A purification preparation method and application of anti-diphtheria toxin mutant antibody and CRM197

Abstract

The present invention proposes a purification preparation method and application of an anti-diphtheria toxin mutant antibody and CRM197. On the basis of developing and preparing CRM197 antigen, the present invention further immunizes mice with diphtheria toxin mutants, fuses mouse spleen cells with mouse myeloma cells, screens hybridoma cell lines that produce specific monoclonal antibodies against diphtheria toxin mutants, and obtains anti-diphtheria toxin mutant antibody sequences after sequencing. After the hybridoma cells are cultured in mice, anti-CRM197 antibodies can be prepared in vitro by chromatography purification and other methods. The antibody has a high affinity with CRM197, can be used for the purification preparation of CRM197 protein, can also be used for the purification preparation of coupled vaccines with CRM197 as a carrier, and further can be used for enzyme-linked immunosorbent assay to detect the content of CRM197 in coupled vaccines with CRM197 as a carrier.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a purification preparation method and application of an anti-diphtheria toxin mutant antibody and CRM197. Background Art

[0002] Diphtheria toxin (DT) is an exotoxin encoded by the tox gene of β-phage after it lysogenizes diphtheria bacteria. DT can inactivate the peptide chain elongation factor (EP) of eukaryotic cells, block their cellular protein synthesis, and thus lead to cell apoptosis.

[0003] CRM197 (cross-reacting materials 197) is a type of diphtheria toxin mutant, which was obtained from a strain of a toxic gene mutant of Corynebacterium diphtheriae in the early stage. Due to a missense mutation, the glycine (Gly) at position 52 in the antigen-binding fragment of diphtheria toxin mutated to glutamic acid (Glu), causing the active site of the diphtheria toxin enzyme to change. The A fragment of CRM197 cannot bind to EF2, and thus cannot have a toxic effect on cells, but the immunogenicity is not significantly different from that of diphtheria toxin. Both CRM197 and DT can bind to the diphtheria toxin receptor (DTR) on the cell membrane. Therefore, CRM197 is often used as an ideal immune protein carrier and is widely used in the field of vaccine development.

[0004] Wang Chun'e et al. separated and purified the prepared CRM197, used it as a protein carrier, and successfully prepared a group A meningococcal conjugate, increasing the carrier types of conjugate vaccines in my country. Zhao Xuena et al. cross-linked G17 with CRM197 to prepare an anti-cancer vaccine, and proved its significant tumor inhibition effect through in vitro efficacy tests. Sticking P et al. found in transdermal immunization studies that DT vaccines using CRM197 as a cross-linking reactant can increase the level of functional antibodies in mice. Broke M et al. systematically studied the biochemical and biological properties of CRM197, pointing out its important use as a vaccine conjugate protein and its potential clinical application. For more than ten years, several widely used conventional pediatric complex sugar vaccines against serious diseases (such as diseases caused by Haemophilus influenzae type B and Streptococcus pneumoniae) have used CRM197 as a protein carrier.

[0005] The inventors have previously developed and applied for a patent (a method for preparing a diphtheria toxin mutant CRM197, patent number: CN104140972B), providing a cheap and efficient CRM197 expression system. On this basis, the inventors further proposed a monoclonal antibody against a diphtheria toxin mutant CRM197 targeting CRM197. Summary of the invention

[0006] The present invention provides an anti-diphtheria toxin mutant (CRM197) antibody, which can bind to CRM197 with high affinity, can be applied to the purification preparation of CRM197, and can also be further applied to the purification of a conjugate vaccine using CRM197 as a carrier and the detection of the CRM197 content in the conjugate vaccine.

[0007] To achieve the above object, the present invention provides an anti-diphtheria toxin mutant antibody, wherein the heavy chain variable region of the antibody has the following complementary determining region CDR:

[0008] VH-CDR1 of SEQ ID NO: 2, VH-CDR2 of SEQ ID NO: 3, and VH-CDR3 of SEQ ID NO: 4;

[0009] The light chain variable region of the antibody has the following complementarity determining region CDR:

[0010] VL-CDR1 of SEQ ID NO: 10, VL-CDR2 of SEQ ID NO: 11, and VL-CDR3 of SEQ ID NO: 12;

[0011] Furthermore, the amino acid sequence of the antibody heavy chain variable region is SEQ ID NO: 1.

[0012] Furthermore, the amino acid sequence of the antibody light chain variable region is SEQ ID NO: 9.

[0013] Furthermore, the antibody is a partially or fully humanized, or fully human monoclonal antibody.

[0014] The present invention also provides a method for preparing an anti-diphtheria toxin mutant antibody, comprising the following steps:

[0015] Step 1: Immunize mice with diphtheria toxin mutants;

[0016] Step 2: Fusion of mouse spleen cells with mouse myeloma cells;

[0017] Step 3: Screening hybridoma cell lines that produce specific monoclonal antibodies against diphtheria toxin mutants;

[0018] Step 4: The hybridoma cells are cultured in mice, the ascites is harvested, and the anti-diphtheria toxin mutant antibodies are prepared by chromatography purification.

[0019] The present invention also provides a method for purifying and preparing CRM197, wherein the purification of CRM197 is achieved through the affinity between the anti-diphtheria toxin mutant antibody and CRM197, and the method comprises the following steps:

[0020] Step 1: coupling the anti-diphtheria toxin mutant antibody to agarose gel to obtain an antibody-rich affinity filler;

[0021] Step 2: Using E. coli to recombinantly express the CRM197 protein to obtain inclusion bodies containing the CRM1967 protein;

[0022] Step 3: The inclusion body refolding solution is loaded onto a chromatography column filled with the affinity filler in step 1, and CRM197 protein with a purity of >95% can be obtained by acid elution.

