CN113912728A

CN113912728A - Affinity purification method for reducing host cell protein content in production of anti-human interleukin-33 monoclonal antibody

Info

Publication number: CN113912728A
Application number: CN202111455041.6A
Authority: CN
Inventors: 戴长松; 朱华杰; 李帅; 徐义超; 唐宇杰; 戴璐; 李梦茹; 李梦杰; 何勇梅; 吴亦亮
Original assignee: Jiangsu Quanxin Biomedical Co ltd
Current assignee: Jiangsu Quanxin Biomedical Co ltd
Priority date: 2021-11-04
Filing date: 2021-12-01
Publication date: 2022-01-11
Anticipated expiration: 2041-12-01
Also published as: CN113912728B

Abstract

The application discloses an affinity purification method for reducing the content of host cell protein in the production of an anti-human interleukin-33 monoclonal antibody, which comprises the following steps: balancing before loading: using buffer solution and balance affinity chromatography medium to obtain well-balanced affinity chromatography medium; loading: combining the fermentation liquor of the anti-human interleukin-33 monoclonal antibody with a well-balanced affinity chromatography medium; and (3) elution: pre-eluting and final eluting the mixed solution to remove host cell protein and obtain a purified anti-human interleukin-33 monoclonal antibody; 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. The method is simple and feasible, can carry out amplification and purification production, does not need pretreatment on cell fermentation supernatant, has high elution sample yield, and keeps HCP residual quantity at a lower level, thereby relieving the pressure of removing HCP in the subsequent purification step.

Description

Affinity purification method for reducing host cell protein content in production of anti-human interleukin-33 monoclonal antibody

Technical Field

The present application relates to the field of biotechnology. In particular, the present application relates to an affinity purification method for reducing the host cell protein content in the production of anti-human interleukin-33 (IL-33) monoclonal antibodies.

Background

The affinity purification in the production process of antibody drugs is a very critical process step, and the process captures and concentrates the antibody in the fermentation liquor to realize the first step of crude purification of the antibody. During the large-scale fermentation process of genetic engineering cell strains such as Chinese Hamster Ovary (CHO) cells and the like, the cells are subjected to apoptosis and lysis in different physiological cycles, and Host Cell Proteins (HCPs) are released. HCP refers to protein components derived from host cells, including host cell structural proteins and transforming proteins (cell secreted growth promoting proteins). The HCP not only can possibly induce the organism to generate anti-HCP antibody to cause anaphylactic reaction, but also can possibly cause the organism to generate antibody to protein medicine by 'adjuvant effect' to influence the treatment effect of the medicine, and the quantitative determination of the HCP remained in the genetic engineering medicine is an important means of quality control, and is helpful to keep the effectiveness and consistency of the purification process. In the antibody affinity purification, HCP generated by engineering cells in the fermentation process needs to be effectively removed while high-efficiency antibody recovery rate is ensured. Therefore, the research of the antibody affinity purification process which can be widely applied to large-scale fermentation of the antibody and is more economical and feasible is very significant for further industrialized popularization of antibody medicines, and the invention relates to the aspect.

At present, many methods for removing HCP residues are available, and each method has the following characteristics: 1) the chromatography mode is as follows: the method comprises Protein A affinity chromatography, anion and cation chromatography, and removal capability to HCP in three chromatography processes, wherein the Protein A affinity chromatography is used as a basis, the removal capability is strong, and the method is a main step for removing HCP, and the anion and cation chromatography is mainly used as a further removal process of subsequent HCP. The process for removing HCP by chromatography is a main means for removing HCP, and various chromatography processes continuously put forward and apply actual production in the continuous improvement process; 2) tangential flow ultrafiltration mode: the removal capability of HCP is limited, the residual quantity is difficult to control, the process control coefficient is not high, and the HCP can only be used as an auxiliary process for removing HCP; 3) polymer precipitation mode: polymers such as PEG, polyacrylic acid and the like have positive charges in a wider pH range, and are combined with antibodies to form precipitates through charge action, while HCPs are not easy to precipitate due to lower isoelectric points. The HCP content of the precipitated antibody samples was significantly reduced, but the process was not suitable for process scale-up, and the precipitation may have some effect on the activity of the antibody. Therefore, many processes for removing HCPs are required, and a comprehensive consideration and selection process is required due to the difference in the sample fermentation process and the properties of the antibody of each purpose.

