CN114014929B

CN114014929B - Preparation method of anti-human interleukin-33 monoclonal antibody concentrated solution

Info

Publication number: CN114014929B
Application number: CN202111300668.4A
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: 2022-07-19
Anticipated expiration: 2041-11-04
Also published as: CN114014929A

Abstract

The application discloses a preparation method of a concentrated solution of an anti-human interleukin-33 monoclonal antibody, which comprises the following steps: and (3) ultrafiltration and concentration: concentrating the solution containing the anti-human interleukin-33 monoclonal antibody to obtain a concentrated sample; ultrafiltration and replacement: replacing the concentrated sample with a replacement buffer solution to obtain an ultrafiltration replacement concentrated solution; and (3) secondary ultrafiltration concentration: and adding the amino acid additive mother liquor into the ultrafiltration displacement concentrated solution, uniformly mixing to obtain a mixed solution, and carrying out ultrafiltration concentration on the mixed solution to obtain the concentrated solution. The preparation method can effectively reduce the viscosity of the ultrafiltration concentrated solution, reduce the aggregation or precipitation of the anti-human interleukin-33 monoclonal antibody and improve the stability of the anti-human interleukin-33 monoclonal antibody.

Description

Preparation method of anti-human interleukin-33 monoclonal antibody concentrated solution

Technical Field

The present application relates to the field of biotechnology. Specifically, the application relates to a preparation method of a concentrated solution of 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.

Therefore, in order to prepare a subcutaneous injection containing a high concentration of the anti-human interleukin-33 monoclonal antibody, it is necessary to develop a low viscosity ultrafiltration concentrate which 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 preparation method of a low-viscosity concentrated solution containing a high-concentration new anti-human interleukin-33 monoclonal antibody, wherein the concentrated solution contains more than 100mg/mL of the anti-human interleukin-33 monoclonal antibody and 10-500 mM of an amino acid additive, and the preparation method can effectively reduce the viscosity of an ultrafiltration concentrated solution, reduce aggregation or precipitation of the anti-human interleukin-33 monoclonal antibody and improve the stability of the ultrafiltration concentrated solution.

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 be specifically combined with IL-33 to prevent immune cells from releasing proinflammatory cytokines, so that aspects of asthma exacerbation prevention, asthma control improvement and the like are achieved.

Currently, no targeted human IL-33 monoclonal antibody medicine is on the market at home and abroad, wherein the REGN3500 developed by cooperation of a regenerant and Xenofei and the Etokimab of AnaptysBio companies have the fastest progress, the clinical research of 2/3 stage is currently carried out, and the intended administration mode is intravenous or subcutaneous injection. From the perspective of reducing the clinical use cost of biological agents and improving the compliance of patients, the preferable dosage form of the antihuman IL-33 monoclonal antibody medicament is a high-protein water injection used for subcutaneous injection, and if QX007N is prepared into subcutaneous injection, the protein content in 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 preparation method of a concentrated solution of an anti-human interleukin-33 monoclonal antibody, which comprises the following steps:

and (3) ultrafiltration concentration: concentrating the solution containing the anti-human interleukin-33 monoclonal antibody to obtain a concentrated sample;

ultrafiltration and replacement: replacing the concentrated sample with a replacement buffer solution to obtain an ultrafiltration replacement concentrated solution;

and (3) secondary ultrafiltration concentration: adding amino acid additive mother liquor into the ultrafiltration displacement concentrated solution, uniformly mixing to obtain a mixed solution, and carrying out ultrafiltration concentration on the mixed solution to obtain a concentrated solution;

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 amino acid sequence shown as SEQ ID NO: 7;

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

In the present application, in the ultrafiltration concentration step, the concentration conditions are: the flow rate is 120-300L/m²H, transmembrane pressure difference is 0.6-1.5 bar;

preferably, in the step of ultrafiltration concentration, the concentration of the anti-human interleukin-33 monoclonal antibody in the concentrated sample is 20-60 mg/mL, preferably 30-50 mg/mL.

In the present application, in the ultrafiltration displacement step, the displacement buffer solution is used in an amount of 7 times or more by weight of the concentrated sample;

preferably, in the step of ultrafiltration and replacement, the concentration of the replacement buffer solution is 5-50 mM, and is preferably 20 mM;

preferably, in the step of ultrafiltration and replacement, the pH of the replacement buffer solution is 5.5-6.5, preferably 5.7-6.3;

preferably, in the ultrafiltration displacement step, the displacement buffer solution is selected from one of histidine-hydrochloric acid buffer solution, citric acid buffer solution, phosphate buffer solution and sodium acetate-acetic acid buffer solution.