[0023] The present invention also proposes a method for purifying and preparing a conjugate vaccine using CRM197 as a carrier, wherein the conjugate vaccine is purified by affinity between the anti-diphtheria toxin mutant antibody and CRM197 in the conjugate vaccine, and the method comprises the following steps:

[0024] Step 1: coupling the anti-diphtheria toxin mutant antibody to agarose gel to obtain an antibody-rich affinity filler;

[0025] Step 2: The coupled mixture is loaded onto a chromatography column filled with the affinity filler in step 1, and the coupled mixture containing only CRM197 is obtained by acid elution;

[0026] The conjugate vaccine is a recombinant hEGF-CRM197 tumor therapeutic vaccine or a combination vaccine obtained by cross-linking other proteins with CRM197.

[0027] The present invention also proposes a method for detecting the content of CRM197 in a conjugate vaccine using CRM197 as a carrier, which uses the affinity between the antibody against the diphtheria toxin mutant and CRM197 in the conjugate vaccine to achieve antigen detection; the method comprises the following steps:

[0028] Step 1: Dilute the conjugate vaccine and CRM197 in multiple ratios and coat them on the ELISA plate;

[0029] Step 2: Block the ELISA plate with blocking solution;

[0030] Step 3: Add HRP-labeled CRM197 antibody to each reaction well and incubate at 37°C for 1 hour;

[0031] Step 4: Add color developing solution to develop color, then add stop solution to terminate the reaction; measure the absorbance at 450nm;

[0032] The conjugate vaccine is a recombinant hEGF-CRM197 tumor therapeutic vaccine or a combination vaccine obtained by cross-linking other proteins with CRM197.

[0033] Compared with the prior art, the advantages of the present invention are: the anti-diphtheria toxin mutant antibody of the present invention can be applied to various fields by utilizing its characteristics, including the purification and preparation of CRM197 protein, the purification and preparation of conjugate vaccines with CRM197 as a carrier, and the detection of the content of CRM197 in conjugate vaccines with CRM197 as a carrier by enzyme-linked immunosorbent assay, etc., further expanding the application of the anti-diphtheria toxin mutant antibody, and the diphtheria toxin mutant antibody preparation method of the present invention is economical and efficient, and has high market applicability.

[0034] The antibody of the present invention has specificity and high affinity. The antibody can specifically bind to the CRM197 protein, can efficiently purify and separate the CRM197 protein, and can specifically identify the CRM197 component in the conjugate vaccine, thereby facilitating the purification and separation of the conjugate vaccine and detecting the CRM197 content in the conjugate vaccine. The anti-diphtheria toxin mutant antibody provided by the present invention has a significantly higher affinity for the CRM197 protein than commercially available diphtheria toxin antibodies and other polyclonal antibodies. In the production of conjugate vaccines related to the CRM197 protein, the vaccine components containing CRM197 can be captured with strong specificity and high affinity, and the effective components of the vaccine can be purified to solve the purification problem of the conjugate vaccine; at the same time, the antibody can be used for the more sensitive enzyme-linked immunosorbent assay to detect the content of CRM197 in the conjugate vaccine, which is better used to evaluate the effective components and batch consistency of the vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 These are the results of screening different hybridoma cells expressing CRM197 antibodies using the ELISA method. The numbers are the numbers of different cell lines.

[0036] Figure 2 A comparison of the affinity of CRM197 antibodies for different cell lines is shown, and the numbers are the numbers of the different cell lines.

[0037] Figure 3 The SDS-PAGE electrophoresis diagram of the anti-CRM197 antibody prepared according to the method of the present invention in a specific embodiment of the present invention is shown.

[0038] Figure 4 This is a study on the binding between the anti-CRM197 antibody of the present invention and CRM197.

[0039] Figure 5 The SDS-PAGE electrophoresis diagram of the CRM197 protein purified and prepared according to the affinity filler and the purification method provided by the present invention in a specific embodiment of the present invention is shown.

[0040] Figure 6The present invention provides a chromatogram of the recombinant hEGF-CRM197 tumor therapeutic vaccine purified by the affinity filler and the purification method provided by the present invention.

[0041] Figure 7 The SDS-PAGE electrophoresis diagram of the recombinant hEGF-CRM197 tumor therapeutic vaccine purified using the affinity filler and the purification method provided by the invention in a specific embodiment of the present invention is shown.

[0042] Figure 8 The Western blot identification results of the recombinant hEGF-CRM197 tumor therapeutic vaccine purified according to the affinity filler and the purification method provided by the present invention in a specific embodiment of the present invention are shown.

[0043] Fig. 9 The results of ELISA determination of CRM197 content in recombinant hEGF-CRM197 tumor therapeutic vaccine. DETAILED DESCRIPTION

[0044] After extensive and in-depth research and a large number of screenings, the inventors have developed an antibody against a diphtheria toxin mutant (CRM197), provided a method for preparing the antibody and an antibody sequence, and further verified the effects of using the antibody to purify and prepare CRM197 protein, a conjugate vaccine containing a CRM197 carrier, and detecting the CRM197 content in the conjugate vaccine. The experimental results show that the anti-CRM197 antibody provided by the present invention has a high affinity with CRM197, and can be purified in one step to prepare a high-purity CRM197 protein. Further, the conjugate containing the CRM197 portion in the mixture can be effectively separated and the CRM197 content in the conjugate can be detected. The present invention was completed on this basis.