Disclosure of Invention

In order to solve the problem of Host Cell Protein (HCP) residue in antibody production, the application discloses a novel method for effectively reducing CHO host cell protein in antibody purification production, which can be widely applied to an antibody affinity purification process, and the used material has low cost and price and is easy to amplify the process. In the application, the effect of reducing the HCP, the antibody and the filler matrix in the pre-elution of affinity chromatography is mainly utilized, namely the acting force between the HCP, the antibody protein and the filler medium can be weakened to different degrees through the active reagent, so that the effect of removing the HCP is achieved, the requirement of large-scale high-quality purification preparation of the antibody drug is met, and the safety of clinical use of the antibody drug is ensured.

The specific technical scheme of the application is as follows:

the application provides an affinity purification method for reducing the content of host cell protein in the production of an anti-human interleukin-33 monoclonal antibody, which comprises the following steps:

balancing before loading: using buffer solution and balance affinity chromatography medium to obtain well-balanced affinity chromatography medium;

loading: combining the anti-human interleukin-33 monoclonal antibody fermentation liquor with the well-balanced affinity chromatography medium, and loading;

and (3) elution: then, pre-elution and final elution are carried out in sequence, so as to remove host cell protein and obtain a purified anti-human interleukin-33 monoclonal antibody;

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.

Preferably, 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.

Preferably, the affinity chromatography medium is selected from one of chromatography media formed by crosslinking ligand on agarose, polyvinyl ether, hydroxylated polyether resin, polyacrylic resin, polystyrene divinyl phenyl resin, polymethacrylic resin, polystyrene resin, hydroxyapatite and glass matrix, and is preferably the chromatography media formed by crosslinking ligand on polyvinyl ether;

preferably, the ligand is Protein A, Protein G or Protein L, and more preferably is Protein A.

Preferably, the buffer solution I is selected from one of phosphate buffer solution, Tris-HCl buffer solution and boric acid-borax buffer solution; in the buffer solution I, the salt concentration is 5 mM-0.25M, and the pH value is 5.5-8.0.

Preferably, a pre-elution buffer is adopted for pre-elution, and the pre-elution buffer is a neutral buffer and/or an acidic buffer;

preferably, the neutral buffer is selected from one of phosphate buffer, tris buffer and glycine buffer;

preferably, the acidic buffer is selected from one of a citric acid-disodium hydrogen phosphate buffer, an acetic acid-sodium acetate buffer and a citric acid-trisodium citrate buffer;

preferably, the pH of the pre-elution buffer is 5.0-7.5, and more preferably 5.5-6.5.

Preferably, when the pre-elution is carried out by adopting a pre-elution buffer solution, a pre-elution active agent is also added into the pre-elution buffer solution;

preferably, the pre-elution active agent is selected from one or more of guanidine hydrochloride, polysorbate 80 and sodium chloride;

preferably, the pre-elution activator is guanidine hydrochloride;

further preferably, the concentration of the guanidine hydrochloride is 0.01-1M.

Preferably, a final elution is performed with a final elution buffer;

the final elution buffer solution is selected from one or two or more of citric acid-disodium hydrogen phosphate buffer solution, acetic acid buffer solution, glycine-HCl buffer solution and citric acid-sodium citrate buffer solution, and preferably is citric acid-disodium hydrogen phosphate buffer solution;

preferably, the pH value of the citric acid-disodium hydrogen phosphate buffer solution is 2.9-3.8.

Preferably, when the anti-human interleukin-33 monoclonal antibody fermentation liquor is combined with the well-balanced affinity chromatography medium, buffer I is used for balancing.

Preferably, the elution step further comprises the step of using a buffer solution to balance the solution after the pre-elution and before the final elution.

Preferably, the buffer II is selected from one of phosphate buffer, Tris-HCl buffer and boric acid-borax buffer;

preferably, the second buffer is phosphate buffer or Tris-HCl buffer;

preferably, the pH value of the second buffer solution is 5.5-8.0.