In the present application, in the second ultrafiltration concentration step, the concentration of the amino acid additive in the mixed solution is 50 to 300mmol/L, preferably 50 to 200mmol/L, and more preferably 100 to 150 mmol/L.

In the application, in the secondary ultrafiltration concentration step, the concentration of the protein in the concentrated solution is 120-300 mg/mL, preferably 120-200 mg/mL, and more preferably 120-160 mg/mL.

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 mixed solution is 100-200 mM;

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

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

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

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

In the present application, the solution containing the anti-human interleukin-33 monoclonal antibody is obtained by subjecting a cell fermentation broth expressing the anti-human interleukin-33 monoclonal antibody to affinity chromatography, low pH inactivation, anion chromatography, cation chromatography and nanofiltration.

ADVANTAGEOUS EFFECTS OF INVENTION

The concentrated solution of the anti-human IL-33 monoclonal antibody prepared by the method of the application contains the anti-human interleukin-33 monoclonal antibody, has low viscosity, can be injected by a syringe easily, and 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/REGN3500) 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 was used for chronic sinusitis (phase II clinic).

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 chart.

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^TMActivity profile of NF-. kappa.B/AP-1 signaling 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 to induce 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.

and (3) ultrafiltration and concentration: concentrating the solution containing the anti-human interleukin-33 monoclonal antibody to obtain a concentrated sample;

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 set forth 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.

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.The human variable regions from the whole antibodies generated by such animals may be further modified, for example by combination with different 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.A., Histology 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 such variable domain sequences can then 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 immunisation, as described by Griffiths, A.D.et al, EMBO J,12: 725-. Finally, non-immune libraries can also be generated synthetically 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 form in which the Flow direction of the liquid is Tangential (parallel) to the surface of the filter membrane, and compared with a conventional Filtration form (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, and the Filtration speed is stable, 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: 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 can bind to interleukin-33 of human and cynomolgus monkey, and can 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, "not to bind" when determining the species specificity of the anti-human IL-33 monoclonal antibody of the present application 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 sequence shown in SEQ ID NO: 7 (EVQLVESGGGLVQPGGSLRLSCAASGFSLSSYHMIWVRQAPGKGLEWVGVIYPNSNIYYATWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTIYVHVYSALSIWGQGTLVTVSS);

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

In one embodiment, in the ultrafiltration concentration step, the concentration conditions are: the flow rate is 120-300L/m²H, e.g. may be 120L/m²·h、135L/m²·h、150L/m²·h、165L/m²·h、180L/m²·h、190L/m²·h、200L/m²·h、225L/m²·h、250L/m²·h、275L/m²·h、300L/m²H, etc.; the transmembrane pressure difference (TMP) is 0.6-1.5 bar, such as 0.6bar, 0.7bar, 0.8bar, 0.9bar, 1.0bar, 1.1bar, 1.2bar, 1.3bar, 1.4bar, 1.5bar, etc.;

in one embodiment, in the step of ultrafiltration concentration, the concentration of the anti-human interleukin-33 monoclonal antibody in the concentrated sample is 20-60 mg/mL, for example, 20mg/mL, 30mg/mL, 40mg/mL, 50mg/mL, 60mg/mL, etc., preferably 30-50 mg/mL.

In one embodiment, in the ultrafiltration-displacement step, the displacement buffer solution is used in an amount of 7 times or more, for example, 7 times, 8 times, 9 times, 10 times, or the like, based on the weight of the concentrated sample.

In the present application, the concentration of the substitution buffer solution in the ultrafiltration-substitution step is not limited in any way, and can be selected by those skilled in the art as desired. In one embodiment, in the ultrafiltration-displacement step, the concentration of the displacement buffer solution is 5 to 50mM, and may be, for example, 5mM, 10mM, 20mM, 30mM, 40mM, 50mM, etc., and is preferably 20 mM.

