New protein a ligand mutant and use thereof

Abstract

Provided is an Fc-binding polypeptide with an improved alkaline stability, containing a mutant of the B domain of protein A. Further provided are a multimer containing a polypeptide, a separation matrix containing a polypeptide or a multimer, and a method for separating a protein containing Fc. The multimer or separation matrix containing the polypeptide has a significantly improved alkaline stability and an enhanced binding ability to IgG during alkaline cleaning.

Description

A novel Protein A ligand mutant and its applications

[0001] Cross-reference of related applications

[0002] This application is based on and claims priority to Chinese patent applications No. 202411942195.1, filed on December 26, 2024, and No. 202510251476.0, filed on March 4, 2025, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0003] This invention relates to the field of protein purification, and in particular to a protein A with improved alkali resistance and its affinity chromatography medium. Background Technology

[0004] In recent years, the biopharmaceutical field has flourished, with antibody drugs gradually becoming the most popular biopharmaceutical products in global research and production. Compared to traditional therapies, antibody drugs offer advantages such as longer half-lives, lower cytotoxicity, and more significant efficacy in treating cancer, autoimmune diseases, and viral infections. With the FDA approving over 100 monoclonal antibody (mAb) drugs, market demand for monoclonal antibodies continues to increase. The high commercialization of antibody drugs has driven major pharmaceutical companies to maximize productivity while controlling costs, and antibody purification processes are crucial in this process.

[0005] The most common purification method currently is affinity chromatography, which relies on affinity chromatography media composed of Staphylococcus aureus protein A. Studies have found that wild-type protein A consists of five homologous domains: E, D, A, B, and C. Each domain contains three antiparallel α-helices, which have strong binding affinity to the constant part (Fc region) of immunoglobulin G (IgG) molecules and weak binding affinity to the variable part (Fab region). This binding is independent of the antigen-binding specificity of antibodies, enabling protein A to efficiently recover immunoglobulins from a variety of samples, including serum, plasma products, and cell cultures, making it the preferred medium for antibody capture and purification.

[0006] However, during the purification of IgG using protein A, the chromatographic medium can adsorb impurities such as host proteins, endotoxins, and residual antibodies. In-situ cleaning (CIP) with NaOH is necessary to maintain the selectivity of the chromatographic medium. The NaOH concentration used in CIP ranges from 0.1M to 1M, depending on the degree and nature of the contamination. This strongly alkaline environment is very demanding for proteins containing affinity ligands, as natural protein A is poorly alkali-resistant; exposure to solutions with a pH higher than 13 may affect the protein structure and damage the chromatographic medium. Generally, repeated in-situ cleaning of the affinity chromatography matrix reduces the binding affinity of protein A ligands to antibodies. Extending the lifespan requires increasing the amount of ligand used throughout the process, which significantly increases purification costs, making it neither economical nor desirable.

[0007] In summary, protein A, as a key reagent in affinity chromatography, plays a crucial role in immunoglobulin purification due to its high efficiency and selectivity. There is a need in this field to develop protein A constructs with improved performance. Summary of the Invention

[0008] The present invention provides a novel B domain construct of protein A, which, compared to the B domain of wild-type protein A, has one or more improved properties, such as: (1) alkali resistance; (2) high binding capacity to IgG, thereby improving the efficiency of IgG production and reducing costs.

[0009] Therefore, in a first aspect, the present invention provides an Fc-binding polypeptide that, compared with the amino acid sequence shown in SEQ ID NO:110, contains or contains only amino acid substitution mutations selected from one or more of the following positions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10): position 1, position 2, position 4, position 5, position 6, position 7, position 8, position 10, position 11, position 13, position 15, position 16, position 17, position 18, position 21, position 24, position 25, position 28, position 31, position 33, position 36, position 40, position 42, position 49, position 53, and position 57;

[0010] Preferably, the polypeptide comprises or contains only amino acid substitution mutations selected from one or more of the following positions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10):

[0011] (i) The amino acid at position 1 is mutated and replaced with leucine (1L);

[0012] (ii) The amino acid at position 2 is mutated to glutamic acid (2E);

[0013] (iii) The amino acid at position 4 is mutated to glutamic acid (4E);

[0014] (iv) The amino acid at position 5 is mutated and replaced with leucine (5L);

[0015] (v) The amino acid at position 6 is mutated to threonine (6T);