ADVANTAGEOUS EFFECTS OF INVENTION

The affinity purification process is simple and feasible, amplification purification production can be carried out, pre-treatment is not needed to be carried out on cell fermentation supernatant, the yield of an elution sample is high, and meanwhile, the HCP residual quantity is kept at a low level (the residual control quantity is not higher than 0.1%), so that the pressure for removing HCP in the subsequent purification step is reduced, and the HCP residual quantity of the final sample of the human interleukin-33 (IL-33) monoclonal antibody is ensured to be at an extremely low level. Meanwhile, in the application, the affinity purification process of the fermentation supernatants of different batches is verified to have good stability.

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

The monoclonal antibody drug (Itepekimab/REGN3500) targeting interleukin-33 developed by Xenoffy company is intended to be used for treating inflammatory diseases such as chronic obstructive pulmonary disease (clinical stage III), asthma (clinical stage II) and the like, and Etokimab/ANB020 developed by AnaptyBio company is used for chronic sinusitis (clinical stage II).

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 represent heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3, respectively.

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, for a review of the methods of obtaining human antibodies from transgenic animals, see Lonberg, N., nat. Biotech. (Nature Biotechnology) 23: 111-1125 (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., 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 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 Biology 178:1-37(2001), and are further described, for example, in McCafferty, J.et al, Nature 348:552-554 (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. USA90: 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 the present application, "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom (regardless of the number of passages). Progeny may not be identical to the parent cell in terms of nucleic acid content, but may contain mutations. Progeny of mutants screened or selected for the originally transformed cell to have the same function or biological activity are included in the present specification.

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 sequence shown in SEQ ID NO: 7(EVQLVESGGGLVQPGGSLRLSCAASGFSLSSYHMIWVRQAPGKGLEW VGVIYPNSNIYYATWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA RTIYVHVYSALSIWGQGTLVTVSS);

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

In a specific embodiment, the affinity chromatography medium is selected from one of chromatography media in which the ligand is crosslinked to agarose, polyvinyl ether, hydroxylated polyether resin, polyacrylic resin, polystyrene divinyl phenyl resin, polymethacrylic resin, polystyrene resin, hydroxyapatite and glass matrix, and is preferably a chromatography medium in which the ligand is crosslinked to polyvinyl ether;

The ligands of the present application can specifically bind to monoclonal antibodies.

In the present application, there is no limitation on the affinity filler, which can be confirmed according to the needs of those skilled in the art, for example, the affinity filler can be Mabselect, Mabselect Sure, of GE healthcare, Protein A Diamond, of Borglong Biotechnology, Inc., of MERCK

A。

In one embodiment, the buffer one includes, but is not limited to, one of phosphate buffer, Tris-HCl buffer, and boric acid-borax buffer. The salt concentration in the first buffer solution is not limited in any way, and can be selected by one skilled in the art according to the need, for example, in one embodiment, the salt concentration in the first buffer solution is 5 mM-0.25M, and can be, for example, 5mM, 10mM, 20mM, 50mM, 0.1M, 0.15M, 0.2M, 0.25M, etc.; the pH of the buffer solution is 5.5-8.0, such as 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, etc.

In one embodiment, the first buffer is a phosphate buffer or a Tris-hydrochloric acid buffer.

In a preferred embodiment, NaCl or Na is added to the first buffer solution₂SO₄To reduce non-specific adsorption between the non-antibody protein and the filler.

In one embodiment, the first buffer is phosphate buffer, and NaCl or Na is added into the first buffer₂SO₄To reduce non-specific adsorption between the non-antibody protein and the filler, the salt concentration of the phosphate buffer is between 5mM and 0.15M, preferably between 10mM and 50mM, and more preferably 20 mM.

The pH of the first buffer is not limited in this application, and can be selected by one skilled in the art according to needs, for example, in a specific embodiment, the pH of the first buffer is 6.5 to 7.5, and preferably 6.9.

For NaCl or Na₂SO₄In the presence of NaCl or Na, which can be determined according to the requirements of the person skilled in the art₂SO₄The concentration of (A) can be selected by one skilled in the art as desired, and in one embodiment, NaCl or Na₂SO₄The concentration of (B) may be 0 to 250mM, preferably 150 mM.