[0045] the term

[0046] In order to more easily understand the present invention, some technical and scientific terms are specifically defined below. Unless otherwise clearly defined in this article, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions can be changed. It should also be understood that the terms used herein are intended only to describe specific embodiments, and are not intended to be restrictive, and the scope of the present invention will be limited only by the appended claims.

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. As used herein, the term "about" when used in reference to a specific recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values ​​therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0048] The three-letter and one-letter codes for amino acids used in the present invention are as described in J. biol. chem, 243, p3558 (1968).

[0049] As used herein, the term "optional" or "optionally" means that the event or situation described subsequently may occur but need not occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable region of a specific sequence may have but need not have, and may be 1, 2 or 3.

[0050] "Sequence identity" of the present invention means the degree of identity between two nucleic acids or two amino acid sequences when optimally compared and compared with mutations such as appropriate substitutions, insertions or deletions. The sequence identity between the sequences described in the present invention and the sequences with the same identity can be at least 85%, 90% or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%.

[0051] As used herein, the terms "diphtheria toxin mutant" and "CRM197 protein" or "CRM197" are used interchangeably, and both refer to the CRM197 protein to which the antibodies of the present invention specifically bind.

[0052] Preferably, the amino acid sequence of the diphtheria toxin mutant CRM197 of the present invention is shown in SEQ ID NO: 19;

[0053] (SEQ ID NO: 19)

[0054] Antibody

[0055] As used herein, the term "antibody" or "immunoglobulin" is a heterotetrameric glycoprotein of about 150,000 daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

[0056] As used herein, the term "variable" means that some parts of the variable region in an antibody are different in sequence, which forms the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three fragments called complementary determining regions (CDRs) or hypervariable regions in the variable regions of light and heavy chains. The more conservative part of the variable region is called the framework region (FR). The variable regions of natural heavy and light chains each contain four FR regions, which are roughly in a b-folding configuration, connected by three CDRs forming a connecting loop, and can form a partial b-folding structure in some cases. The CDRs in each chain are closely together through the FR region and together with the CDRs of another chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Volume I, 647-669 pages (1991)). The constant region does not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody's antibody-dependent cytotoxicity.

[0057] The "light chains" of vertebrate antibodies (immunoglobulins) can be classified into one of two distinct classes (called kappa and l) based on the amino acid sequence of their constant regions. Immunoglobulins can be divided into different classes based on the amino acid sequence of their heavy chain constant regions. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant regions corresponding to the different classes of immunoglobulins are called a, d, e, g, and m, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known in the art.

[0058] Generally, the antigen binding properties of an antibody can be described by three specific regions located in the variable regions of the heavy and light chains, called variable regions (CDRs). This segment is divided into four framework regions (FRs). The amino acid sequences of the four FRs are relatively conservative and do not directly participate in the binding reaction. These CDRs form a ring structure, and the β-folds formed by the FRs in between are close to each other in spatial structure. The CDRs on the heavy chain and the CDRs on the corresponding light chains constitute the antigen binding site of the antibody. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR region.

[0059] In the present invention, "VH-CDR1" and "CDR-H1" are used interchangeably, both referring to the CDR1 of the heavy chain variable region; "VH-CDR2" and "CDR-H2" are used interchangeably, both referring to the CDR2 of the heavy chain variable region; "VH-CDR3" and "CDR-H3" are used interchangeably, both referring to the CDR3 of the heavy chain variable region. "VL-CDR1" and "CDR-L1" are used interchangeably, both referring to the CDR1 of the light chain variable region; "VL-CDR2" and "CDR-L2" are used interchangeably, both referring to the CDR2 of the light chain variable region; "VL-CDR3" and "CDR-L3" are used interchangeably, both referring to the CDR3 of the light chain variable region.

[0060] The present invention includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.

[0061] The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-consecutive amino acids in a unique spatial conformation.

[0062] The terms "specific binding", "selective binding", "selectively binds" and "specifically binds" refer to the binding of an antibody to a predetermined epitope on an antigen. -7 M, for example, less than about 10 -8 M, 10 -9 M or l0 -10 Binds with an affinity (KD) of M or less.

[0063] As used herein, the term "antigenic determinant" refers to a discrete three-dimensional site on an antigen that is recognized by the antibodies or antigen-binding fragments of the present invention.

[0064] In the present invention, the antibody can specifically recognize the CRM197 protein, and the affinity is significantly higher than that of commercially available antibodies.

[0065] In the present invention, antibodies include murine, chimeric, humanized or fully human antibodies prepared by techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including human and non-human parts, can be obtained by standard DNA recombinant techniques, and they are all useful antibodies. A chimeric antibody is a molecule in which different parts are from different animal species, such as a chimeric antibody having a variable region from a monoclonal antibody from a mouse, and a constant region from a human immunoglobulin (see, for example, U.S. Pat. No. 4,816,567 and U.S. Pat. No. 4,816,397, which are hereby incorporated by reference in their entirety). A humanized antibody refers to an antibody molecule derived from a non-human species, having one or more complementary determining regions (CDRs) derived from a non-human species and a framework region derived from a human immunoglobulin molecule (see U.S. Pat. No. 5,585,089, which is hereby incorporated by reference in its entirety). These chimeric and humanized monoclonal antibodies can be prepared using DNA recombinant techniques well known in the art.

[0066] In the present invention, the antibodies may be monospecific, bispecific, trispecific, or more multispecific.

