Immunoglobulin binding proteins and uses thereof

Abstract

There is provided an isolated polypeptide consisting of a mutated B domain of Protein A, a non-native immunoglobulin binding protein comprising the mutated B domain, a conjugate comprising the mutated B domain or non-native immunoglobulin binding protein, and related uses.

Description

Immunoglobulin binding protein and its application Technical Field

[0001] The present invention relates to the field of protein purification. Specifically, the present invention provides an isolated polypeptide consisting of a mutated B domain of Protein A, an immunoglobulin-binding protein comprising the mutated B domain, a conjugate comprising the mutated B domain or a non-natural immunoglobulin-binding protein, and related uses. Background Art

[0002] Protein A, short for staphylococcus protein A, originates from a strain of Staphylococcus aureus. It contains five highly homologous single domains: E, D, A, B, and C, starting from the N-terminus. Each domain contains approximately 58 amino acid residues, forming a three-stranded antiparallel α-helix structure. Each of these single domains can specifically bind to the Fc (crystallizable) and Fab (antigen-binding) segments of IgG (immunoglobulin G) molecules. The binding region to the Fc segment is located at the junction of the second and third constant regions (CH2 and CH3), while the binding region to the Fab segment is located in the variable region of the heavy chain (VH, limited to the VH3 gene family).

[0003] Recombinant Protein A has the following advantages as a ligand material for antibody molecule purification: 1) low expression cost and can be expressed on a large scale using the E. coli prokaryotic system; 2) strong specificity and strong affinity for human IgG1, IgG2, IgG4 and mouse IgG2; 3) good regeneration and can be repeatedly regenerated and recycled under alkaline conditions.

[0004] However, most recombinant Protein A fillers currently on the market bind to both the Fc and VH regions. Bispecific or multispecific antibodies, which typically contain multiple VH regions, bind to Protein A with significantly higher strength than monoclonal antibodies. This results in more stringent elution conditions during antibody purification, such as lower pH values. These conditions themselves pose a potential risk to antibody quality and increase the number of subsequent purification steps and costs.

[0005] Summary of the Invention

[0006] The immunoglobulin-binding protein comprising a mutated B domain of Protein A provided by the present invention has high binding activity to the Fc domain (e.g., the IgG Fc domain) while eliminating binding activity to the antibody heavy chain variable region. It can provide milder elution conditions during the purification of bispecific or multispecific antibodies (e.g., bispecific or multispecific antibodies containing an IgG Fc domain), effectively ensuring sample quality and reducing the steps and costs of subsequent fine purification. This effectively solves the problems encountered when using traditional Protein A to purify bispecific or multispecific antibodies, such as harsh elution conditions (e.g., low pH values) and significant impact on antibody quality.

[0007] Therefore, in one aspect, the present application provides an isolated polypeptide consisting of a mutant B domain of Protein A or a variant thereof; wherein the mutant B domain comprises one or more (e.g., 1, 2, 3 or 4) amino acid substitutions selected from N23T, D36V, D37R, Q40V compared to the B domain of wild-type Protein A;

[0008] wherein the variant has at least 90%, such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mutated B domain; or, has one or several (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9) amino acid substitutions (preferably conservative substitutions), additions, or deletions, and retains the function of the mutated B domain; and;

[0009] The variant and the mutated B domain have the same amino acid residues at positions 1, 23, 29, 36, 37 and 40 corresponding to the B domain of wild-type Protein A.

[0010] In certain embodiments, the mutated B domain further comprises the amino acid substitutions: (i) G29A, and / or, (ii) A1V, compared to the B domain of wild-type Protein A.

[0011] In certain embodiments, the mutated B domain comprises amino acid substitutions compared to the B domain of wild-type Protein A: (a) N23T, G29A, D36V, D37R, and Q40V, or (b) A1V, N23T, D36V, D37R, and Q40V.

[0012] In certain embodiments, the mutated B domain comprises the following amino acid substitutions compared to the B domain of wild-type Protein A: A1V, N23T, G29A, D36V, D37R, and Q40V.

[0013] In certain embodiments, the mutated B domain further comprises an amino acid substitution K58A compared to the B domain of wild-type Protein A.

[0014] In certain embodiments, the mutated B domain comprises the following amino acid substitutions compared to the B domain of wild-type Protein A: N23T, D36V, D37R, Q40V, and K58A.

[0015] In certain embodiments, the mutated B domain comprises amino acid substitutions compared to the B domain of wild-type Protein A: (a) N23T, G29A, D36V, D37R, Q40V, and K58A, or (b) A1V, N23T, D36V, D37R, Q40V, and K58A.

[0016] In certain embodiments, the mutated B domain comprises the following amino acid substitutions compared to the B domain of wild-type Protein A: A1V, N23T, G29A, D36V, D37R, Q40V, and K58A.

[0017] In certain embodiments, the B domain of the wild-type Protein A has the sequence shown in SEQ ID NO:4.

[0018] In certain embodiments, the mutated B domain has an amino acid sequence as shown in SEQ ID NO: 5 or 17. The sequence shown here does not include an amino acid (such as methionine (Met)) encoded by a start codon (such as ATG) at its N-terminus. Those skilled in the art understand that in the process of preparing proteins by genetic engineering, due to the effect of the start codon, the first position of the resulting polypeptide chain is often an amino acid (such as Met) encoded by the start codon. The mutated B domain of the present invention not only encompasses an amino acid sequence that does not include an amino acid (such as Met) encoded by a start codon at its N-terminus, but also encompasses an amino acid sequence that includes an amino acid (such as Met) encoded by a start codon at its N-terminus. Therefore, a sequence that further includes an amino acid (such as Met) encoded by a start codon at the N-terminus of the above-mentioned amino acid sequence is also within the scope of protection of the present invention.

[0019] In certain embodiments, the isolated polypeptide specifically binds to the IgG Fc domain in the range of pH 7.0-8.0 (e.g., pH 7.0-7.6, pH 7.0-7.4); and / or, the isolated polypeptide dissociates from the IgG Fc domain in the range of pH 2.7-4.5 (e.g., pH 2.7-4.0, pH 3.5-4.5, or pH 3.8-4.3).

[0020] In another aspect, the present application also provides a recombinant protein comprising the isolated polypeptide as described above.

[0021] In certain embodiments, the recombinant protein does not comprise the amino acid sequence of the B domain of wild-type Protein A.