In one embodiment, the phosphate buffer may be one or both of a disodium hydrogen phosphate buffer and a sodium dihydrogen phosphate buffer.

In one embodiment, buffer one is used for equilibration when the anti-human interleukin-33 monoclonal antibody fermentation broth is bound to the equilibrated affinity chromatography medium.

In one embodiment, the pre-elution is performed using a pre-elution buffer, which is a neutral buffer and/or an acidic buffer.

In a specific embodiment, the neutral buffer is selected from one of phosphate buffer, tris buffer and glycine buffer, and the acidic buffer is selected from one of citric acid-disodium hydrogen phosphate buffer, acetic acid-sodium acetate buffer and citric acid-trisodium citrate buffer.

The pH of the pre-elution buffer is not limited in this application, and can be selected by one skilled in the art according to needs, for example, in a specific embodiment, the pH of the pre-elution buffer is 5.0 to 7.5, and for example, may be 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and the like, and further preferably is 5.5 to 6.5.

In one embodiment, when a pre-elution buffer is used for pre-elution, a pre-elution active agent is also added to the pre-elution buffer; the pre-elution active agent includes, but is not limited to, one or more of guanidine hydrochloride, polysorbate 80 and sodium chloride, preferably, the pre-elution active agent is guanidine hydrochloride.

The concentration of guanidine hydrochloride is not limited in any way, and can be selected by one skilled in the art according to needs, for example, in one embodiment, the concentration of guanidine hydrochloride is 0.01 to 1M, such as 0.01M, 0.05M, 0.1M, 0.15M, 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1M, etc., preferably 0.05 to 0.15M, and more preferably 0.1M.

The salt concentration in the pre-elution buffer is not limited in this application, and can be selected by one skilled in the art according to the need, for example, in one embodiment, the salt concentration in the pre-elution buffer is 0-0.5M, preferably 0.1M.

In one embodiment, a final elution is performed with a final elution buffer; the final elution buffer includes, but is not limited to, one or two or more of a citric acid-disodium hydrogen phosphate buffer, an acetic acid buffer, a glycine-HCl buffer, and a citric acid-sodium citrate buffer, and preferably a citric acid-disodium hydrogen phosphate buffer.

The pH of the final elution buffer is not limited herein, and can be selected by one skilled in the art according to the need, for example, in a specific embodiment, the pH of the final elution buffer is 2.9 to 3.8, and can be, for example, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, etc.

The salt concentration in the final elution buffer is not limited in any way, and can be selected by those skilled in the art according to the need, for example, in one embodiment, the salt concentration in the second elution solution is 5 to 100mM, such as 5mM, 10mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, etc., preferably 10 to 50 mM.

For example, the salt concentration in the second elution solution may be 5mM, 10mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, etc.

In one embodiment, the affinity medium may be regenerated after the final elution is performed using a regeneration buffer, preferably including, but not limited to, citric acid-disodium hydrogen phosphate buffer, hydrochloric acid, glycine, NaOH, preferably citric acid-disodium hydrogen phosphate buffer and NaOH solution.

In one embodiment, the elution step further comprises, after the pre-elution and before the final elution, equilibrating the pre-eluted solution using a buffer.

In a specific embodiment, the second buffer is a phosphate buffer, a Tris-HCl buffer, or a boric acid-borax buffer.

In a preferred embodiment, the second buffer is one of phosphate buffer, Tris-HCl buffer and boric acid-borax buffer.

In a preferred embodiment, NaCl or Na is added to buffer II₂SO₄To maintain conductance while retaining some of the buffering capacity.

The salt concentration in the second buffer and the pH of the second buffer are not limited in this application, and can be selected by those skilled in the art as needed. For example, in one embodiment, the salt concentration in the second buffer is 5 mM-0.15M, preferably 10-50 mM, and more preferably 20 mM; the pH value of the second buffer solution is 5.5-8.0, preferably 6.5-7.5, and further preferably 7.2.

For NaCl or Na₂SO₄In the presence of NaCl or Na, which can be determined according to the requirements of the person skilled in the art₂SO₄The concentration of (A) can be selected by one skilled in the art as desired, and in one embodiment, NaCl or Na₂SO₄The concentration of (B) is 0 to 250mM, preferably 10 mM.