In the present application, in the ultrafiltration-displacement step, the pH of the displacement buffer solution is not limited in any way as long as it is a partial acidity. In one embodiment, in the ultrafiltration-displacement step, the pH of the displacement buffer solution is 5.5 to 6.5, for example, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, etc., preferably 5.7 to 6.3.

In a specific embodiment, in the ultrafiltration displacement step, the displacement buffer solution is selected from one of histidine-hydrochloric acid buffer solution, citric acid buffer solution, phosphate buffer solution and sodium acetate-acetic acid buffer solution, and is preferably histidine-hydrochloric acid buffer solution. Specifically, the citric acid buffer solution may be a citric acid-sodium citrate buffer solution. Specifically, the phosphate buffer solution may be a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution.

In one embodiment, the displacement buffer (the buffer to be displaced) is pumped into the concentrated sample using a peristaltic pump, and the ultrafiltration is continued while adjusting the pumping rate of the displacement buffer to correspond to the permeation flow rate, whereby the displacement is accomplished when the pumped amount of the displacement buffer is 7 times or more the weight of the concentrated sample.

In one embodiment, in the second ultrafiltration concentration step, the concentration of the amino acid additive in the mixed solution is 50 to 300mmol/L, for example, 50mmol/L, 100mmol/L, 150mmol/L, 200mmol/L, 250mmol/L, 300mmol/L, etc., preferably 50 to 200mmol/L, more preferably 100 to 150 mmol/L; the concentration of the protein in the concentrated solution is 120 to 300mg/mL, and 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, etc., preferably 120 to 200mg/mL, more preferably 120 to 160 mg/mL.

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 mixed solution is 100-200 mM, and may be, for example, 100mM, 110mM, 120mM, 130mM, 140mM, 150mM, 160mM, 170mM, 180mM, 190mM, 200mM, etc.

In one embodiment, the amino acid additive comprises histidine, and the concentration of histidine in the mixed solution 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 mixed solution 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, or the like.

In one embodiment, the amino acid additive comprises arginine hydrochloride, histidine and methionine, wherein in the mixed solution, the concentration of arginine hydrochloride is 100-200 mM, the concentration of histidine is 10-50 mM, and the concentration of methionine is 2-50 mM.

In one embodiment, the amino acid additive comprises arginine hydrochloride, histidine and methionine, wherein the concentration of arginine hydrochloride is 100-200 mM, the concentration of histidine is 10-50 mM, and the concentration of methionine is 2-50 mM.

In a specific embodiment, in the secondary ultrafiltration concentration step, calculating the protein concentration in the obtained ultrafiltration displacement concentrated solution, calculating the theoretical volume of the ultrafiltration displacement concentrated solution (the total amount of the initial protein for ultrafiltration/the protein concentration in the ultrafiltration displacement concentrated solution), adding an amino acid additive mother solution, uniformly mixing, preferably, the volume of the amino acid additive is 1/9 of the volume of the ultrafiltration displacement concentrated solution, continuing ultrafiltration concentration, and performing concentration according to the concentration required by the raw solution (generally, the concentration is about 30% higher than the concentration of the raw solution) to obtain a concentrated solution, wherein the concentration of the anti-human interleukin-33 monoclonal antibody is 100-200 mg/mL, and the viscosity of the concentrated solution is less than or equal to 15 cP. If the amino acid additive adopted by the application is not directly subjected to over-concentration, the viscosity value of a concentrated solution can reach 25-50 cP, and the back pressure is too high, so that the membrane bag and equipment cannot be used for over-concentration and recovery.

In a specific embodiment, the viscosity of the concentrated solution is below 15cP when the concentration of the anti-human interleukin-33 monoclonal antibody in the concentrated solution is above 180 mg/mL.

In a specific embodiment, the viscosity of the concentrated solution is below 10cP when the concentration of the anti-human interleukin-33 monoclonal antibody in the concentrated solution is above 150 mg/mL.

In one embodiment, the solution containing the anti-human interleukin-33 monoclonal antibody is obtained by subjecting a cell fermentation broth expressing the anti-human interleukin-33 monoclonal antibody to affinity chromatography, low pH inactivation, anion chromatography, cation chromatography and nanofiltration.