[0022] In certain embodiments, the recombinant protein does not comprise the amino acid sequence shown in SEQ ID NO:4.

[0023] In certain embodiments, the recombinant protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7 or 8) isolated polypeptides as described above. In certain embodiments, the recombinant protein comprises 3, 4, 5 or 6 isolated polypeptides as described above.

[0024] In certain embodiments, the recombinant protein comprises a plurality (e.g., 2, 3, 4, 5, 6, 7 or 8) of the isolated polypeptides described above, and the plurality of isolated polypeptides are different, partially identical or completely identical.

[0025] In certain embodiments, each of the isolated polypeptides is optionally connected by the same or different peptide linkers.In certain embodiments, the peptide linkers each independently have an amino acid sequence as shown in SEQ ID NO: 6 or 18.

[0026] In certain embodiments, the recombinant protein further comprises cysteine ​​at its N-terminus or C-terminus. In certain embodiments, the recombinant protein further comprises cysteine ​​at its C-terminus. In certain embodiments, the cysteine ​​is optionally connected to the isolated polypeptide via a linker (e.g., a flexible peptide comprising one or more glycine (G), such as GGGG).

[0027] In certain embodiments, the recombinant protein comprises or consists of an amino acid sequence as shown in any one of SEQ ID NOs: 2, 7, 11, 13, 15, 19-21. The sequence shown here does not contain an amino acid (such as methionine (Met)) encoded by a start codon (such as ATG) at its N-terminus. Those skilled in the art understand that in the process of preparing proteins by genetic engineering, due to the effect of the start codon, the first position of the produced polypeptide chain is often an amino acid (such as Met) encoded by the start codon. The recombinant protein of the present invention encompasses not only an amino acid sequence that does not contain an amino acid (such as Met) encoded by a start codon at its N-terminus, but also an amino acid sequence that contains an amino acid (such as Met) encoded by a start codon at its N-terminus. Therefore, a sequence that further contains an amino acid (such as Met) encoded by a start codon at the N-terminus of the above-mentioned amino acid sequence is also within the scope of protection of the present invention.

[0028] In certain embodiments, the recombinant protein is further linked to another polypeptide, and the other polypeptide is selected from a tag, an enzyme cleavage site, a signal peptide or a leader peptide, a detectable label, or any combination thereof.

[0029] Herein, protein tags are well known in the art, examples of which include but are not limited to His, Flag, GST, MBP, HA, Myc, GFP or biotin, and those skilled in the art know how to select a suitable protein tag according to the desired purpose (e.g., purification, detection or tracing).

[0030] In certain embodiments, the recombinant protein specifically binds to the IgG Fc domain in the range of pH 7.0-8.0 (eg, pH 7.0-7.6, pH 7.0-7.4).

[0031] In certain embodiments, the recombinant protein dissociates from the IgG Fc domain within the pH range of 2.7-4.5 (eg, pH 2.7-4.0, pH 3.5-4.5, or pH 3.8-4.3).

[0032] On the other hand, the application also provides dimers or polymers comprising two or more (e.g., 2, 3, 4, 5, 6, 7 or 8) isolated polypeptides as described above. In certain embodiments, the dimer or polymer comprises 3, 4, 5 or 6 isolated polypeptides as described above.

[0033] In certain embodiments, the two or more (e.g., 2, 3, 4, 5, 6, 7, or 8) isolated polypeptides are different, partially identical, or completely identical.

[0034] In certain embodiments, the dimer or multimer does not comprise the amino acid sequence of the B domain of wild-type Protein A.

[0035] In certain embodiments, the dimer or multimer does not comprise the amino acid sequence shown in SEQ ID NO:4.

[0036] In certain embodiments, the dimer or multimer comprises at least two polypeptide chains, and the two or more separate polypeptides are present in at least two polypeptide chains.

[0037] In certain embodiments, the isolated polypeptides are reversibly or irreversibly linked via linkers to form dimers or multimers.

[0038] In certain embodiments, at least one polypeptide chain in the dimer or multimer further comprises a cysteine ​​at its N-terminus or C-terminus. In certain embodiments, the polypeptide chain further comprises a cysteine ​​at its C-terminus. In certain embodiments, the cysteine ​​is optionally connected to the isolated polypeptide via a linker (e.g., a flexible peptide comprising one or more glycine (G), such as GGGG).

[0039] In certain embodiments, at least one polypeptide chain in the dimer or multimer is further linked to another polypeptide, and the other polypeptide is selected from a tag, an enzyme cleavage site, a signal peptide or a leader peptide, a detectable label, or any combination thereof.

[0040] Herein, protein tags are well known in the art, examples of which include but are not limited to His, Flag, GST, MBP, HA, Myc, GFP or biotin, and those skilled in the art know how to select a suitable protein tag according to the desired purpose (e.g., purification, detection or tracing).

[0041] In certain embodiments, the dimer or multimer specifically binds to the IgG Fc domain in the range of pH 7.0-8.0 (eg, pH 7.0-7.6, pH 7.0-7.4).

[0042] In certain embodiments, the dimer or multimer dissociates from the IgG Fc domain in the range of pH 2.7-4.5 (e.g., pH 2.7-4.0, pH 3.5-4.5, or pH 3.8-4.3).

[0043] In another aspect, the present application also provides a conjugate comprising a solid support and the isolated polypeptide, recombinant protein, and / or dimer or multimer as described above linked to the solid support.

[0044] In certain embodiments, the isolated polypeptide, recombinant protein, dimer or multimer is directly or indirectly, reversibly or irreversibly linked to a solid support.

[0045] In certain embodiments, the isolated polypeptide, recombinant protein, dimer or multimer is linked to the solid support via covalent and / or non-covalent bonds.

[0046] In certain embodiments, the solid supports include those based on synthetic polymers, for example, polyvinyl ethers, polyvinyl alcohols, polymethacrylates, polyacrylates, polystyrenes, polyacrylamides, polymethacrylamides, and polycarbonates.

[0047] In certain embodiments, the solid support is in the form of, but not limited to, beads (spherical or irregularly shaped), hollow fibers, solid fibers, pads, gels, membranes, cartridges, columns, chips, slides, plates, or monoliths.

[0048] In certain embodiments, the solid support is selected from agarose microparticles, polymer microspheres, silica microspheres or magnetic beads.