In one embodiment, the phosphate buffer may be, for example, a buffer of disodium hydrogen phosphate and sodium dihydrogen phosphate.

The affinity purification process is simple and feasible, amplification purification production can be carried out, pre-treatment is not needed to be carried out on cell fermentation supernate, the yield of an elution sample is high, and meanwhile the HCP residual quantity is kept at a low level (the residual control quantity is not higher than 0.1%), so that the pressure for removing HCP in the subsequent purification step is reduced, and the HCP residual quantity of the final sample of the antibody is ensured to be at an extremely low level. Meanwhile, the affinity purification process of the fermentation supernatants of different batches is verified, so that the affinity purification process has 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; reuse of HEK Blue^TMDetection by IL-33 reporter gene cell methodTo test, a clone having an inhibitory activity against human interleukin-33 was selected. 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 germ 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 the 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 the 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 amino acid sequence of the humanized heavy chain variable region is shown in SEQ ID NO: 7 is shown in the specification; the amino acid sequence of the humanized light chain variable region is shown as SEQ ID NO: shown in fig. 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 heavy chain variable region is about 480bp, and the light chain full length is about 781 bp.

The correct heavy chain expression plasmid pQX2.1-78VH-Hu25 (the full-length amino acid sequence of the expressed heavy chain is shown as SEQ ID NO: 10) and the light chain expression plasmid pQX2.2-78VK-Hu3 (the full-length amino acid sequence of the expressed light chain is shown as SEQ ID NO: 11) were co-transfected into ExpCHO-S cells. One day before transfectionExpicHO-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 Protein A. 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)

The affinity of QX007N (HZD78-70) for human interleukin-33 was measured using Biacore T200, 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 serve 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 affinity of anti-human Interleukin-33 antibodies for binding to human Interleukin-33

Sample name	k_a(10⁵M^-1S^-1)	k_d(10^-4S^-1)	K_D(10^-10M)
				QX007N	3.92	4.81	12.28
ANB 020	4.24	3.69	8.69

Furthermore, based on the same detection method as described above, we also found that Etokimab/ANB020 binds to interleukin-33 of cynomolgus monkey and rhesus monkey, whereas QX007N (HZD78-70) binds to interleukin-33 of cynomolgus monkey but not to interleukin-33 of rhesus monkey.

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 to trigger a signaling cascade leading to NF-. kappa.B/AP-1 signalingAnd produces 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 results of fig. 4 show that: QX007N (HZD78-70) can inhibit recombinant human interleukin-33 from inducing HEK Blue^TMNF- κ B/AP-1 Signal transduction in IL-33 cells, IC thereof₅₀6.67 ng/ml; in contrast, for Etokimab/ANB020, IC was measured in the same manner₅₀6.05 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 3%7 ℃ 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_630nmData were analyzed using 4-parameter curve fitting using SoftMax Pro software (fig. 5) to analyze antibody neutralization activity.

The results of fig. 5 show that: QX007N (HZD78-70) can inhibit natural human interleukin-33 from inducing HEK Blue^TMNF- κ B/AP-1 Signal transduction in IL-33 cells, IC thereof₅₀3.91 ng/ml; in contrast, for Etokimab/ANB020, IC was measured in the same manner₅₀It was 2.5 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 plates⁵Individual cells/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 of 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), IC thereof₅₀5.87 ng/ml; in contrast, for Etokimab/ANB020, IC was measured in the same manner ₅₀44 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 plating (100. mu.L) from healthy volunteers was usedPerwell), 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_450nmData were analyzed using 4-parameter curve fitting using SoftMax Pro software (fig. 7) to analyze antibody neutralization activity.

The results of 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; in contrast, for Etokimab/ANB020, IC was measured in the same manner₅₀31.9 ng/ml.

