of the ultrafiltration process is facilitated, and the operability of the process is improved.

The foregoing is directed to preferred embodiments of the present application, other than the limiting examples of the present application, and variations of the present application may be made by those skilled in the art using the foregoing teachings. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present application still belong to the protection scope of the technical solution of the present application.

Sequence listing

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Ile Asp Asn Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

Claims

1. A liquid preparation comprising an anti-human interleukin-33 monoclonal antibody,

wherein the protein concentration of the human interleukin-33 resisting monoclonal antibody is 120-300 mg/mL, and the amino acid concentration of the amino acid additive is 10-500 mM;

the anti-human interleukin-33 monoclonal antibody comprises three heavy chain complementarity determining regions CDR-H1, CDR-H2 and CDR-H3 and three light chain complementarity determining regions CDR-L1, CDR-L2 and CDR-L3, wherein:

the amino acid sequence of CDR-H1 is shown in SEQ ID NO:1 is shown in the specification;

the amino acid sequence of CDR-H2 is shown in SEQ ID NO:2 is shown in the specification;

the amino acid sequence of CDR-H3 is shown in SEQ ID NO:3 is shown in the specification;

the amino acid sequence of CDR-L1 is shown in SEQ ID NO:4 is shown in the specification;

the amino acid sequence of CDR-L2 is shown in SEQ ID NO:5 is shown in the specification;

the amino acid sequence of CDR-L3 is shown in SEQ ID NO: and 6.

2. The liquid preparation according to claim 1, wherein the protein concentration of the anti-human interleukin-33 monoclonal antibody is 120 to 200 mg/mL.

3. The liquid preparation according to claim 1, wherein the protein concentration of the anti-human interleukin-33 monoclonal antibody is 120 to 160 mg/mL.

4. The liquid formulation of claim 1, wherein the anti-human interleukin-33 monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein,

the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 7 is shown in the specification;

the amino acid sequence of the light chain variable region is shown as SEQ ID NO: shown in fig. 8.

5. The liquid preparation according to any one of claims 1 to 4, wherein the amino acid additive is one or two or more selected from arginine hydrochloride, histidine, lysine, methionine and proline.

6. The liquid formulation of any one of claims 1 to 4, wherein the amino acid additive comprises one or two or three of arginine hydrochloride, histidine and methionine.

7. The liquid formulation of any one of claims 1 to 4, wherein the amino acid additive comprises arginine hydrochloride, and the concentration of arginine hydrochloride in the liquid formulation is 100 to 200 mM.

8. The liquid formulation of any one of claims 1 to 4, wherein the amino acid additive comprises histidine, and the concentration of histidine in the liquid formulation is 10 to 50 mM.

9. The liquid formulation of any one of claims 1 to 4, wherein the amino acid additive comprises methionine, and the concentration of methionine in the liquid formulation is 2 to 50 mM.

10. The liquid preparation according to any one of claims 1 to 4, further comprising a surfactant in an amount of 0.01 to 5 mg/mL.

11. The liquid preparation according to claim 10, wherein the surfactant is contained in an amount of 0.1 to 2 mg/mL.

12. The liquid preparation according to claim 10, wherein the surfactant is contained in an amount of 0.2 to 1 mg/mL.

13. The liquid formulation of claim 10, wherein the surfactant is polysorbate 80.

14. The liquid preparation according to any one of claims 1 to 4, wherein the viscosity of the liquid preparation is 15cP or less at a concentration of 180mg/mL or more of the anti-human interleukin-33 monoclonal antibody in the liquid preparation.

15. The liquid preparation according to any one of claims 1 to 4, wherein the viscosity of the liquid preparation is 10cP or less at a concentration of the anti-human interleukin-33 monoclonal antibody of 150mg/mL or more in the liquid preparation.

16. The liquid preparation according to any one of claims 1 to 4, further comprising sucrose, wherein the concentration of sucrose is 20 to 140 mg/mL.

17. The liquid formulation of claim 16, wherein the sucrose is at a concentration of 50-90 mg/mL.

18. The liquid preparation according to any one of claims 1 to 4, further comprising sorbitol, wherein the concentration of sorbitol is 10 to 200 mg/mL.

19. The liquid formulation of claim 18, wherein the concentration of sorbitol is 20-100 mg/mL.

20. The liquid formulation of claim 18, wherein the concentration of sorbitol is 50-80 mg/mL.

21. The liquid preparation according to any one of claims 1 to 4, further comprising sodium chloride, wherein the concentration of the sodium chloride is 20 to 300 mg/mL.

22. The liquid formulation of claim 21, wherein the sodium chloride is present at a concentration of 100-200 mg/mL.

23. The liquid preparation according to any one of claims 1 to 4, wherein the pH of the liquid preparation is 5.5 to 6.5.