[0049] Any suitable technology can be used for being connected to the polypeptide of separation as herein described, recombinant protein or dimer or polymer to solid support.For example, in some embodiments, can utilize amino and / or carboxyl group existing in the polypeptide of separation, recombinant protein or dimer or polymer by conventional coupling technology to be connected to solid support.For example, diepoxide (bisepoxide), epichlorohydrin, CNBr, N-hydroxysuccinimide (NHS) are known coupling reagents.In some embodiments, can introduce interval between solid support and the polypeptide of separation, recombinant protein or dimer or polymer, thereby it improves the availability of the polypeptide of separation, recombinant protein or dimer or polymer, perhaps, promotes the polypeptide of separation, recombinant protein or dimer or polymer chemical coupling to upholder.

[0050] In certain embodiments, more than one site on the isolated polypeptide, recombinant protein, or dimer or multimer can be attached to a solid support (ie, by multipoint attachment).

[0051] In certain embodiments, the isolated polypeptide, recombinant protein, dimer or multimer is linked to the solid support via a disulfide bond formed between cysteine ​​residues therein.

[0052] In certain embodiments, the conjugates are used to purify immunoglobulins.

[0053] In certain embodiments, the conjugate is used to purify samples containing IgG Fc domains.

[0054] In certain embodiments, the sample containing the IgG Fc domain is an IgG antibody or a heavy chain thereof, a bispecific or multispecific antibody containing an IgG Fc domain, and / or a nanobody containing an IgG Fc domain.

[0055] In certain embodiments, the IgG Fc domain-containing sample is a bispecific or multispecific antibody containing an IgG Fc domain.

[0056] In certain embodiments, the conjugate is an affinity chromatography medium.

[0057] In certain embodiments, the conjugate specifically binds to the IgG Fc domain in the range of pH 7.0-8.0 (e.g., pH 7.0-7.6, pH 7.0-7.4).

[0058] In certain embodiments, the conjugate dissociates from the IgG Fc domain within the range of pH 2.7-4.5 (eg, pH 2.7-4.0, pH 3.5-4.5, or pH 3.8-4.3).

[0059] In certain embodiments, the isolated polypeptide or recombinant protein or dimer or multimer described above specifically binds to the Fc domain of an immunoglobulin (such as an IgG Fc domain) and does not bind (e.g., does not detectably bind) to the variable region of an immunoglobulin (e.g., Fab).

[0060] In another aspect, the present application also provides an isolated nucleic acid molecule encoding the isolated polypeptide or recombinant protein or dimer or multimer as described above or the polypeptide chain contained therein.

[0061] In another aspect, the present application also provides a vector comprising the nucleic acid molecule as described above. In certain embodiments, the vector is a cloning vector or an expression vector.

[0062] In another aspect, the present application also provides a host cell comprising the nucleic acid molecule or vector as described above.

[0063] Such host cells include, but are not limited to, prokaryotic cells such as bacterial cells (such as E. coli cells), and eukaryotic cells such as fungal cells (such as yeast cells), insect cells, plant cells and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.). In certain embodiments, the host cell is a microorganism.

[0064] The isolated polypeptide, recombinant protein, dimer or multimer of the present invention or the polypeptide chain contained therein can be prepared by various methods known in the art, for example, by chemical synthesis or genetic engineering recombinant technology. For example, a DNA molecule encoding the isolated polypeptide, recombinant protein, dimer or multimer of the present invention or the polypeptide chain contained therein can be obtained by chemical synthesis or PCR amplification. The resulting DNA molecule is inserted into an expression vector and then transfected into a host cell. The transfected host cell is then cultured under specific conditions to express the isolated polypeptide, recombinant protein, dimer or multimer of the present invention or the polypeptide chain contained therein.

[0065] On the other hand, the present application also provides a method for preparing the isolated polypeptide or recombinant protein or dimer or multimer or the polypeptide chain contained therein as described above, which comprises culturing the host cell as described above under conditions allowing protein expression, and recovering the isolated polypeptide or the recombinant protein or the dimer or multimer or the polypeptide chain contained therein from the cultured host cell culture.

[0066] On the other hand, the present application also provides a method for preparing the conjugate as described above, which comprises the step of linking the isolated polypeptide, recombinant protein, and / or dimer or multimer as described above to a solid support.

[0067] In certain embodiments, the method comprises contacting the isolated polypeptide, recombinant protein, and / or dimer or multimer with the solid support, thereby forming a connection between the isolated polypeptide, recombinant protein, and / or dimer or multimer and the solid support.

[0068] In certain embodiments, the linkage is direct or indirect, reversible or irreversible.

[0069] In certain embodiments, the linkage is covalent or non-covalent.

[0070] In certain embodiments, the solid support is selected from agarose microparticles, polymer microspheres, silica microspheres or magnetic beads.

[0071] In certain embodiments, the linkage comprises a disulfide bond.

[0072] In another aspect, the present application also provides the use of the isolated polypeptide, or recombinant protein, or dimer or multimer, or conjugate as described above for preparing a purification reagent for purifying immunoglobulins.

[0073] In certain embodiments, the purification reagent is used to purify a sample containing an IgG Fc domain.

[0074] In certain embodiments, the sample containing the IgG Fc domain is an IgG antibody or a heavy chain thereof, a bispecific or multispecific antibody containing an IgG Fc domain, and / or a nanobody containing an IgG Fc domain.

[0075] In certain embodiments, the IgG Fc domain-containing sample is a bispecific or multispecific antibody containing an IgG Fc domain.

[0076] In another aspect, the present application also provides a method for purifying immunoglobulins, which comprises using the conjugate as described above.

[0077] In certain embodiments, the purification is affinity chromatography.In certain embodiments, the affinity chromatography comprises using a conjugate as described above as the stationary phase.

[0078] In certain embodiments, the methods are used to purify samples containing IgG Fc domains.

[0079] In certain embodiments, the method comprises the following steps:

[0080] (1) allowing the sample containing the IgG Fc domain to bind to the conjugate in a first solvent to form a complex;

[0081] (2) dissociating the complex in a second solvent; and,

[0082] (3) Collecting the dissociation product of the sample containing the IgG Fc domain.

[0083] In certain embodiments, the pH of the first solvent is 7.0-8.0; for example, the pH of the first solvent is 7.0-7.6 or 7.0-7.4. In certain embodiments, the first solvent is a buffer (eg, Tris buffer).