In a specific embodiment, the affinity chromatography is performed using Protein a. Specifically, in the ultrafiltration concentration step and the ultrafiltration displacement step, the materials of the ultrafiltration membrane used include, but are not limited to, modified Polyethersulfone (PES), polyvinylidene fluoride (PVDF), Cellulose Acetate (CA), etc., and the pore diameter thereof is usually 30kDa or 50kDa, and preferably, the ultrafiltration membrane is Pellicon2/Pellicon3 (type a mesh, 30kDa) of mercuric millipore, and the ultrafiltration membrane of sydows and pell corporation.

The preparation method can reduce the viscosity of the high-concentration antibody liquid medicine and improve the stability of the high-concentration antibody liquid medicine in the ultrafiltration liquid-changing process, is simple and easy to implement, can carry out amplification production, can ensure the high purity of the sample and can obtain higher recovery rate. Meanwhile, the verification of high-concentration antibodies of different subtypes proves that the method for reducing the viscosity by adding the amino acid additive mother liquor has stronger applicability and good stability.

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 (hIL-33) is purchased from Shanghai near-shore science and technology Limited, is used for immunizing New Zealand rabbits, and is subjected to B cell cloning technology to obtain antigen binding specific antibody clones, so that monoclonal antibodies which are bound with the human interleukin-33 and have human interleukin-33 inhibitory activity are screened. Firstly, detecting cell supernatant by Binding ELISA, and selecting a clone combined with human interleukin-33; then HEK Blue^TMIL-33 reporter gene cell assay was performed to select clones with human interleukin-33 inhibitory activity. The above immunization and screening process was delegated to a commercial company.

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, or a pharmaceutically acceptable salt thereof.

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.

Except for this, when QX007N (HZD78-70) was compared with the affinity of Etokimab/ANB020, a monoclonal antibody against human interleukin-33 developed by AnaptyBio, the method for detecting the known antibody was the same as that for detecting 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 affinity of antibodies for binding to human interleukin-33

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 signal cascade leading to 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 cells were assayed for the neutralizing activity of QX007N (HZD78-70) 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 under conditions overnight. 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 four parameter curve fitting using softMaxPro software (figure 4) to further 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-kB/AP-1 Signal transduction in IL-33 cells

Preparing natural human interleukin-33, and verifying the neutralizing activity of QX007N (HZD78-70) on natural human interleukin-33. 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, the data were analyzed using 4-parameter curve fitting using SoftMax Pro software (fig. 5) to further analyze the neutralizing activity of the antibodies.

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 of QX007N (HZD78-70) is further characterized by taking mononuclear cells in human whole blood as a determination basis and IFN-gamma as a determination indexAnd (4) activity. 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, fermentation is carried out on a bioreactor with the scale of 2L, the antibody QX007N obtained in the example 1 is produced, clarified fermentation liquor is obtained by a centrifuge or a deep membrane filtration, Protein A chromatography is adopted to capture target Protein, and impurities are removed by low pH value virus inactivation and anion and cation chromatography, thus obtaining an intermediate sample to be ultrafiltered.

And (3) ultrafiltration concentration: performing ultrafiltration by using the intermediate, wherein the ultrafiltration equipment is a Labscale small-sized ultrafiltration instrument (clamping two 50cm blocks)²Pellicon 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 to obtain a concentrated sample.

Ultrafiltration and replacement: and (3) selecting a 20mM histidine-hydrochloric acid buffer solution as a replacement buffer solution, controlling the pH value to be 6.0, pumping the histidine-hydrochloric acid buffer solution into the concentrated sample by using a peristaltic pump, mixing to obtain a mixed solution, continuously performing ultrafiltration on the mixed solution, and simultaneously adjusting the pumping speed of the histidine-hydrochloric acid buffer solution to be consistent with the permeation flow rate, namely keeping the weight of the sample constant, and finishing the replacement when the volume of the histidine-hydrochloric acid buffer solution is 7 times of the weight of the concentrated sample to obtain an ultrafiltration replacement concentrated solution.

And (3) secondary ultrafiltration concentration: 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 to obtain a concentrated solution with the protein concentration of table 2.

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 sample concentration 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 of Sharp-Olson, which was operated as follows:

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

2. placing the injector into a pipe groove for fixation, clicking an instrument host interface, setting a shearing rate, clicking 'Run' for detection after the setting is finished, recording a viscosity value, and generally measuring for multiple times and taking 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.

The results of measuring the antibody concentration and viscosity value of the concentrated solution are shown in Table 2.