[0016] (vi) The amino acid at position 7 is mutated to glutamic acid (7E) or arginine (7R);

[0017] (vii) The amino acid at position 8 is mutated and replaced with alanine (8A);

[0018] (viii) The amino acid at position 10 is mutated and replaced with lysine (10K);

[0019] (ix) The amino acid at position 11 is mutated to aspartic acid (11D);

[0020] (x) The amino acid at position 13 is mutated and replaced with leucine (13L);

[0021] (xi) The amino acid at position 15 is substituted and mutated to lysine (15K) or alanine (15A);

[0022] (xii) The amino acid at position 16 is mutated and replaced with leucine (16L);

[0023] (xiii) The amino acid at position 17 is mutated and replaced with methionine (17M);

[0024] (xiv) describes the amino acid at position 18 being mutated to lysine (18K);

[0025] (xv) The amino acid at position 21 is mutated and replaced with histidine (21H);

[0026] (xvi) The amino acid at position 24 is mutated to aspartic acid (24D);

[0027] (xvii) The amino acid at position 25 is mutated to alanine (25A);

[0028] (xviii) The amino acid at position 28 is mutated to alanine (28A) or glutamine (28Q);

[0029] The amino acid at position 31 of (xix) is mutated to leucine (31L);

[0030] (xx) The amino acid at position 33 is substituted and mutated to lysine (33K) or threonine (33T);

[0031] (xxi) The amino acid at position 36 is mutated and replaced with glutamic acid (36E);

[0032] (xxii) The amino acid at position 40 is substituted and mutated to lysine (40K) or alanine (40A);

[0033] (xxiii) The amino acid at position 42 is mutated and replaced with alanine (42A);

[0034] (xxiv) The amino acid at position 49 was mutated to glutamine (49Q);

[0035] The amino acid at position 53 in (xxv) is mutated to alanine (53A);

[0036] The amino acid at position 57 in (xxvi) is mutated and replaced with lysine (57K);

[0037] The mutated amino acid sites are the natural sequence sites relative to the amino acid sequence shown in SEQ ID NO:110.

[0038] As will be readily understood by those skilled in the art, the statement "each mutated amino acid site is a natural sequence site relative to the amino acid sequence shown in SEQ ID NO:110" means that the amino acid site in the polypeptide is located at the position corresponding to the amino acid site in the amino acid sequence shown in SEQ ID NO:110. For example, "position 1" means the amino acid site in the polypeptide that corresponds to position 1 of SEQ ID NO:110; "position 2" means the amino acid site in the polypeptide that corresponds to position 2 of SEQ ID NO:110.

[0039] As used herein, the expression "the amino acid site in the polypeptide that corresponds to the first amino acid residue of SEQ ID NO:110" means, when the sequence of the polypeptide is optimally aligned with SEQ ID NO:110, that is, when the sequence of the polypeptide is aligned with SEQ ID NO:110 to obtain the highest percentage identity, the amino acid site in the sequence of the polypeptide being compared is located at the same position as the first amino acid residue of SEQ ID NO:110.

[0040] Unless otherwise specified or there is a clear contradiction between the preceding and following statements, the meaning of the remaining similar expressions in this document shall be defined in a manner similar to that described above.

[0041] As used herein, the mutation “1L” means that the amino acid residue in the polypeptide corresponding to the first position of SEQ ID NO:110 is replaced with L; the mutation “2E” means that the amino acid residue in the polypeptide corresponding to the second position of SEQ ID NO:110 is replaced with E.

[0042] Unless otherwise specified or there is a clear contradiction between the preceding and following statements, the meaning of the remaining similar expressions in this document shall be defined in a manner similar to that described above.

[0043] In some embodiments, the polypeptide is selected from any of the following, compared to the amino acid sequence shown in SEQ ID NO:110:

[0044] (1) The polypeptide contains or contains only an amino acid substitution mutation selected from any of the following positions: position 1, position 2, position 4, position 5, position 6, position 7, position 8, position 10, position 11, position 13, position 15, position 16, position 17, position 18, position 21, position 24, position 25, position 28, position 33, position 36, position 40, position 42, position 49, position 53, position 57;

[0045] Preferably, the polypeptide contains or contains only an amino acid substitution mutation selected from any of the following positions: 1L, 2E, 4E, 5L, 6T, 7E or 7R, 8A, 10K, 11D, 13L, 15K or 15A, 16L, 17M, 18K, 21H, 24D, 25A, 28A or 28Q, 33K or 33T, 36E, 40K or 40A, 42A, 49Q, 53A, 57K;