[0084] In certain embodiments, the IgG Fc domain-containing sample is provided in the first solvent, for example, by providing the first solvent containing the IgG Fc domain-containing sample as a mobile phase for affinity chromatography.

[0085] In certain embodiments, the pH of the second solvent is 2.7-4.5; for example, the pH of the second solvent is 2.7-4.0, 3.5-4.5 or 3.8-4.3. In certain embodiments, the second solvent is a buffer (eg, glycine buffer).

[0086] In certain embodiments, the second solvent is provided in the mobile phase of the affinity chromatography.

[0087] In another aspect, the present application also provides a kit comprising the isolated polypeptide, or recombinant protein, or dimer or multimer, or conjugate as described above.

[0088] In certain embodiments, the kit further comprises reagents for protein purification (eg, a first solvent as described above, and / or a second solvent as described above). In certain embodiments, the purification is affinity chromatography.

[0089] In certain embodiments, the first solvent is a buffer (eg, Tris buffer).

[0090] In certain embodiments, the second solvent is a buffer (eg, a glycine buffer).

[0091] In certain embodiments, the kit is used to purify immunoglobulins.

[0092] In certain embodiments, the kit is used to purify a sample containing an IgG Fc domain.

[0093] In certain embodiments, the sample containing the IgG Fc domain is an IgG antibody or a heavy chain thereof, a bispecific or multispecific antibody containing an IgG Fc domain, and / or a nanobody containing an IgG Fc domain.

[0094] In certain embodiments, the IgG Fc domain-containing sample is a bispecific or multispecific antibody containing an IgG Fc domain.

[0095] Definition of terms

[0096] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. At the same time, in order to better understand the present invention, the definitions and explanations of relevant terms are provided below.

[0097] When the terms "for example," "such as," "including," "including," "comprising," or variations thereof are used herein, these terms will not be considered as limiting terms, but will be interpreted to mean "but not limited to" or "not limited to."

[0098] The terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0099] As used herein, when referring to the amino acid sequence of the wild-type Protein A B domain, the sequence set forth in SEQ ID NO: 4 is used for description. However, those skilled in the art will appreciate that naturally occurring mutations may occur in the amino acid sequence of the wild-type Protein A B domain without affecting its biological function. Therefore, in the present invention, the term "wild-type Protein A B domain" and similar expressions include all such sequences, including, for example, the sequence set forth in SEQ ID NO: 4 and its natural variants. Furthermore, when describing a specific amino acid position in the wild-type Protein A B domain, it includes not only the specific amino acid position in SEQ ID NO: 4, but also the corresponding amino acid position in its natural variants. For example, the expression "the amino acid residue at position 1 of the wild-type Protein A B domain" includes the amino acid residue at position 1 of SEQ ID NO: 4, as well as the amino acid residues at the corresponding positions in its natural variants. According to the present invention, the expression "amino acid residues at corresponding positions" refers to the amino acid residues at equivalent positions in the compared sequences when the sequences are optimally aligned, ie when the sequences are aligned to obtain the highest percentage identity.

[0100] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as an antibody and its antigen. The strength or affinity of a specific binding interaction can be measured by the equilibrium dissociation constant (K) of the interaction. D ) indicates. In the present invention, the term "K D ” refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen.

[0101] The specific binding properties between two molecules can be determined using methods known in the art. One method involves measuring the rate of formation and dissociation of the antigen binding site / antigen complex. Both the "association rate constant" (ka or kon) and the "dissociation rate constant" (kdis or koff) can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361: 186-187). The ratio of kdis / kon is equal to the dissociation constant K D (See Davies et al., Annual Rev Biochem, 1990; 59: 439-473). K can be measured by any effective method. D, kon and kdis values. In certain embodiments, the dissociation constant can be measured in Biacore using surface plasmon resonance (SPR). In addition, the dissociation constant can be measured using bioluminescence interferometry or Kinexa.

[0102] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, it is referred to as an expression vector. A vector can be introduced into a host cell via transformation, transduction, or transfection, allowing the genetic material it carries to be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages, such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomas (such as SV40). A vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain an origin of replication.

[0103] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells. A host cell may include a single cell or a cell population.

[0104] As used herein, the term "identity" refers to the match between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions match). Typically, two sequences are compared when they are aligned for maximum identity. Such an alignment can be achieved, for example, by using the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed using a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol Biol. 48:444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0105] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions of residues physically or functionally similar to corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10): 879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94: 412-417 (1997), which are incorporated herein by reference).

[0106] The twenty conventional amino acids referred to herein are written in accordance with conventional usage. See, for example, Immunology—A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also, in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0107] As used herein, the term "chromatography" refers to a separation technique that uses a mobile phase and a stationary phase to separate one type of molecule (e.g., immunoglobulin) from other molecules (e.g., contaminants) in a sample. The liquid mobile phase is a mixture containing the specific type of molecule to be separated and other molecules. The specific type of molecule to be separated and other molecules are transported across or through the stationary phase (e.g., a solid matrix) by the mobile phase. Because different molecules in the mobile phase interact differently with the stationary phase, the specific type of molecule to be separated and other molecules in the mobile phase can be separated.

[0108] As used herein, the term "affinity chromatography" refers to a chromatographic method that relies on specific binding between molecules, wherein a ligand coupled to a stationary phase interacts with a molecule (e.g., an immunoglobulin) in a mobile phase (sample), i.e., the ligand has a specific affinity for the molecule to be purified. As understood in the context of the present invention, affinity chromatography involves adding a sample containing an immunoglobulin to a stationary phase containing a ligand (e.g., a mutated B domain of the present invention, or an immunoglobulin binding protein comprising said mutated B domain).

[0109] Advantageous Effects of the Invention

[0110] The immunoglobulin-binding protein comprising a mutated B domain of Protein A provided by the present invention has high binding activity to Fc while eliminating binding activity to the variable region of the antibody heavy chain. It can provide milder elution conditions during the purification of bispecific or multispecific antibodies, effectively guaranteeing sample quality and reducing the steps and costs of subsequent fine purification. This can effectively solve the problems encountered when using traditional Protein A to purify bispecific or multispecific antibodies, such as harsh elution conditions (for example, low pH values) and significant impact on antibody quality.