EXAMPLE 7 comparison of the Effect of different buffers on the elimination of HCP from the fermentation broth of recombinant humanized anti-human interleukin-33 monoclonal antibody (QX007N)

Preparing fermentation liquor of the anti-human interleukin-33 monoclonal antibody:

the antibody QX007N obtained in example 1 was produced using CHO cells as host cells and Eden-B600 as fermentation basal medium. And (3) performing cell culture by using a conventional cell culture process, starting harvesting when the cell viability is lower than 80% or the culture is carried out for 18 days, performing deep filtration on a harvest solution by adopting a primary filter MD0HC10FS1 and a secondary filter MX0HC10FS1, and collecting a clarified cell culture supernatant so as to obtain a fermentation broth (a fermentation broth intermediate for short) of the anti-human interleukin-33 monoclonal antibody.

Using buffer solution and balance affinity chromatography medium to obtain well-balanced affinity chromatography medium; then combining the anti-human interleukin-33 monoclonal antibody fermentation liquor with the well-balanced affinity chromatography medium, which comprises the following specific steps:

buffer solution I (12mmol/L Na) is adopted₂HPO₄，8mmol/L NaH₂PO₄0.15mol/L NaCl) and the Protein a chromatographic column (AT Protein a Diamond Plus, 0.15L) was equilibrated and the QX007N broth intermediate was loaded onto the equilibrated Protein a chromatographic column to bind thereto, the loading being set AT 55mg/mL, and then equilibrated with buffer one until the broth completely flowed through the column;

then, a pre-elution was carried out using a pre-elution buffer having the composition shown in Table 2Followed by buffer II (6mmol/L Na)₂HPO₄、4mmol/L NaH₂PO₄pH 7.2);

then, final elution is carried out to collect samples, and the collected samples are subjected to antibody concentration and host protein (HCP) residual content determination, wherein the final elution adopts a final elution buffer solution of 7mmol/L Na₂HPO₄15mmol/L citric acid, pH 3.1. And regenerating the chromatographic column by using a regeneration buffer solution (100mmol/L NaOH and 1mol/L NaCl). And collecting eluted samples to determine the concentration of the antibody and the residual content of HCP.

TABLE 2 Pre-elution buffer composition

The antibody concentration was determined as follows:

1. the wavelength of the spectrophotometer was adjusted to 280nm and the final elution buffer was used as a control for zero calibration.

2. Diluting a sample to be detected by using a final elution buffer solution, 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 results obtained are shown in table 3.

HCP content determination method:

1. sample dilution: the dilution factor is typically selected based on an estimate of HCP in the sample, such that the final HCP concentration falls within the range of the standard curve (typically 10ng/mL to 80 ng/mL). The dilution factor of the sample in one step is not more than 10 times, and the minimum sampling amount is not less than 5 mu L.

2. Anti-CHO HRP 100. mu.L was added to each well of the take-out strip.

3. Loading: adding standard sample, and standard sample (each having two wells, the standard sample does not need to have multiple wells), 50 μ L/well, and sealing plate. The mixture was placed on a horizontal shaker at room temperature for 2 hours at 180rpm in the dark.

4. Washing the plate: discarding the liquid in the hole, adding the washing liquid 300 muL/hole by using a multichannel pipettor, standing for 30 seconds, then throwing off the liquid, patting on absorbent paper, and washing the plate for 4 times. After the last plate washing is finished, residual washing liquid in the holes needs to be patted dry as much as possible.

5. Color development and end reading: TMB reagent (TMB Substrate) was added at 100. mu.L/well, and the mixture was allowed to stand for 30 minutes to develop color, protected from light. After 30 minutes, 100. mu.l/well of Stop Solution (Stop Solution) was added, and the reading was taken by a microplate reader at 450nm, with 650nm as reference.

6. And selecting analysis software to analyze data, and drawing a four-parameter standard curve by taking the OD value of the standard substance as a vertical coordinate and the concentration as a horizontal coordinate. And substituting the OD value measured by the sample into the standard curve to obtain the actual measurement value of the added sample HCP.

CHO cell protein residual amount (%). The sample average measured value (ng/mL). times.dilution factor/undiluted sample protein content (mg/mL) was determined^-4(%), the results are shown in Table 3.