TABLE 2 summary of viscosity reduction results of different additives (protective agents) for anti-human IL-33 antibody

As shown in the results in Table 2, the viscosity of the anti-human IL-33 antibody increased exponentially with the increase of the protein concentration, and the viscosity of the monoclonal antibody reached 31.1cP at a concentration of 200mg/mL without the addition of additives, so that the concentration process could not be realized by using a conventional ultrafiltration membrane. 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 150mg/mL in conclusion.

Example 8 comparison of different methods

The preparation of a solution containing anti-human interleukin-33 monoclonal antibody QX007N (UF0), the ultrafiltration concentration step and the ultrafiltration displacement step were performed in the same manner as in example 7.

And (3) secondary ultrafiltration concentration: dividing the ultrafiltration displacement concentrated solution obtained in the ultrafiltration displacement step into two parts, and respectively carrying out a concentration process:

A. sampling from the first part, determining the concentration of protein in the ultrafiltration displacement concentrated solution, calculating the theoretical volume of the ultrafiltration displacement concentrated solution at the moment, adding 1/9 hydrochloric acid arginine mother liquor (1.5mol/L) with the theoretical volume, uniformly mixing, and concentrating by using a merck Millipore 30kDa ultrafiltration centrifugal tube until the concentration of the protein is 150mg/mL, and marking as concentrated solution 1;

B. directly concentrating the second part by adopting an ultrafiltration concentration step until the protein concentration is 150mg/mL, and marking as a concentrated solution 2;

then, the concentrated solution 1 and the concentrated solution 2 are respectively and uniformly mixed with mother liquor of polysorbate 80 as a surfactant, and the mixture is filtered by using a 0.2 mu m filter membrane to respectively obtain stock solution 1(U1F2) and stock solution 2(U2F 2). 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 viscosities and purities of stock solution 1(U1F2) and stock solution 2(U2F2) were measured, respectively, in the same manner as in example 7, and the purities were measured 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 under the condition of flow rate of 5mL/min, screwing a quaternary pump switch, connecting a chromatographic column, and flushing the chromatographic column by 100% mobile phase for 30 minutes under the condition of flow rate of 1.0 mL/min;

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 adaptability of the system 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 of sample injection), then 1 needle of blank sample is analyzed, and then 2 samples are analyzed.

4. And sequentially integrating chromatographic peaks of each sample according to the sequence of polymers, main peaks, degradation products and the like, integrating all chromatographic peaks which are inconsistent with the retention time of the blank sample, and calculating the proportion of each peak by an area normalization method.

The measurement results are shown in Table 3.

TABLE 3 arginine ultrafiltration concentration for reducing anti-human IL-33 monoclonal antibody viscosity number and protection

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

<110> Jiangsu Quanxin biomedicine GmbH

<120> preparation method of anti-human interleukin-33 monoclonal antibody concentrated solution

<130> TPE01860

<160> 11

<170> PatentIn version 3.5

<210> 1

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial Synthesis

<400> 1

Ser Tyr His Met Ile

1 5

<210> 2

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial Synthesis

<400> 2

Val Ile Tyr Pro Asn Ser Asn Ile Tyr Tyr Ala Thr Trp Ala Lys Gly

1 5 10 15

<210> 3

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial Synthesis

<400> 3

Thr Ile Tyr Val His Val Tyr Ser Ala Leu Ser Ile

1 5 10

<210> 4

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial Synthesis

<400> 4

Gln Ala Ser Glu Ser Val Leu Asn Glu Val Ser

1 5 10

<210> 5

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial Synthesis

<400> 5

Phe Ala Ser Lys Leu Ala Ser

1 5

<210> 6

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial Synthesis

<400> 6

Gln Gln Asp Trp Ser Met Asp Asn Ile Asp Asn Ala

1 5 10

<210> 7

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial Synthesis

<400> 7

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Ser Tyr

20 25 30

His Met Ile Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Val Ile Tyr Pro Asn Ser Asn Ile Tyr Tyr Ala Thr Trp Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu

65 70 75 80

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

85 90 95

Arg Thr Ile Tyr Val His Val Tyr Ser Ala Leu Ser Ile Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 8

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial Synthesis

<400> 8

Ala Phe Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Glu Ser Val Leu Asn Glu