[0111] Moreover, in certain embodiments, the immunoglobulin binding protein comprising a mutated B domain of Protein A provided by the present invention has a lowered isoelectric point, so that the immunoglobulin binding protein can be relatively stable in a weakly alkaline environment, which is beneficial for subsequent coupling experiments, thereby effectively ensuring that the Protein A protein is coupled to the filler matrix and improving the utilization rate of the Protein A filler.

[0112] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are intended only to illustrate the present invention and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0113] FIG1 shows the modification sites of the Protein A mutant of the present application.

[0114] Figure 2 shows the SDS-Page protein electrophoresis results of the Protein A mutant of the present application; wherein, lane M is a Marker protein sample, lane 1 is a sample of the engineered bacteria of the Protein A mutant before induction, lane 2 is a sample of the whole bacterial lysate of the Protein A mutant after induction, lane 3 is a sample of the supernatant of the bacterial lysate of the Protein A mutant after induction, and lane 4 is a sample of the precipitate of the bacterial lysate of the Protein A mutant after induction.

[0115] FIG3 shows the HPLC-SEC results of the Protein A mutants of the present application.

[0116] FIG4 shows the affinity performance test results of the Protein A mutants of the present application and antibodies.

[0117] FIG5 shows the affinity determination results of the chromatography medium prepared from the Protein A mutants of the present application with VHH-Fc, Fc alone, and VHH alone.

[0118] FIG6 shows that the chromatography medium prepared from the Protein A mutant of the present application is suitable for milder elution conditions.

[0119] FIG7 shows the HPLC-SEC results of Protein A mutants 2, 3 and 4 of the present application.

[0120] FIG8 shows the results of affinity determination of Protein A mutants 2, 3, and 4 of the present application to Fc.

[0121] Sequence information

[0122] A description of the sequences involved in this application is provided in the table below.

[0123] Table 1: Sequence information DETAILED DESCRIPTION

[0124] The invention will now be described with reference to the following examples which are intended to illustrate the invention but not to limit it.

[0125] Example 1: Expression and performance of Protein A mutants

[0126] 1. Synthesis of Protein A mutant expression vector

[0127] The nucleotide sequence of the Protein A mutant was artificially synthesized using a full-sequence DNA synthesis method (Suzhou Jinweizhi Biotechnology Co., Ltd.). The nucleotide sequence is SEQ ID NO:3, and its corresponding amino acid sequence is SEQ ID NO:2, with a theoretical size of approximately 30 kDa. The nucleotide sequence was then constructed into a pET-28a vector linearized by Nco I / Hind III double enzyme digestion to obtain an expression plasmid for the Protein A mutant. The mutation sites of the Protein A mutant relative to wild-type Protein A are shown in Figure 1.

[0128] In addition, to facilitate protein expression and purification, the Protein A mutant used in this example contains methionine at the N-terminus and an 8×His (HHHHHHHH) tag is fused to the C-terminus.

[0129] 2. Construction of Protein A mutant engineered bacteria

[0130] Take 1 μL of the plasmid from step 1 above and add it to 100 μL of commercially available BL21(DE3) competent cells. Incubate on ice for 30 minutes. Then, heat shock the plasmid and competent cell mixture in a 42°C water bath for 90 seconds. Remove and incubate on ice for 2 minutes. Next, add 450 μL of LB to the mixture and incubate at 37°C, 220 rpm for 45 minutes. Finally, remove the majority of the medium by centrifugation, spread the bacteria onto an LB plate containing kanamycin, and incubate in a 37°C incubator overnight. Transformants that emerge are identified as positive by sequencing using universal primers for the pET-28a vector.

[0131] 3. Small-scale expression of Protein A mutants

[0132] Select a positive single clone from step 2 above and culture it in 10 mL of LB medium containing kanamycin (37°C, 220 rpm) for approximately 14 hours to prepare the seed solution. Add 10 mL of the seed solution to 1000 mL of LB medium containing kanamycin and culture at 37°C, 220 rpm for 2 hours (OD600 reading is 0.6). Add 1 M IPTG to the culture medium at 0.1% of the culture volume and induce expression at 25°C, 150 rpm, for 16 hours.

[0133] The fermentation broth was centrifuged (6000 rpm, 4°C) for 30 minutes to remove the culture medium. The bacterial sludge was suspended in 50 mL of Tris buffer (20 mM Tris-HCl, 150 mM NaCl, pH 7.4) and ultrasonically disrupted at 25% power for 5 seconds on and 5 seconds off for 30 minutes. The lysate was centrifuged at 12000 rpm and 4°C for 30 minutes, and the supernatant and precipitate were separated and subjected to SDS-PAGE protein electrophoresis. The results of SDS-PAGE protein electrophoresis are shown in Figure 2, which shows that the Protein A mutant was successfully expressed.

[0134] 4. Purification of Protein A mutants

[0135] The bacterial lysate supernatant obtained in step 3 above was purified using an AKTA system over a Ni column, specifically: the protein purifier and Ni column were rinsed with an equilibration solution (20mM Tris-HCl, 150mM NaCl, pH 7.4) until the baseline was balanced; the lysate supernatant was loaded at an appropriate flow rate so that the sample was retained in the purification column for 5 minutes, and impurities were removed with a wash solution (20mM Tris-HCl, 150mM NaCl, 5mM imidazole, pH 7.4) until the baseline was balanced; finally, the protein was eluted with an elution solution (20mM Tris-HCl, 150mM NaCl, 250mM imidazole, pH 7.4). The eluted protein was reduced with 5mM DTT and its components were detected by liquid chromatography (SEC). The HPLC-SEC test results are shown in Figure 3. As shown in Figure 3, the purity of the reduced protein was high, close to 90%. The nonspecific adsorption between the His tag at the C-terminus of the protein and the chromatographic column caused a certain degree of tailing of the protein characteristic peak. In addition, the characteristic peak of the reducing agent DTT can be observed at a high retention time.

[0136] 5. Determination of affinity between Protein A mutants and antibodies

[0137] ForteBio affinity determination was performed according to existing methods (see: Estep, P et al., Solution-based high-throughput antibody-antigen affinity and epitope fractionation measurements, MAbs, 2013.5(2): p.270-8). Briefly, the sensor was equilibrated offline in assay buffer for 30 minutes, followed by online detection for 60 seconds to establish a baseline. The biotin-labeled bispecific antibody Ava-2GS-NSD (see publication number CN114106190A) was then loaded online onto the SA sensor. The sensor was then exposed to 100 nM of a protein A mutant for 110 seconds, after which the sensor was transferred to PBS for dissociation for 5 minutes. Kinetic analysis was performed using a 1:1 binding model.