[0067] In the present invention, the antibody of the present invention also includes its conservative variants, which refer to polypeptides formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids with amino acids of similar or similar properties compared with the amino acid sequence of the antibody of the present invention. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table A.

[0068] Table A

[0069] Initial residue Representative replacement Preferred substitutions Ala (A) Val; Leu; Ile Val Arg I Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp (D) Glu Glu CysI Ser Ser Gln (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro; Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu ; Val ; Met ; Ala ; Phe Leu Leu (L) Ile ; Val ; Met ; Ala ; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Leu; Val; Ile; Ala; Tyr Leu Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile ; Leu ; Met ; Phe ; Ala Leu

[0070] In the present invention, the antibody is an antibody that specifically binds to CRM197. The present invention provides a highly specific and high affinity antibody against CRM197, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region (VH) amino acid sequence, and the light chain comprises a light chain variable region (VL) amino acid sequence.

[0071] The heavy chain variable region (VH) has a complementarity determining region CDR selected from the following group:

[0072] VH-CDR1 shown in SEQ ID NO: 2, VH-CDR2 shown in SEQ ID NO: 3, and VH-CDR3 shown in SEQ ID NO: 4;

[0073] The light chain variable region (VL) has a complementarity determining region (CDR) selected from the group consisting of:

[0074] VL-CDR1 shown in SEQ ID NO: 10, VL-CDR2 shown in SEQ ID NO: 11, and VL-CDR3 shown in SEQ ID NO: 12; wherein any one of the above amino acid sequences also includes a derivative sequence that is optionally added, deleted, modified and / or substituted with at least one amino acid and can retain the binding affinity to CRM197.

[0075] In another preferred embodiment, the sequence formed by adding, deleting, modifying and / or replacing at least one amino acid sequence is preferably an amino acid sequence with a homology or sequence identity of at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95%.

[0076] Methods for determining sequence homology or identity known to those of ordinary skill in the art include, but are not limited to: Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM and Griffin, HG, ed., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987 and Sequence Analysis Primer, Gribskov, M. and Devereux, J., ed., Stockton Press, New York, 1991 and Carillo, H. and Lipman, D., SIAM J. Applied Math., 48:1073 (1988). The preferred method for determining identity is to obtain the largest match between the sequences tested. Methods for determining identity are compiled in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include, but are not limited to, the GCG program package (Devereux, J. et al., 1984), BLASTP, BLASTN and FASTA (Altschul, S, F. et al., 1990). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al., 1990). The well-known Smith Waterman algorithm can also be used to determine identity.

[0077] Preferably, the antibody described herein is one or more of a full-length antibody protein, an antigen-antibody binding domain protein fragment, a bispecific antibody, a multispecific antibody, a single chain antibody (scFv), a single domain antibody (sdAb), and a single region antibody (Signle-domain antibody), as well as a monoclonal antibody or polyclonal antibody prepared from the above antibodies. The monoclonal antibody can be prepared by a variety of approaches and techniques, including hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc. The mainstream is to prepare monoclonal antibodies from wild-type or transgenic mice by hybridoma technology.

[0078] The full-length antibody protein is a conventional full-length antibody protein in the art, which includes a heavy chain variable region, a light chain variable region, a heavy chain constant region and a light chain constant region. The heavy chain variable region and light chain variable region of the protein and the human heavy chain constant region and the human light chain constant region constitute a full-length human antibody protein. Preferably, the full-length antibody protein is IgG1, IgG2, IgG3 or IgG4; more preferably, IgG1.

[0079] The antibody of the present invention may be a double-chain or single-chain antibody, and may be selected from animal-derived antibodies, chimeric antibodies, and humanized antibodies, more preferably humanized antibodies, human-animal chimeric antibodies, and more preferably fully humanized antibodies.

[0080] The antibody derivatives of the present invention can be single-chain antibodies and / or antibody fragments, such as Fab, Fab', (Fab')2 or other known antibody derivatives in the field, as well as any one or more of IgA, IgD, IgE, IgG and IgM antibodies or other subtypes of antibodies.

[0081] The single-chain antibody is a conventional single-chain antibody in the art, which includes a heavy chain variable region, a light chain variable region and a short peptide of 15 to 20 amino acids.

[0082] Wherein, the animal is preferably a mammal, such as a mouse.

[0083] The antibody of the present invention may be a chimeric antibody, a humanized antibody, a CDR-grafted and / or modified antibody targeting CRM197.

[0084] In the above content of the present invention, the number of added, deleted, modified and / or substituted amino acids is preferably not more than 40% of the total number of amino acids in the initial amino acid sequence, more preferably not more than 35%, more preferably 1-33%, more preferably 5-30%, more preferably 10-25%, more preferably 15-20%.

[0085] In the above content of the present invention, more preferably, the number of the added, deleted, modified and / or substituted amino acids may be 1-7, more preferably 1-5, more preferably 1-3, more preferably 1-2.

[0086] In another preferred embodiment, the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO: 1.

[0087] In another preferred example, the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO: 9.

[0088] In a more preferred embodiment, each antibody or recombinant protein of the present invention specifically includes the following VL and VH sequences, as well as CDR and FR sequences.