TABLE 3QX007N affinity purification yield and HCP residual amount results

The results in table 3 show that the HCP residue in the fermentation broth intermediate is greater than 25%, and the yields are greater than 95% after the treatment of the affinity purification process; for the removal of HCP, an experiment group containing guanidine hydrochloride is adopted, the HCP residue of a sample after affinity chromatography is lower than 0.1%, the load of the subsequent purification process step for removing HCP is obviously reduced, the pH of the sample is adjusted, and when the sample is loaded in the subsequent chromatography process step (anion exchange chromatography), the sample is kept relatively clear and can be directly injected without other treatment, so that the simplicity of the process is enhanced, and the process time is saved; and by adopting polysorbate 80 or NaCl experimental group, HCP residue of the sample after affinity chromatography is still more than 0.1%, and the HCP residue removal effect is worse than that of guanidine hydrochloride.

EXAMPLE 8 comparison of the Effect of HCP clearance by different batches of recombinant humanized anti-IL-33 monoclonal antibody (QX007N) fermentation broth

Preparation of fermentation broth intermediate and affinity chromatography method as in example 7, three batches of fermentation broth intermediate were prepared, and pre-elution was performed using pre-elution buffer containing guanidine hydrochloride: 0.1mol/L sodium citrate, 0.1mol/L guanidine hydrochloride and 11mmol/L citric acid, and the pH value is 5.8. The affinity process yield and HCP residual content were measured as described in example 7, and the results are shown in table 4.

TABLE 4 affinity purification yield and HCP residual amount results for different batches of QX007N

The results in Table 4 show that the HCP residues of the fermentation liquor intermediate of QX007N of different batches are all over 20 percent, and the cell culture process is stable; the yield of the affinity process of the three batches of samples is more than 95 percent, and the yield meets the process requirement; the HCP residues of the samples after the affinity chromatography are all lower than 0.1 percent, and the removal of the HCP is kept stable by adopting guanidine hydrochloride and elution.

In summary, the present application adopts the affinity purification method described above, the residual amount of HCP in the obtained monoclonal antibody is kept at a low level, the residual control amount is not higher than 0.1%, thereby reducing the pressure for removing HCP in the subsequent purification step, and the affinity purification process described herein has good stability.

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> affinity purification method for reducing host cell protein content in production of anti-human interleukin-33 monoclonal antibody

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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. An affinity purification method for reducing the content of host cell protein in the production of anti-human interleukin-33 monoclonal antibody, which comprises the following steps:

loading: combining the anti-human interleukin-33 monoclonal antibody fermentation liquor with the well-balanced affinity chromatography medium;

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

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

3. Affinity purification method according to claim 1 or 2, wherein the affinity chromatography medium is selected from one of ligand-crosslinked chromatography media on agarose, polyvinyl ether, hydroxylated polyether resins, polyacrylic resins, polystyrene divinyl phenyl resins, polymethacrylic resins, polystyrene resins, hydroxyapatite, glass matrices, preferably ligand-crosslinked chromatography media on polyvinyl ether;

4. The affinity purification method according to any one of claims 1 to 3, wherein the buffer one is selected from one of phosphate buffer, Tris-HCl buffer and boric acid-borax buffer; in the buffer solution I, the salt concentration is 5 mM-0.25M, and the pH value is 5.5-8.0.

5. The affinity purification method according to any one of claims 1 to 4, wherein a pre-elution buffer is used for pre-elution, wherein the pre-elution buffer is a neutral buffer and/or an acidic buffer;

6. The affinity purification method according to any one of claims 1 to 5, wherein when the pre-elution is performed using a pre-elution buffer, a pre-elution active agent is further added to the pre-elution buffer;

preferably, the pre-elution activator is guanidine hydrochloride;

7. The affinity purification method according to any one of claims 1 to 6, wherein a final elution is performed with a final elution buffer;

8. The affinity purification method according to any one of claims 1 to 7, wherein a buffer solution I is used for equilibration when the anti-human interleukin-33 monoclonal antibody fermentation broth is bound to the well-balanced affinity chromatography medium.

9. The affinity purification method according to any one of claims 1 to 8, wherein the elution step further comprises, after the pre-elution and before the final elution, equilibrating the pre-eluted solution using a buffer solution.

10. The affinity purification method according to claim 9, wherein the buffer II is selected from one of phosphate buffer, Tris-HCl buffer and boric acid-borax buffer;

preferably, the second buffer is phosphate buffer or Tris-HCl buffer;

preferably, the pH value of the second buffer solution is 5.5-8.0.