[0046] (2) The polypeptide contains or contains only amino acid substitution mutations selected from any two of the following positions: position 7, position 8, position 11, position 15, position 21, position 28, position 33, position 36, position 53;

[0047] Preferably, the polypeptide contains or contains only amino acid substitution mutations selected from any two of the following positions: 7R, 8A, 11D, 15K, 21H, 28A or 28Q, 33K, 36E, 53A;

[0048] (3) The polypeptide contains or contains only amino acid substitution mutations selected from any three of the following positions: position 11, position 13, position 15, position 18, position 21, position 28, position 33, position 49, and position 53;

[0049] Preferably, the polypeptide contains or contains only amino acid substitution mutations selected from any three of the following positions: 11D, 13L, 15K or 15A, 18K, 21H, 28A or 28Q, 33K, 49Q, 53A;

[0050] (4) The polypeptide contains or contains only amino acid substitution mutations selected from any four of the following positions: position 5, position 7, position 11, position 13, position 15, position 18, position 21, position 25, position 28, position 33, position 40, position 42, position 49, position 53, and position 57.

[0051] Preferably, the polypeptide contains or contains only amino acid substitution mutations selected from any four of the following positions: 5L, 7R, 11D, 13L, 15K, 18K, 21H, 25A, 28Q, 33K, 40K or 40A, 42A, 49Q, 53A, 57K.

[0052] (5) The polypeptide contains or contains only amino acid substitution mutations selected from any five of the following positions: position 2, position 5, position 7, position 10, position 15, position 17, position 21, position 24, position 28, position 31, position 33, position 40, position 42, position 49, position 53, and position 57;

[0053] Preferably, the polypeptide contains or contains only amino acid substitution mutations selected from any five of the following positions: 2E, 5L, 7E, 10K, 15K, 17M, 21H, 24D, 28Q, 31L, 33K, 40K or 40A, 42A, 49Q, 53A, 57K.

[0054] (6) The polypeptide contains or contains only amino acid substitution mutations selected from any six of the following positions: position 2, position 5, position 10, position 15, position 21, position 24, position 33, position 40, position 42, position 49, and position 53;

[0055] Preferably, the polypeptide contains or contains only amino acid substitution mutations selected from any six of the following positions: 2E, 5L, 10K, 15K, 21H, 24D, 33K, 40A, 42A, 49Q, 53A;

[0056] (7) The polypeptide contains or contains only amino acid substitution mutations selected from any seven of the following positions: position 5, position 10, position 15, position 21, position 24, position 33, position 40, position 42, position 49, and position 53.

[0057] Preferably, the polypeptide contains or contains only amino acid substitution mutations selected from any seven of the following positions: 5L, 10K, 15K, 21H, 24D, 33K, 40A, 42A, 49Q, 53A;

[0058] The mutated amino acid sites are the natural sequence sites relative to the amino acid sequence shown in SEQ ID NO:110.

[0059] In some embodiments, the polypeptide comprises or only comprises, compared to the amino acid sequence shown in SEQ ID NO:110:

[0060] (1) There is an amino acid substitution mutation at least at position 21; preferably, the amino acid at position 21 is substituted with histidine (21H);

[0061] And optional location,

[0062] (2) The polypeptide further comprises or contains only amino acid substitution mutations selected from one or more of the following positions (e.g., 1, 2, 3, 4, 5 or 6): position 2, position 5, position 7, position 8, position 10, position 11, position 13, position 15, position 17, position 18, position 24, position 25, position 28, position 31, position 33, position 36, position 40, position 42, position 49, position 53, position 57;

[0063] Preferably, the polypeptide further comprises or contains only amino acid substitution mutations selected from one or more of the following positions (e.g., 1, 2, 3, 4, 5 or 6): 2E, 5L, 7E or 7R, 8A, 10K, 11D, 13L, 15K or 15A, 17M, 18K, 24D, 25A, 28A or 28Q, 31L, 33K, 36E, 40K or 40A, 42A, 49Q, 53A, 57K;

[0064] The mutated amino acid sites are the natural sequence sites relative to the amino acid sequence shown in SEQ ID NO:110.