[0089] Table B Summary of antibody sequences of the present invention

[0090] name sequence SEQ ID NO: Heavy chain variable region (VH) <![CDATA[QVQLKESGPGLVAPSQSLSITCTVS GFSLTGYG INWIRQPPGKDLEWLGM IWGDGST DYNSALKSRLSISKDDSKSQVFLKMNSLQTDDTARYYC ARGNYFDF WGQGTTLTVSS]]> 1 VH-CDR1 GFSLTGYG 2 VH-CDR2 IWGDGST 3 VH-CDR3 ARGNYFDF 4 VH-FR1 QVQLKESGPGLVAPSQSLSITCTVS 5 VH-FR2 INWIRQPPGKDLEWLGM 6 VH-FR3 DYNSALKSRLSISKDDSKSQVFLKMNSLQTDDTARYYC 7 VH-FR4 WGQGTTLTVSS 8 Light chain variable region (VL) <![CDATA[DIVLTQSPASLAVSLGQRATISCRAS ESVNNYGISF MNWFQQKPGQPPKLLIY AAS NQGSGVPARFSGSGSGTDFSLNIHPMEEDDTAMYFC QQSKEFPRT FGGGTKVEIK]]> 9 VL-CDR1 ESVNNYGISF 10 VL-CDR2 AAS 11 VL-CDR3 QQSKEFPRT 12 VL-FR1 DIVLTQSPASLAVSLGQRATISCRAS 13 VL-FR2 MNWFQQKPGQPPKLLIY 14 VL-FR3 NQGSGVPARFSGSGSGTDFSLNIHPMEEDDTAMYFC 15 VL-FR4 FGGGTKVEIK 16 VH coding sequence CAGGTGCAGCTGAAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCATCACATGCACCGTCTCAGGGTTCTCATTAACCGGCTATGGTATAAACTGGATTCGCCAGCCTCCAGGAAAGGATCTGGAGTGGCTGGGAATGATATGGGGTGATGGCAGCACAGACTATAATTCAGCTCTCAAATCCAGACTGAGCATCAGCAAGGACGACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCAGGTACTACTGTGCCAGAGGGAACTACTTTGACTTCTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA 17 Coding sequence of VL GACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCTCCTGCAGAGCCAGCGAAAGTGTTAATAATTATGGCATTAGTTTTATGAACTGGTTCCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATGCTGCATCCAACCAAGGATCCGGGGTCCCTGCCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCAGCCTCAACATCCATCCTATGGAGGAGGATGATACTGCAATGTATTTCTGTCAGCAAAGTAAGGAGTTTCCTCGGACGTTCGGTGGAGGCACCAAGGTGGAAATCAAA 18

[0091] Polynucleotide encoding

[0092] The present invention also provides a polynucleotide encoding the above-mentioned antibody or its heavy chain variable region or light chain variable region.

[0093] Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in the sequence listing SEQ ID NO: 17; and / or, the nucleotide sequence of the nucleic acid encoding the light chain variable region is shown in the sequence listing SEQ ID NO: 18.

[0094] More preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in the sequence listing SEQ ID NO: 17; and the nucleotide sequence of the nucleic acid encoding the light chain variable region is shown in the sequence listing SEQ ID NO: 18.

[0095] The method for preparing the nucleic acid is a conventional method in the art, and preferably comprises the following steps: obtaining a nucleic acid molecule encoding the above protein by gene cloning technology, or obtaining a nucleic acid molecule encoding the above protein by artificial full sequence synthesis.

[0096] Those skilled in the art know that the base sequence encoding the amino acid sequence of the above-mentioned protein can be appropriately introduced with substitution, deletion, change, insertion or addition to provide a polynucleotide homologue. The polynucleotide homologue of the present invention can be prepared by replacing, deleting or adding one or more bases of the gene encoding the protein sequence within the range of maintaining the antibody activity.

[0097] Antibody preparation

[0098] The sequence of the DNA molecule of the antibody of the present invention or its fragment can be obtained by conventional techniques, such as PCR amplification or genomic library screening. In addition, the coding sequences of the light chain and the heavy chain can be fused together to form a single-chain antibody.

[0099] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0100] In addition, artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is shorter. Usually, a long fragment of sequence can be obtained by synthesizing multiple small fragments first and then connecting them.

[0101] At present, the DNA sequence encoding the antibody (or its fragment, or its derivative) of the present invention can be obtained completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.

[0102] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0103] The host cell can be a prokaryotic cell, such as a bacterial cell, a lower eukaryotic cell, such as a yeast cell, or a higher eukaryotic cell, such as a mammalian cell. Preferred animal cells include (but are not limited to): CHO cells, HEK-293 cells.

[0104] Typically, the transformed host cells are cultured under conditions suitable for the expression of the antibodies of the present invention, and then purified using conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, and other conventional separation and purification methods well known to those skilled in the art to obtain the antibodies of the present invention.

[0105] The resulting monoclonal antibodies can be identified by conventional means. For example, the binding specificity of the monoclonal antibodies can be determined by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of the monoclonal antibodies can be determined, for example, by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

[0106] The antibodies of the present invention can be expressed in cells, on cell membranes, or secreted outside cells. If necessary, the antibodies can be separated and purified by various separation methods using their physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with protein precipitants (salting out method), centrifugation, osmotic sterilization, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

[0107] application

[0108] The present invention also provides uses of the antibodies of the present invention, such as for separating and purifying CRM197 protein and purifying and separating conjugate vaccines. Furthermore, the antibodies can be used to detect the content of CRM197 in conjugate vaccines.

[0109] Preferably, the conjugate vaccine is a recombinant hEGF-CRM197 tumor therapeutic vaccine; and / or other conjugate vaccines using CRM197 as a cross-linking reactant.