20 25 30

Val Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asp Trp Ser Met Asp Asn

85 90 95

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

100 105 110

<210> 9

<211> 159

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial Synthesis

<400> 9

Ser Ile Thr Gly Ile Ser Pro Ile Thr Glu Tyr Leu Ala Ser Leu Ser

1 5 10 15

Thr Tyr Asn Asp Gln Ser Ile Thr Phe Ala Leu Glu Asp Glu Ser Tyr

20 25 30

Glu Ile Tyr Val Glu Asp Leu Lys Lys Asp Glu Lys Lys Asp Lys Val

35 40 45

Leu Leu Ser Tyr Tyr Glu Ser Gln His Pro Ser Asn Glu Ser Gly Asp

50 55 60

Gly Val Asp Gly Lys Met Leu Met Val Thr Leu Ser Pro Thr Lys Asp

65 70 75 80

Phe Trp Leu His Ala Asn Asn Lys Glu His Ser Val Glu Leu His Lys

85 90 95

Cys Glu Lys Pro Leu Pro Asp Gln Ala Phe Phe Val Leu His Asn Met

100 105 110

His Ser Asn Cys Val Ser Phe Glu Cys Lys Thr Asp Pro Gly Val Phe

115 120 125

Ile Gly Val Lys Asp Asn His Leu Ala Leu Ile Lys Val Asp Ser Ser

130 135 140

Glu Asn Leu Cys Thr Glu Asn Ile Leu Phe Lys Leu Ser Glu Thr

145 150 155

<210> 10

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial Synthesis

<400> 10

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Ser Tyr

20 25 30

His Met Ile Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Val Ile Tyr Pro Asn Ser Asn Ile Tyr Tyr Ala Thr Trp Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu

65 70 75 80

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

85 90 95

Arg Thr Ile Tyr Val His Val Tyr Ser Ala Leu Ser Ile Trp Gly Gln

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Gly Lys

450

<210> 11

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial Synthesis

<400> 11

Ala Phe Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Glu Ser Val Leu Asn Glu

20 25 30

Val Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asp Trp Ser Met Asp Asn

85 90 95

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 preparation method of a concentrated solution of an anti-human interleukin-33 monoclonal antibody comprises the following steps:

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:6 is shown in the specification;

the pH value of the replacement buffer solution is 5.5-6.5;

the concentration of the replacement buffer solution is 5-50 mM;

the replacement buffer solution is selected from one of histidine-hydrochloric acid buffer solution, citric acid buffer solution, phosphate buffer solution and sodium acetate-acetic acid buffer solution;

the amino acid additive is selected from one of arginine hydrochloride, lysine and proline;

the concentration of the amino acid additive in the mixed solution is 100-200 mM;

the concentration of protein in the concentrated solution is 120-200 mg/mL;

the viscosity of the concentrated solution is below 10 cP.

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

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.

3. The production method according to claim 1 or 2, wherein in the ultrafiltration concentration step, the concentration conditions are: the flow rate is 120-300L/m²H, transmembrane pressure difference of 0.6 to 1.5 bar.

4. The method according to claim 1 or 2, wherein in the ultrafiltration concentration step, the concentration of the anti-human interleukin-33 monoclonal antibody in the concentrated sample is 20 to 60 mg/mL.

5. The method according to claim 1 or 2, wherein in the ultrafiltration concentration step, the concentration of the anti-human interleukin-33 monoclonal antibody in the concentrated sample is 30 to 50 mg/mL.

6. The production method according to claim 1 or 2, wherein in the ultrafiltration-substitution step, the substitution buffer solution is used in an amount of 7 times or more by weight as much as the weight of the concentrated sample.

7. The production method according to claim 1 or 2, characterized in that the concentration of the substitution buffer solution is 20 mM.

8. The method according to claim 1 or 2, wherein the pH of the substitution buffer solution is 5.7 to 6.3.

9. The method according to claim 1 or 2, wherein the concentration of the amino acid additive in the mixed solution is 100 to 150 mM.

10. The method according to claim 1 or 2, wherein the concentration of the protein in the concentrated solution is 120 to 160 mg/mL.

11. The production method according to claim 1 or 2, wherein the solution containing the anti-human interleukin-33 monoclonal antibody is obtained by subjecting a cell fermentation broth expressing the anti-human interleukin-33 monoclonal antibody to affinity chromatography, low pH inactivation, anion chromatography, cation chromatography, and nanofiltration.