[0065] In some embodiments, the polypeptide comprises or only comprises, compared to the amino acid sequence shown in SEQ ID NO:110:

[0066] (1) It has an amino acid substitution mutation at at least position 21; preferably, the amino acid at position 21 is substituted with histidine (21H); and

[0067] (2) The polypeptide further comprises, or comprises only, any one of the following:

[0068] (i) The polypeptide further comprises or contains only an amino acid substitution mutation selected from any of the following positions: position 7, position 8, position 11, position 15, position 28, position 33, position 36, position 53; preferably, the polypeptide further comprises or contains only an amino acid substitution mutation selected from any of the following positions: 7R, 8A, 11D, 15K, 28A or 28Q, 33K, 36E, 53A;

[0069] (ii) The polypeptide further comprises or contains only amino acid substitution mutations selected from any two of the following positions: position 11, position 13, position 15, position 28, position 33, position 49, position 53; preferably, the polypeptide further comprises or contains only amino acid substitution mutations selected from any two of the following positions: 11D, 13L, 15K or 15A, 28A or 28Q, 33K, 49Q, 53A;

[0070] (iii) The polypeptide further comprises or contains only amino acid substitution mutations selected from any three of the following positions: position 5, position 7, position 11, position 13, position 15, position 18, position 25, position 28, position 33, position 40, position 42, position 49, position 53, and position 57; preferably, the polypeptide further comprises or contains only amino acid substitution mutations selected from any three of the following positions: 5L, 7R, 11D, 13L, 15K, 18K, 25A, 28Q, 33K, 40K or 40A, 42A, 49Q, 53A, and 57K;

[0071] (iv) The polypeptide further comprises or contains only amino acid substitution mutations selected from any four of the following positions: position 2, position 5, position 7, position 10, position 15, position 17, position 24, position 28, position 31, position 33, position 40, position 42, position 49, position 53, and position 57; preferably, the polypeptide further comprises or contains only amino acid substitution mutations selected from any four of the following positions: 2E, 5L, 7E, 10K, 15K, 17M, 24D, 28Q, 31L, 33K, 40K, or 40A, 42A, 49Q, 53A, and 57K;

[0072] (v) The polypeptide further comprises or contains only amino acid substitution mutations selected from any five of the following positions: position 2, position 5, position 10, position 15, position 24, position 33, position 40, position 42, position 49, and position 53; preferably, the polypeptide further comprises or contains only amino acid substitution mutations selected from any five of the following positions: 2E, 5L, 10K, 15K, 24D, 33K, 40A, 42A, 49Q, and 53A;

[0073] (vi) The polypeptide further comprises or contains only amino acid substitution mutations selected from any six of the following positions: position 5, position 10, position 15, position 24, position 33, position 40, position 42, position 49, and position 53; preferably, the polypeptide further comprises or contains only amino acid substitution mutations selected from any six of the following positions: 5L, 10K, 15K, 24D, 33K, 40A, 42A, 49Q, and 53A;

[0074] The mutated amino acid sites are the natural sequence sites relative to the amino acid sequence shown in SEQ ID NO:110.

[0075] In some embodiments, the polypeptide comprises or only comprises, compared to the amino acid sequence shown in SEQ ID NO:110:

[0076] (1) It has an amino acid substitution mutation at at least position 21; preferably, the amino acid at position 21 is substituted with histidine (21H); and

[0077] (2) There is an amino acid substitution mutation at least at position 15; preferably, the amino acid at position 15 is substituted with lysine (15K);

[0078] And optional location,

[0079] (3) The polypeptide further comprises or contains only amino acid substitution mutations selected from one or more of the following positions (e.g., 1, 2, 3, 4 or 5): position 2, position 5, position 7, position 10, position 11, position 13, position 15, position 17, position 18, position 24, position 25, position 28, position 31, position 33, position 40, position 42, position 49, position 53, position 57;

[0080] Preferably, the polypeptide further comprises or contains only amino acid substitution mutations selected from one or more of the following positions (e.g., 1, 2, 3, 4 or 5): 2E, 5L, 7E or 7R, 10K, 11D, 13L, 15K, 17M, 18K, 24D, 25A, 28Q or 28A, 31L, 33K, 40K or 40A, 42A, 49Q, 53A, 57K;

[0081] The mutated amino acid sites are the natural sequence sites relative to the amino acid sequence shown in SEQ ID NO:110.