[0110] The main advantages of the present invention include:

[0111] 1) Specificity. The antibody can specifically bind to the CRM197 protein, can efficiently purify and separate the CRM197 protein, and can specifically identify the CRM197 component in the conjugate vaccine, thereby facilitating the purification and separation of the conjugate vaccine and the detection of the CRM197 content in the conjugate vaccine.

[0112] 2) High affinity. The anti-diphtheria toxin mutant antibody provided by the present invention has a significantly higher affinity for CRM197 protein than commercially available diphtheria toxin antibodies and other polyclonal antibodies. In the production of conjugate vaccines related to the CRM197 protein, the vaccine components containing CRM197 can be captured with strong specificity and high affinity, and the effective components of the vaccine can be purified, thus solving the purification problem of the conjugate vaccine; at the same time, the antibody can be used for the more sensitive enzyme-linked immunosorbent assay to detect the content of CRM197 in the conjugate vaccine, which can be better used to evaluate the effective components and batch consistency of the vaccine.

[0113] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0114] Example 1: Preparation and screening of anti-CRM197 antibody monoclonal cell lines

[0115] CRM197 protein (the preparation method refers to the patent application of the inventor: Preparation method of diphtheria toxin mutant CRM197, patent authorization announcement number: CN 104140972 B) was used to immunize BALB / c mice multiple times to obtain mouse spleen cells that highly expressed antibodies. The spleen cells of the immunized mice were fused with SP2 / 0 myeloma cells to prepare hybridoma cells. Five monoclonal cell lines were finally screened using ELISA and monoclonal methods, and their ELISA binding curves and ED50 values ​​were determined.

[0116] 1. Materials and instruments

[0117] 1. Mouse: BALB / c

[0118] 2. Myeloma cells: SP2 / 0 (ATCC)

[0119] 3. Immunogen: CRM197 protein (1 mg / ml)

[0120] 4. Cell growth medium: 1640 + 10% FBS (Gibco) + OPI Media Supplement (Sigma)

[0121] 5. Detection antibody: M-HRP-anti-mouse IgG

[0122] 6. Cell fusion instrument: BTX ECM 2001 multifunctional cell fusion instrument

[0123] (II) Animal Immunization Methods

[0124] 1. Mouse lymph node immunization: CRM197 protein was injected into the inguinal lymph nodes at a dose of 50 μg / mouse, once every two weeks for a total of three immunizations. The tail vein blood was collected for the last immunization to determine the titer. Hybridomas were prepared when the titer was above 1:100,000.

[0125] 2. Preparation of hybridoma cells: Hybridoma cells were prepared using ECM2001 multifunctional cell fusion instrument.

[0126] (III) ELISA detection method (indirect method)

[0127] 1. CRM197 protein was diluted to 1 μg / ml with PBS, 100 μl / well was coated at 4°C overnight;

[0128] 2. Wash the plate once with PBST, block with 200 μl / well 3% BSA-PBS, 37°C, 1 hour;

[0129] 3. Wash the plate once with PBST;

[0130] 4. Dilute each antibody to 1 μg / ml with 1% BSA-PBS, 2-fold dilution, 12 gradients in total, 100 μl / well, 37°C, 1 hour;

[0131] 5. Wash the plate 3 times with PBST;

[0132] 6. Dilute the anti-mouse IgG secondary antibody in 1% BSA-PBS, add 100 μl / well, 37°C, 1 hour;

[0133] 7. Wash the plate 5 times with PBST;

[0134] 8. Develop color with TMB and read the OD450 value on a microplate reader.

[0135] (IV) Results

[0136] Five hybridoma cell lines with the best growth status and the highest titer were screened, and their ELISA binding curves are shown in Figure 1 .

[0137] Furthermore, the affinity of the hybridoma cells was compared with that of CRM197. Figure 2 , it can be seen that cell line 1 has the best affinity. At the same time, we selected commercially available diphtheria toxin monoclonal antibodies for peer comparison testing and found that the antibody produced by cell line 1 has a binding ability to CRM197 that is about 30 times that of commercially available antibodies, which is much better than the existing diphtheria toxin antibodies.

[0138] Example 2: Preparation of the CRM197 antibody of the present invention

[0139] Experimental method: The hybridoma cell line No. 1 in Example 1 was monocloned using the limiting dilution method to obtain anti-CRM197 monoclonal antibody cells. The cells were cultured and expanded and then inoculated into the peritoneal cavity of Balb / c mice. The mice were observed for ascites production within 7-14 days after injection and the ascites was collected.

[0140] The collected ascites was first precipitated with ammonium sulfate, and then the precipitate was resuspended in buffer A (20mM PBS, pH 7.0) and loaded onto the Protein A antibody purification column that had been equilibrated with buffer A. After loading, the column was rinsed with buffer A until the A280 value and conductivity returned to the baseline, and 100% buffer B (20mM citric acid, pH 2.7) was used for one-step elution, the A280 value of the eluate was monitored, and the eluted fractions were collected as the preliminary purification product of the anti-CRM197 antibody.

[0141] The pH of the preliminary purified product was adjusted to 5.0-6.0 using buffer C (10 mM citric acid, pH 5.5), loaded onto an already balanced cation exchange chromatography column, and then the NaCl concentration was linearly increased until 40% elution, and the largest eluted fraction was collected, which was the anti-CRM197 antibody.