[0138] The results are shown in FIG4 . As shown in FIG4 , the Protein A mutant of the present application has a strong binding activity with the bispecific antibody, and the Kd value reaches 9.0×10 -11 M, can be used for affinity purification of antibodies.

[0139] 6. Preparation of Protein A Mutant Affinity Media for Purification

[0140] The specific steps are as follows:

[0141] (1) Transfer the chromatographic medium to be coupled into a centrifuge tube and centrifuge it. Remove the supernatant and equilibrate it with 0.1 M phosphate buffer solution (pH 8.0). Centrifuge to remove the phosphate buffer solution and repeat this process three times.

[0142] (2) The Protein A mutant protein was replaced with the above-mentioned phosphate buffer solution and added to the above-mentioned medium for coupling at 37°C, 130 rpm, for 24 h;

[0143] (3) After the coupling is completed, the supernatant is removed by centrifugation and ethanolamine is added for blocking at 37°C, 130 rpm, for 6 h;

[0144] (4) Wash the filler three times with 0.1 M phosphate buffer solution (pH 7.0) and store it at 2-8°C until use.

[0145] 7. Affinity determination of chromatographic media prepared from Protein A mutants with VHH-Fc, Fc alone, and VHH alone

[0146] ForteBio affinity determination was performed according to existing methods (see: Estep, P et al., Solution-based high-throughput antibody-antigen affinity and epitope fractionation measurements, MAbs, 2013. 5(2): p. 270-8). Briefly, the sensor was equilibrated offline in assay buffer for 30 min, followed by online detection for 60 s to establish a baseline, and the biotin-labeled Protein A mutant of the present invention was loaded online onto the SA sensor. The sensor was then exposed to 100 nM VHH-Fc (VHH-Fc amino acid sequence as shown in SEQ ID NO: 8), Fc (Fc amino acid sequence as shown in SEQ ID NO: 9), or VHH (VHH amino acid sequence as shown in SEQ ID NO: 10) for 5 min, after which the sensor was transferred to PBS for dissociation for 5 min. Kinetic analysis was performed using a 1:1 binding model.

[0147] The results are shown in Figure 5 . As shown in Figure 5 , the chromatography medium prepared from the Protein A mutant of the present application retains its binding activity to Fc while eliminating its binding activity to the antibody heavy chain variable region. This indicates that the chromatography medium prepared from the Protein A mutant of the present application can effectively prevent the adverse effects of binding between the antibody heavy chain variable region and wild-type Protein A on the antibody purification process.

[0148] 8. Chromatographic media prepared from Protein A mutants for the purification of bispecific antibodies

[0149] The target protein Bi-088 was purified using a Protein A affinity chromatography column, MabSelect PrismA (Cytiva, Catalog No. 17519901) or a Protein A mutant filler described herein. The target protein (see Publication No. CN114195900A) is a bispecific antibody, with a single molecule containing four VHH regions (all from the VH3 gene family). The purification column was equilibrated with 5 to 10 column volumes of equilibration buffer (20mM Tris-HCl, 150mM NaCl, pH 7.4) until the conductivity and pH of the effluent remained constant and sample loading began. After loading, the column was rinsed with equilibration buffer until the UV value of the effluent no longer decreased. The sample was eluted sequentially using elution buffer (20mM glycine-HCl, pH 4.0 or pH 3.4) and the effluent was collected. After the UV value stabilized, the column was regenerated with 0.1M NaOH solution.

[0150] The results are shown in FIG6 . As can be seen from FIG6 , the chromatographic medium prepared from the Protein A mutant of the present application can provide milder elution conditions during the purification of bispecific or multispecific antibodies, effectively guarantee the sample quality, and reduce the steps and costs of subsequent fine purification.

[0151] Specifically, with the same sample load (22 mg), the protein A affinity chromatography column of the present application (column volume 0.7 mL) can recover 14.6 mg of protein under pH 4.0 elution conditions, and no additional protein flows out under pH 3.4 conditions, with a total recovery rate of 66.4%; the MabSelect PrismA chromatography column only recovered 1.1 mg of protein under pH 4.0 elution conditions.

[0152] Example 2: Further optimization of Protein A mutant performance

[0153] In order to lower the isoelectric point of the Protein A mutant, three new mutants were designed based on the Protein A mutant (i.e., the initial mutant) of Example 1. Among them, Mutant 2 mutated the lysine at the C-terminus of the mutant B domain (the amino acid sequence of the mutant B domain is shown in SEQ ID NO: 5) to alanine, and the amino acid sequence of Mutant 2 is SEQ ID NO: 11, and the corresponding nucleotide sequence is SEQ ID NO: 12; Mutant 3 mutated the lysine on the linker (the amino acid sequence of the linker is shown in SEQ ID NO: 6) to alanine, and the amino acid sequence of Mutant 3 is SEQ ID NO: 13, and the corresponding nucleotide sequence is SEQ ID NO: 14; Mutant 4 mutated the lysine at the above two positions to alanine at the same time, and the amino acid sequence of Mutant 4 is SEQ ID NO: 15, and the corresponding nucleotide sequence is SEQ ID NO: 16.

[0154] The theoretical isoelectric points of the amino acids of each mutant are shown in Table 2 (calculated by the Expasy tool: https: / / web.expasy.org / cgi-bin / protparam / protparam). The isoelectric point pI value of the initial mutant (i.e., the mutant shown in Example 1, the amino acid sequence is shown in SEQ ID NO: 2) is reduced from 7.90 to 5.91 (mutant 2, the amino acid sequence is shown in SEQ ID NO: 11), 6.17 (mutant 3, the amino acid sequence is shown in SEQ ID NO: 13) and 5.37 (mutant 4, the amino acid sequence is shown in SEQ ID NO: 15), respectively, making the immunoglobulin binding protein relatively stable in a weakly alkaline environment.

[0155] Table 2 Theoretical isoelectric point values ​​of each mutant

[0156] The construction, expression, and purification processes of each mutant can be found in Example 1. The HPLC-SEC analysis results for each mutant are shown in Figure 7. As shown in Figure 7, the purity of the reduced proteins was high, exceeding 90%. Furthermore, a certain degree of tailing in the characteristic peaks may be due to nonspecific adsorption between the His tag at the C-terminus of the protein and the chromatographic column.