[0082] In some embodiments, the polypeptide comprises or only comprises, compared to the amino acid sequence shown in SEQ ID NO:110:

[0083] (1) There is an amino acid substitution mutation at least at position 21; preferably, the amino acid at position 21 is substituted with histidine (21H);

[0084] (2) It has an amino acid substitution mutation at at least position 15; preferably, the amino acid at position 15 is substituted with lysine (15K); and

[0085] (3) The polypeptide further comprises, or comprises only, any one of the following:

[0086] (i) The polypeptide further comprises or only comprises an amino acid substitution mutation selected from any of the following positions: position 11, position 13, position 28, position 49, position 53; preferably, the polypeptide further comprises or only comprises an amino acid substitution mutation selected from any of the following positions: 11D, 13L, 28A, 49Q, 53A;

[0087] (ii) The polypeptide further comprises or contains only amino acid substitution mutations selected from any two of the following positions: position 5, position 7, position 11, position 13, position 18, position 25, position 28, position 33, position 40, position 42, position 49, position 53; preferably, the polypeptide further comprises or contains only amino acid substitution mutations selected from any two of the following positions: 5L, 7R, 11D, 13L, 18K, 25A, 28Q, 33K, 40K or 40A, 42A, 49Q, 53A;

[0088] (iii) The polypeptide further comprises or comprises only amino acid substitution mutations selected from any three of the following positions: position 2, position 5, position 7, position 10, position 17, position 24, position 28, position 31, position 33, position 40, position 42, position 49, position 53, and position 57; preferably, the polypeptide further comprises or comprises only amino acid substitution mutations selected from any three of the following positions: 2E, 5L, 7E, 10K, 17M, 24D, 28Q, 31L, 33K, 40K or 40A, 42A, 49Q, 53A, and 57K;

[0089] (iv) The polypeptide further comprises or contains only amino acid substitution mutations selected from any four of the following positions: position 2, position 5, position 10, position 24, position 33, position 40, position 42, position 49, and position 53; preferably, the polypeptide further comprises or contains only amino acid substitution mutations selected from any four of the following positions: 2E, 5L, 10K, 24D, 33K, 40A, 42A, 49Q, and 53A;

[0090] (iv) The polypeptide further comprises or contains only amino acid substitution mutations selected from any five of the following positions: position 5, position 10, position 24, position 33, position 40, position 42, position 49, and position 53; preferably, the polypeptide further comprises or contains only amino acid substitution mutations selected from any four of the following positions: 5L, 10K, 24D, 33K, 40A, 42A, 49Q, and 53A;

[0091] The mutated amino acid sites are the natural sequence sites relative to the amino acid sequence shown in SEQ ID NO:110.

[0092] In some embodiments, the polypeptide comprises or only comprises, compared to the amino acid sequence shown in SEQ ID NO:110:

[0093] (1) There is an amino acid substitution mutation at least at position 21; preferably, the amino acid at position 21 is substituted with histidine (21H);

[0094] (2) It has an amino acid substitution mutation at at least position 15; preferably, the amino acid at position 15 is substituted with lysine (15K); and

[0095] (3) There is an amino acid substitution mutation at least at position 49; preferably, the amino acid at position 49 is substituted with glutamine (49Q);

[0096] And optional location,

[0097] (4) The polypeptide further comprises or contains only amino acid substitution mutations selected from one or more of the following positions (e.g., 1, 2, 3 or 4): position 5, position 10, position 24, position 33, position 40, position 42, position 53;

[0098] Preferably, the polypeptide further comprises or contains only amino acid substitution mutations selected from one or more of the following positions (e.g., 1, 2, 3 or 4): 5L, 10K, 24D, 33K, 40A, 42A, 53A;

[0099] The mutated amino acid sites are the natural sequence sites relative to the amino acid sequence shown in SEQ ID NO:110.

[0100] In some embodiments, the polypeptide comprises or only comprises, compared to the amino acid sequence shown in SEQ ID NO:110:

[0101] (1) There is an amino acid substitution mutation at least at position 21; preferably, the amino acid at position 21 is substituted with histidine (21H);

[0102] (2) There is an amino acid substitution mutation at least at position 15; preferably, the amino acid at position 15 is substituted with lysine (15K);

[0103] (3) It has an amino acid substitution mutation at least at position 49; preferably, the amino acid at position 49 is mutated to glutamine (49Q); and

[0104] (4) The polypeptide further comprises, or comprises only, any one of the following:

[0105] (i) The polypeptide further comprises or contains only an amino acid substitution mutation selected from any one of the following positions: position 33, position 40; preferably, the polypeptide further comprises or contains only an amino acid substitution mutation selected from any one of the following positions: 33K, 40A;

[0106] (ii) The polypeptide further comprises or contains only amino acid substitution mutations selected from any two of the following positions: position 33 and position 53; preferably, the polypeptide further comprises or contains only amino acid substitution mutations selected from any two of the following positions: 33K and 53A;

[0107] (iii) The polypeptide further comprises or contains only amino acid substitution mutations selected from any three of the following positions: position 10, position 33, position 42, and position 53; preferably, the polypeptide further comprises or contains only amino acid substitution mutations selected from any three of the following positions: 10K, 33K, 42A, and 53A.

[0108] (iv) The polypeptide further comprises or contains only amino acid substitution mutations selected from any four of the following positions: position 5, position 10, position 24, position 33, position 40, and position 53; preferably, the polypeptide further comprises or contains only amino acid substitution mutations selected from any four of the following positions: 5L, 10K, 24D, 33K, 40A, and 53A.

[0109] The mutated amino acid sites are the natural sequence sites relative to the amino acid sequence shown in SEQ ID NO:110.

[0110] In some embodiments, the polypeptide comprises or only comprises, compared to the amino acid sequence shown in SEQ ID NO:110:

[0111] (1) There is an amino acid substitution mutation at least at position 21; preferably, the amino acid at position 21 is substituted with histidine (21H);

[0112] (2) It has an amino acid substitution mutation at at least position 33; preferably, the amino acid at position 33 is substituted with lysine (33K); and

[0113] (3) There is an amino acid substitution mutation at least at position 53; preferably, the amino acid at position 53 is substituted with alanine (53A);

[0114] And optional location,

[0115] (4) The polypeptide further comprises or contains only amino acid substitution mutations selected from one or more of the following positions (e.g., 1, 2, 3 or 4): position 2, position 5, position 7, position 10, position 15, position 17, position 24, position 28, position 31, position 33, position 40, position 42, position 49, position 53, position 57;

[0116] Preferably, the polypeptide further comprises or contains only amino acid substitution mutations selected from one or more of the following positions (e.g., 2, 3 or 4): 2E, 5L, 7E, 10K, 15K, 17M, 24D, 28Q, 31L, 33K, 40K or 40A, 42A, 49Q, 53A, 57K.

[0117] The mutated amino acid sites are the natural sequence sites relative to the amino acid sequence shown in SEQ ID NO:110.

[0118] In some embodiments, the polypeptide comprises or comprises only the following amino acid sequence compared to that shown in SEQ ID NO:110:

[0119] (1) There is an amino acid substitution mutation at least at position 21; preferably, the amino acid at position 21 is substituted with histidine (21H);

[0120] (2) There is an amino acid substitution mutation at least at position 33; preferably, the amino acid at position 33 is substituted with lysine (33K);

[0121] (3) It has an amino acid substitution mutation at at least position 53; preferably, the amino acid at position 53 is mutated to alanine (53A); and

[0122] (4) The polypeptide further comprises, or comprises only, any one of the following:

[0123] (i) The polypeptide further comprises or only comprises an amino acid substitution mutation selected from any of the following positions: position 15, position 28, position 40, position 49, position 57; preferably, the polypeptide further comprises or only comprises an amino acid substitution mutation selected from any of the following positions: 15K, 28Q, 40K, 49Q, 57K;

[0124] (ii) The polypeptide further comprises or comprises only amino acid substitution mutations selected from any two of the following positions: position 2, position 5, position 7, position 10, position 15, position 17, position 24, position 28, position 31, position 40, position 42, position 49, position 57; preferably, the polypeptide further comprises or comprises only amino acid substitution mutations selected from any two of the following positions: 2E, 5L, 7E, 10K, 15K, 17M, 24D, 28Q, 31L, 40K or 40A, 42A, 49Q, 57K;

[0125] (iii) The polypeptide further comprises or contains only amino acid substitution mutations selected from any three of the following positions: position 2, position 5, position 10, position 15, position 24, position 40, position 42, and position 49; preferably, the polypeptide comprises or contains only amino acid substitution mutations selected from any three of the following positions: 2E, 5L, 10K, 15K, 24D, 40A, 42A, and 49Q;

[0126] (iv) The polypeptide further comprises or contains only amino acid substitution mutations selected from any four of the following positions: position 5, position 10, position 15, position 24, position 40, position 42, and position 49; preferably, the polypeptide further comprises or contains only amino acid substitution mutations selected from any four of the following positions: 5L, 10K, 15K, 24D, 40A, 42A, and 49Q;

[0127] The mutated amino acid sites are the natural sequence sites relative to the amino acid sequence shown in SEQ ID NO:110.