[0142] Experimental results:

[0143] Ammonium sulfate treatment can quickly and effectively remove impurities in ascites. The treated crude extract can capture anti-CRM197 antibodies in one step by passing through a Protein A antibody column, and then finely purified by a cation exchange column to prepare high-purity anti-CRM197 antibodies. The purified anti-CRM197 antibodies were analyzed by SDS-PAGE electrophoresis. The results are as follows: Figure 3 , showing two protein bands with molecular weights of approximately 50kD and 25kD, containing typical heavy and light chain structures.

[0144] Example 3: The CRM197 antibody of the present invention can specifically recognize the CRM197 protein

[0145] The antibody of the present invention and the commercially available diphtheria toxin antibody were used as primary antibodies to perform Western-blot experiments respectively to verify the effectiveness of the CRM197 antibody.

[0146] Experimental methods:

[0147] Take the CRM197 protein for SDS-PAGE electrophoresis, transfer the protein on the gel to a nitrocellulose membrane, and block it with 10% skim milk powder. Take the CRM197 antibody (Anti-CRM197, 0.1 mg / ml, 1:5000 dilution) prepared in Example 2 and the commercially available diphtheria toxin antibody (Anti-Diphtheria Toxin, abcam, 0.1 mg / ml, 1:1000) as the primary antibody to incubate the nitrocellulose membrane respectively; after washing the unbound primary antibody on the membrane with TTBS, add the anti-mouse IgG secondary antibody (1 mg / ml, 1:10000 dilution) for incubation. After the incubation is completed, wash the unbound antibody with TTBS again, and add the substrate buffer for color development.

[0148] Experimental results:

[0149] The results are as follows Figure 4 As shown, clear CRM197 protein bands can be seen on both membranes. This shows that both anti-CRM197 antibodies and diphtheria toxin antibodies can specifically recognize CRM197 protein. In addition, under the same experimental conditions, the working concentration of the antibody invented by the present invention is much lower than that of the commercially available diphtheria toxin antibody, indicating that the CRM197 antibody prepared by the present invention has a better affinity with the CRM197 protein.

[0150] Example 4: Purification of CRM197 protein using the antibody provided by the present invention

[0151] At present, the preparation of recombinant CRM197 protein often adopts the E. coli expression system, which needs to undergo inclusion body transformation and renaturation, and the renaturation solution needs to be purified by anion exchange column, molecular sieve column purification or a combination thereof. The purification process is relatively cumbersome, and the anti-CRM197 antibody of the present invention can effectively capture the CRM197 protein in the renaturation mixture by utilizing the specificity of "antigen-antibody" binding, and then a high-purity CRM197 protein can be obtained by a one-step acid elution.

[0152] Experimental methods:

[0153] The anti-CRM197 antibody purified and prepared in Example 2 was cross-linked to NHS-activated Sepharose 4 Fast Flow or CNBr-activated Sepharose 4 Fast Flow or CNBr-activated Bestarose 4 Fast Flow or Epoxy-activated Bestarose 6B.

[0154] E. coli that successfully expressed the CRM197 protein was broken into inclusion bodies. After denaturation and renaturation of the inclusion bodies, the pH was adjusted with buffer D (20mM Tris / HCl, pH 8.0) and loaded onto the CRM197 antibody affinity chromatography column that had been equilibrated with buffer D. After loading, the column was rinsed with buffer D until the A280 value and conductivity returned to the baseline, and 100% buffer E (20mM citric acid, pH 3.0) was used for one-step elution. The A280 value of the eluate was monitored, and the eluted fractions were collected as the CRM197 protein.

[0155] Experimental results:

[0156] from Figure 5 It can be seen that the purity of the CRM197 protein obtained by one-step purification with the CRM197 antibody affinity column can reach 100%, which significantly improves the purity and preparation efficiency of the target protein and effectively shortens the preparation time of the CRM197 protein.

[0157] Example 5: Using the antibody purification conjugate vaccine provided by the present invention

[0158] After conjugation (protein A-CRM197, protein A is any conjugable protein), the conjugate vaccine with CRM197 as the carrier has a relatively complex composition (protein A, CRM197, protein A-protein A, protein A-CRM197, CRM197-CRM197) and the properties of each component are not uniform, so it is impossible to effectively distinguish the conjugate containing CRM197 protein, and there is a certain difficulty in purification preparation. The anti-CRM197 antibody of the present invention can utilize the specificity of "antigen-antibody" binding to effectively identify the CRM197 component in the conjugate vaccine, thereby separating the vaccine component containing the CRM197 component, thereby achieving the purpose of purification.

[0159] Experimental methods:

[0160] The anti-CRM197 antibody purified and prepared in Example 2 was cross-linked to NHS-activated Sepharose 4 Fast Flow or CNBr-activated Sepharose 4 Fast Flow or CNBr-activated Bestarose 4 Fast Flow or Epoxy-activated Bestarose 6B.

[0161] After the coupling reaction of hEGF protein and CRM197 protein, the reaction solution was adjusted with buffer D (20mM Tris / HCl, pH 8.0) for pH and loaded onto the CRM197 antibody affinity chromatography column that had been equilibrated with buffer D. After loading, the column was rinsed with buffer D until the A280 value and conductivity returned to the baseline, and 100% buffer E (20mM citric acid, pH 3.0) was used for one-step elution, the A280 value of the eluate was monitored, and the eluted fractions were collected as the effective conjugate vaccine containing the CRM197 component.