[0157] Affinity determination of protein A mutants and antibodies

[0158] ForteBio affinity assays were performed according to existing methods (see: Estep, P et al., Solution-based high-throughput antibody-antigen affinity and epitope fractionation measurements, MAbs, 2013. 5(2): p. 270-8). Briefly, the sensor was equilibrated offline in assay buffer for 30 min, followed by online detection for 60 s to establish a baseline. Biotin-labeled mutants were then loaded online onto the SA sensor. The sensor was then exposed to 100 nM Fc protein (SEQ ID NO: 9) for 200 s, after which the sensor was transferred to PBS for dissociation for 5 min. Kinetic analysis was performed using a 1:1 binding model.

[0159] The results are shown in FIG8 , which shows that the binding activities of the mutants to Fc protein are comparable.

[0160] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings published, and these changes are all within the scope of protection of the present invention. The entire invention is given by the appended claims and any equivalents thereof.

Claims

1. An isolated polypeptide consisting of a mutant B domain of Protein A or a variant thereof, wherein: The mutated B domain comprises one or more (e.g., 1, 2, 3 or 4) amino acid substitutions selected from N23T, D36V, D37R, and Q40V compared to the B domain of wild-type Protein A; wherein the variant has at least 90%, such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mutated B domain; or, has one or several (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9) amino acid substitutions (preferably conservative substitutions), additions, or deletions, and retains the function of the mutated B domain; and; The variant and the mutated B domain have the same amino acid residues at positions 1, 23, 29, 36, 37 and 40 corresponding to the B domain of wild-type Protein A.

2. The isolated polypeptide according to claim 1, wherein The mutant B domain further comprises the amino acid substitutions compared to the B domain of wild-type Protein A: (i) G29A, and / or, (ii) A1V; Preferably, the mutated B domain comprises the following amino acid substitutions compared to the B domain of wild-type Protein A: (a) N23T, G29A, D36V, D37R and Q40V, or, (b) A1V, N23T, D36V, D37R and Q40V; Preferably, the mutated B domain comprises the following amino acid substitutions compared to the B domain of wild-type Protein A: A1V, N23T, G29A, D36V, D37R and Q40V.

3. The isolated polypeptide according to claim 1 or 2, wherein The mutant B domain further comprises an amino acid substitution K58A compared to the B domain of wild-type Protein A; Preferably, the mutated B domain comprises the following amino acid substitutions compared to the B domain of wild-type Protein A: N23T, D36V, D37R, Q40V and K58A; Preferably, the mutated B domain comprises the following amino acid substitutions compared to the B domain of wild-type Protein A: (a) N23T, G29A, D36V, D37R, Q40V and K58A, or, (b) A1V, N23T, D36V, D37R, Q40V and K58A; Preferably, the mutated B domain comprises the following amino acid substitutions compared to the B domain of wild-type Protein A: A1V, N23T, G29A, D36V, D37R, Q40V and K58A.

4. The isolated polypeptide according to any one of claims 1 to 3, wherein The B domain of the wild-type Protein A has the sequence shown in SEQ ID NO:

4.

5. The isolated polypeptide according to any one of claims 1 to 4, wherein The mutated B domain has an amino acid sequence as shown in SEQ ID NO: 5 or 17.

6. The isolated polypeptide according to any one of claims 1 to 5, wherein The isolated polypeptide specifically binds to the IgG Fc domain in the range of pH 7.0-8.0 (e.g., pH 7.0-7.6, pH 7.0-7.4); and / or, the isolated polypeptide dissociates from the IgG Fc domain in the range of pH 2.7-4.5 (e.g., pH 2.7-4.0, pH 3.5-4.5, or pH 3.8-4.3).

7. A recombinant protein comprising the isolated polypeptide according to any one of claims 1 to 6; Preferably, the recombinant protein does not contain the amino acid sequence of the B domain of wild-type Protein A; Preferably, the recombinant protein does not comprise the amino acid sequence shown in SEQ ID NO: 4; Preferably, the recombinant protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7 or 8) isolated polypeptides according to any one of claims 1 to 6; Preferably, the recombinant protein comprises a plurality (e.g., 2, 3, 4, 5, 6, 7 or 8) of the isolated polypeptides according to any one of claims 1 to 6, and the plurality of isolated polypeptides are different, partially identical or completely identical; Preferably, each of the isolated polypeptides is optionally connected by the same or different peptide linkers; for example, the peptide linkers each independently have an amino acid sequence as shown in SEQ ID NO: 6 or 18; Preferably, the recombinant protein further comprises a cysteine ​​at its N-terminus or C-terminus; preferably, the recombinant protein further comprises a cysteine ​​at its C-terminus; preferably, the cysteine ​​is optionally linked to the isolated polypeptide via a linker (e.g., a flexible peptide comprising one or more glycine (G), such as GGGG); Preferably, the recombinant protein comprises or consists of an amino acid sequence as shown in any one of SEQ ID NOs: 2, 7, 11, 13, 15, 19-21; Preferably, the recombinant protein is further linked to another polypeptide, and the other polypeptide is selected from a tag, an enzyme cleavage site, a signal peptide or a leader peptide, a detectable marker, or any combination thereof; Preferably, the recombinant protein specifically binds to the IgG Fc domain in the range of pH 7.0-8.0 (e.g., pH 7.0-7.6, pH 7.0-7.4); Preferably, the recombinant protein dissociates from the IgG Fc domain within the pH range of 2.7-4.5 (eg, pH 2.7-4.0, pH 3.5-4.5, or pH 3.8-4.3).