[0128] In some embodiments, the polypeptide comprises or only comprises, compared to the amino acid sequence shown in SEQ ID NO:110:

[0129] (1) There is an amino acid substitution mutation at least at position 21; preferably, the amino acid at position 21 is substituted with histidine (21H);

[0130] (2) There is an amino acid substitution mutation at least at position 33; preferably, the amino acid at position 33 is substituted with lysine (33K);

[0131] (3) It has an amino acid substitution mutation at at least position 53; preferably, the amino acid at position 53 is mutated to alanine (53A); and

[0132] (4) There is an amino acid substitution mutation at least at position 15; preferably, the amino acid at position 15 is substituted with lysine (15K);

[0133] And optional location,

[0134] (5) The polypeptide further comprises or contains only amino acid substitution mutations selected from one or more of the following positions (e.g., 2 or 3): position 2, position 5, position 7, position 10, position 15, position 17, position 24, position 28, position 31, position 33, position 40, position 42, position 49, position 53, position 57;

[0135] Preferably, the polypeptide further comprises or contains only amino acid substitution mutations selected from one or more of the following positions (e.g., 2 or 3): 2E, 5L, 7E, 10K, 15K, 17M, 24D, 28Q, 31L, 33K, 40A or 40K, 42A, 49Q, 53A, 57K.

[0136] The mutated amino acid sites are the natural sequence sites relative to the amino acid sequence shown in SEQ ID NO:110.

[0137] In some embodiments, the polypeptide comprises or only comprises, compared to the amino acid sequence shown in SEQ ID NO:110:

[0138] (1) There is an amino acid substitution mutation at least at position 21; preferably, the amino acid at position 21 is substituted with histidine (21H);

[0139] (2) There is an amino acid substitution mutation at least at position 33; preferably, the amino acid at position 33 is substituted with lysine (33K);

[0140] (3) There is an amino acid substitution mutation at least at position 53; preferably, the amino acid at position 53 is substituted with alanine (53A);

[0141] (4) It has an amino acid substitution mutation at at least position 15; preferably, the amino acid at position 15 is substituted with lysine (15K); and

[0142] (5) The polypeptide further comprises, or comprises only, any one of the following:

[0143] (i) The polypeptide further comprises or contains only an amino acid substitution mutation selected from any of the following positions: position 2, position 5, position 7, position 10, position 17, position 24, position 28, position 31, position 40, position 42A, position 49Q, position 57; preferably, the polypeptide further comprises or contains only an amino acid substitution mutation selected from any of the following positions: 2E, 5L, 7E, 10K, 17M, 24D, 28Q, 31L, 40A or 40K, 42A, 49Q, 57K;

[0144] (ii) The polypeptide further comprises or contains only amino acid substitution mutations selected from any two of the following positions: position 2, position 5, position 10, position 24, position 40, position 42, position 49; preferably, the polypeptide further comprises or contains only amino acid substitution mutations selected from any two of the following positions: 2E, 5L, 10K, 24D, 40A, 42A, 49Q;

[0145] (iii) The polypeptide further comprises or contains only amino acid substitution mutations selected from any three of the following positions: position 5, position 10, position 24, position 40, position 42, and position 49; preferably, the polypeptide further comprises or contains only amino acid substitution mutations selected from any three of the following positions: 5L, 10K, 24D, 40A, 42A, and 49Q.

[0146] The mutated amino acid sites are the natural sequence sites relative to the amino acid sequence shown in SEQ ID NO:110.

[0147] In some embodiments, the polypeptide contains, or contains only, one or more mutations selected from the group consisting of, compared to the amino acid sequence shown in SEQ ID NO:110:

[0148] (1)N21H; (2)E11D; (3)H18K; (4)I31L; (5)N28A; (6)N28Q; (7)V1L; (8)D2E; (9) K4E; (10)D6T; (11)K7E; (12)K7R; (13)E8A; (14)Q10K; (15)E15K; (16)E15A; (17 )I16L; (18)L17M; (19)S33K; (20)S33T; (21)D36E; (22)V40K; (23)V40A; (24)K 42A; (25)D53A; (26)P57K; (27)E25A; (28)F5L; (29)F13L; (30)K49Q; (31)E24D.