[0162] Experimental results:

[0163] from Figure 6 It can be seen that when the conjugate vaccine mixture is loaded onto the affinity chromatography column of the CRM197 antibody, an obvious ultraviolet absorption peak can be seen using acid elution, indicating that the components containing CRM197 in the mixture can be specifically recognized by the CRM197 antibody and effectively separated and purified. Figure 7 SDS-PAGE in the middle shows that the purified sample has effectively removed the small molecule components that failed to cross-link CRM197, such as Figure 8 As shown, and further analyzed by western-blot, it can be seen that the purified sample is mainly a successfully cross-linked conjugate. Therefore, the method can be used to effectively purify and separate the conjugate vaccine.

[0164] discuss:

[0165] Immunoaffinity chromatography mainly utilizes the high specific affinity characteristics between antigen and antibody. The antibody is fixed on the stationary phase, and when the mixture containing the antigen is passed through the chromatographic column, it can be specifically captured, while other substances will not be captured and flow through, thereby effectively separating the target protein. The binding of antigen and antibody is reversible, that is, after the antigen and antibody bind to form a complex, they can dissociate under certain conditions. The dissociated antigen and antibody can still maintain the original physicochemical properties and biological activity. The CRM197 protein in the refolding solution or the CRM197 protein in the CRM197 conjugate vaccine specifically binds to the CRM197 antibody under neutral conditions, and then dissociates under acidic conditions. Therefore, the buffer E used in the present invention is an acidic buffer.

[0166] Example 6: Using the antibody provided by the present invention to detect the CRM197 content in the conjugate vaccine

[0167] Enzyme-linked immunosorbent assay (ELISA) is based on the specific binding characteristics of antigen and antibody, and is commonly used to detect antigen / antibody content, that is, the antigen is diluted in a certain ratio and coated on a solid phase carrier, and then the diluted specific enzyme-labeled antibody is added, and after incubation, the substrate is added to develop color and the result is interpreted. It is intended to use the CRM197 antibody provided by the present invention to be coupled with horseradish peroxidase (HRP) to prepare an enzyme-labeled antibody, directly detect CRM197 coated in an ELISA plate, and use CRM197 protein with a known protein content as a reference to quantitatively detect the content of CRM197 in the coupled vaccine.

[0168] Experimental methods:

[0169] 1. hEGF-CRM197 conjugate vaccine (800ng / ml) and CRM197 (800ng / ml) were diluted in multiples and coated on the ELISA plate at 100μl / well. After sealing the plate, let it stand at 4℃ overnight, then discard the liquid in the wells of the ELISA plate. Wash the plate 3 times with washing solution.

[0170] 2. Add 200 μl / well of blocking solution to the ELISA plate, seal the plate, incubate at 37℃±1℃ for 2 hours, discard the blocking solution, and wash the plate 3 times with washing solution.

[0171] 3. Add 100 μl HRP-labeled CRM197 monoclonal antibody (1:1000) to each well. Mix at 37°C for 10 minutes, seal the plate and incubate at 37°C ± 1°C for 1 hour. Wash the plate 5 times.

[0172] 4. Add 100 μl / well of color development solution to the ELISA plate. After color development at room temperature and away from light for 15 minutes, add 50 μl / well of stop solution to the ELISA plate to terminate the reaction. Use an ELISA reader to measure the absorbance (OD450) at 450 nm.

[0173] Experimental results:

[0174] like Figure 9 As shown, different concentrations of CRM197 protein and antibodies can present an obvious "S" curve. The software calculated that the EC50 value of the CRM197 standard is 10.29ng / ml, and the EC50 value of the hEGF-CRM197 conjugate is 13.62ng / ml. It can be converted that every 1μg / ml hEGF-CRM197 conjugate contains about 0.75μg / ml of CRM197. The above is only a preferred embodiment of the patent of the present invention and does not have any limiting effect on the present invention. Any technician in the relevant technical field, without departing from the scope of the technical scheme of the present invention, makes any form of equivalent replacement or modification to the technical scheme and technical content disclosed in the present invention, which does not depart from the content of the technical scheme of the present invention and still belongs to the protection scope of the present invention.

Claims

1. An anti-diphtheria toxin mutant antibody, characterized in that: The corresponding antibody was prepared using CRM197 as an immunogen, and the heavy chain variable region of the antibody had the following complementary determining region CDR: VH-CDR1 as set forth in SEQ ID NO: 2, VH-CDR2 as set forth in SEQ ID NO: 3, and VH-CDR3 as set forth in SEQ ID NO: 4; The light chain variable region of the antibody has the following complementarity determining region CDR: VL-CDR1 as shown in SEQ ID NO: 10, VL-CDR2 as shown in SEQ ID NO: 11 and VL-CDR3 as shown in SEQ ID NO:

12.

2. The antibody according to claim 1, characterized in that The amino acid sequence of the antibody heavy chain variable region is SEQ ID NO:

1.

3. The antibody according to claim 1, characterized in that The amino acid sequence of the antibody light chain variable region is SEQ ID NO:

9.

4. The antibody according to claim 1, characterized in that The antibody is a partially or fully humanized, or fully human monoclonal antibody.

5. A method for purifying and preparing CRM197, using the anti-diphtheria toxin mutant antibody according to any one of claims 1 to 4, characterized in that: Purification of CRM197 is achieved through the affinity between the anti-diphtheria toxin mutant antibody and CRM197, comprising the following steps: Step 1: coupling the anti-diphtheria toxin mutant antibody to agarose gel to obtain an antibody-rich affinity filler; Step 2: Using E. coli to recombinantly express the CRM197 protein to obtain inclusion bodies containing the CRM197 protein; Step 3: The inclusion body refolding solution is loaded onto a chromatography column filled with the affinity filler in step 1, and CRM197 protein with a purity of >95% can be obtained by acid elution.

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