8. A dimer or multimer comprising two or more (e.g., 2, 3, 4, 5, 6, 7, or 8) isolated polypeptides according to any one of claims 1 to 6; Preferably, the two or more (e.g., 2, 3, 4, 5, 6, 7 or 8) isolated polypeptides are different, partially identical or completely identical; Preferably, the dimer or multimer does not contain the amino acid sequence of the B domain of wild-type Protein A; Preferably, the dimer or multimer does not comprise the amino acid sequence shown in SEQ ID NO: 4; Preferably, the dimer or multimer comprises at least two polypeptide chains, and the two or more separate polypeptides are present in at least two polypeptide chains; Preferably, the isolated polypeptides are reversibly or irreversibly linked via a linker to form a dimer or multimer; Preferably, at least one polypeptide chain in the dimer or multimer further comprises a cysteine ​​at its N-terminus or C-terminus; preferably, the polypeptide chain further comprises a cysteine ​​at its C-terminus; preferably, the cysteine ​​is optionally connected to the isolated polypeptide via a linker (e.g., a flexible peptide comprising one or more glycine (G), such as GGGG); Preferably, at least one polypeptide chain in the dimer or multimer is further connected to another polypeptide, and the other polypeptide is selected from a tag, an enzyme cleavage site, a signal peptide or a leader peptide, a detectable marker, or any combination thereof; Preferably, the dimer or multimer specifically binds to the IgG Fc domain in the range of pH 7.0-8.0 (e.g., pH 7.0-7.6, pH 7.0-7.4); Preferably, the dimer or multimer dissociates from the IgG Fc domain in the range of pH 2.7-4.5 (eg, pH 2.7-4.0, pH 3.5-4.5, or pH 3.8-4.3).

9. A conjugate comprising a solid support and the isolated polypeptide according to any one of claims 1 to 6, the recombinant protein according to claim 7, and / or the dimer or multimer according to claim 8, linked to the solid support; Preferably, the isolated polypeptide, recombinant protein or dimer or multimer is directly or indirectly, reversibly or irreversibly linked to a solid support; Preferably, the isolated polypeptide, recombinant protein or dimer or multimer is linked to the solid support via covalent and / or non-covalent bonds; Preferably, the solid support is selected from agarose microparticles, polymer microspheres, silica microspheres or magnetic beads; Preferably, the isolated polypeptide, recombinant protein, dimer or multimer is linked to the solid support via a disulfide bond formed between cysteine ​​residues therein; Preferably, the conjugate is used to purify immunoglobulins; Preferably, the conjugate is used to purify a sample containing an IgG Fc domain; preferably, the sample containing an IgG Fc domain is an IgG antibody or its heavy chain, a bispecific or multispecific antibody containing an IgG Fc domain, and / or a nanobody containing an IgG Fc domain; Preferably, the sample containing the IgG Fc domain is a bispecific or multispecific antibody containing the IgG Fc domain; Preferably, the conjugate is an affinity chromatography medium; Preferably, the conjugate specifically binds to the IgG Fc domain in the range of pH 7.0-8.0 (e.g., pH 7.0-7.6, pH 7.0-7.4); Preferably, the conjugate dissociates from the IgG Fc domain in the range of pH 2.7-4.5 (eg, pH 2.7-4.0, pH 3.5-4.5, or pH 3.8-4.3).

10. An isolated nucleic acid molecule encoding the isolated polypeptide according to any one of claims 1 to 6, the recombinant protein according to claim 7, or the dimer or multimer according to claim 8, or a polypeptide chain comprised thereof.

11. A vector comprising the nucleic acid molecule according to claim 10; preferably, the vector is a cloning vector or an expression vector.

12. A host cell comprising the nucleic acid molecule of claim 10 or the vector of claim 11.

13. A method for preparing the isolated polypeptide according to any one of claims 1 to 6, the recombinant protein according to claim 7, the dimer or multimer according to claim 8, or the polypeptide chain contained therein, comprising culturing the host cell according to claim 12 under conditions permitting protein expression, and recovering the isolated polypeptide, the recombinant protein, the dimer or multimer, or the polypeptide chain contained therein from the cultured host cell culture.

14. A method for preparing the conjugate according to claim 9, comprising the step of linking the isolated polypeptide according to any one of claims 1 to 6, the recombinant protein according to claim 7, and / or the dimer or multimer according to claim 8 to a solid support; Preferably, the method comprises: contacting the isolated polypeptide, recombinant protein, and / or dimer or multimer with the solid support, thereby forming a connection between the isolated polypeptide, recombinant protein, and / or dimer or multimer and the solid support; Preferably, the connection is direct or indirect, reversible or irreversible; Preferably, the linkage is covalent or non-covalent; Preferably, the solid support is selected from agarose microparticles, polymer microspheres, silica microspheres or magnetic beads; Preferably, the linkage comprises a disulfide bond.

15. Use of the isolated polypeptide according to any one of claims 1 to 6, or the recombinant protein according to claim 7, or the dimer or multimer according to claim 8, or the conjugate according to claim 9 for preparing a purification reagent for purifying immunoglobulins; Preferably, the purification reagent is used to purify a sample containing an IgG Fc domain; Preferably, the sample containing the IgG Fc domain is an IgG antibody or its heavy chain, a bispecific or multispecific antibody containing an IgG Fc domain, and / or a nanobody containing an IgG Fc domain; Preferably, the sample containing the IgG Fc domain is a bispecific or multispecific antibody containing the IgG Fc domain.

16. A method for purifying immunoglobulins, comprising using the conjugate according to claim 9; Preferably, the purification is affinity chromatography; preferably, the affinity chromatography comprises using the conjugate according to claim 9 as a stationary phase; Preferably, the method is used to purify a sample containing an IgG Fc domain; Preferably, the method comprises the following steps: (1) allowing the sample containing the IgG Fc domain to bind to the conjugate in a first solvent to form a complex; (2) dissociating the complex in a second solvent; and, (3) collecting the dissociation product of the sample containing the IgG Fc domain; Preferably, the pH of the first solvent is 7.0-8.0; for example, the pH of the first solvent is 7.0-7.6 or 7.0-7.4; Preferably, the pH of the second solvent is 2.7-4.5; for example, the pH of the second solvent is 2.7-4.0, 3.5-4.5 or 3.8-4.

3.

17. A kit comprising the isolated polypeptide according to any one of claims 1 to 6, or the recombinant protein according to claim 7, or the dimer or multimer according to claim 8, or the conjugate according to claim 9; Preferably, the kit further comprises reagents for protein purification (e.g., a first solvent as defined in claim 16, and / or a second solvent as defined in claim 16); preferably, the purification is affinity chromatography; Preferably, the kit is used for purifying immunoglobulins; Preferably, the kit is used to purify a sample containing an IgG Fc domain; Preferably, the sample containing the IgG Fc domain is an IgG antibody or its heavy chain, a bispecific or multispecific antibody containing an IgG Fc domain, and / or a nanobody containing an IgG Fc domain; Preferably, the sample containing the IgG Fc domain is a bispecific or multispecific antibody containing the IgG Fc domain.

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