[0149] The mutated amino acid sites are the natural sequence sites relative to the amino acid sequence shown in SEQ ID NO:110.

[0150] As used herein, the mutation “N21H” means that the amino acid residue N at the position corresponding to the 21st position of SEQ ID NO:110 in the polypeptide is replaced with H; the mutation “E11D” means that the amino acid residue E at the position corresponding to the 11th position of SEQ ID NO:110 in the polypeptide is replaced with D.

[0151] Unless otherwise specified or there is a clear contradiction between the preceding and following statements, the meaning of the remaining similar expressions in this document shall be defined in a manner similar to that described above.

[0152] In some embodiments, compared to the amino acid sequence shown in SEQ ID NO:110, the polypeptide contains or only contains amino acid substitution mutations selected from any combination of the following positions:

[0153] 21st, 33rd, 21st, 53rd, 21st, 28th, 21st, 8th, 21st, 11th, 21st, 36th, 21st, 7th, 21st, 15th, 21st, 15th, 53rd, 15th, 18th, 33rd, 21st, 15th, 28th, 15th, 28th, 33rd, 21st, 13th, 15th, 21st, 11th, 1 5th, 15th, 28th, 53rd, 21st, 33rd, 53rd, 21st, 15th, 49th, 21st, 28th, 33rd, 21st, 28th, 53rd, 21st, 15th, 28th, 21st, 13th, 33rd, 21st, 13th, 53rd, 21st, 13th, 28th, 21st, 5th, 15th, 53rd, 21st, 11th, 15th 53rd, 21st, 15th, 33rd, 49th, 21st, 13th, 15th, 28th, 21st, 15th, 40th, 49th, 21st, 7th, 15th, 33rd, 21st, 15th, 18th, 53rd, 21st, 15th, 28th, 42nd, 21st, 13th, 15th, 33rd, 21st, 13th, 15th, 53rd, 21st, 15th, 25th 53rd digit, 21st, 15th, 40th, 53rd digit, 21st, 11th, 13th, 15th digit, 21st, 13th, 28th, 53rd digit, 21st, 15th, 28th, 33rd digit, 21st, 15th, 28th, 53rd digit, 21st, 15th, 33rd, 53rd digit, 21st, 10th, 21st, 15th, 28 ..., 21st, 10th, 21st, 15th, 28th, 33rd, 53rd, 21st, 10th 15, 33, 53rd position, 21st, 15, 33, 42nd, 53rd position, 21st, 5, 15, 33, 53rd position, 21st, 15, 24th, 33, 53rd position, 21st, 2, 15, 33, 53rd position, 21st, 15, 17th, 33rd, 53rd position, 21st, 15, 33rd, 49th, 53rd position, 21st, 15, 33rd, 40th, 5th position 3rd digit, 21st, 10th, 15th, 40th, 53rd digit, 21st, 15th, 33rd, 53rd, 57th digit, 21st, 15th, 33rd, 40th, 53rd digit, 21st, 15th, 31st, 33rd, 53rd digit, 21st, 7th, 15th, 33rd, 53rd digit, 21st, 10th, 15th, 33rd, 42nd, 53rd digit, 21st, 5th, 10th, 15th, 33rd, 53rd digit The 21st, 5th, 15th, 33rd, 42nd, and 53rd positions; the 21st, 10th, 15th, 24th, 33rd, and 53rd positions; the 21st, 10th, 15th, 33rd, 40th, and 53rd positions; the 21st, 10th, 15th, 33rd, 49th, and 53rd positions; the 21st, 15th, 24th, 33rd, and 42nd positions; the 21st, 5th, 15th, 24th, 33rd, and 53rd positions; the 21st, 2nd... 15, 33, 42, 53, 21, 15, 33, 42, 49, 53, 21, 5, 10, 15, 33, 42, 53, 21, 10, 15, 24, 33, 42, 53, 21, 5, 10, 15, 24, 33, 53, 21, 5, 10, 15, 33, 49, 53, 21Amino acid substitution mutations at positions 10, 15, 33, 40, 49, 53; positions 21, 10, 15, 24, 33, 40, 53; positions 21, 10, 15, 24, 33, 49, 53; positions 21, 33, 49, 53; positions 21, 33, 53, 57; positions 21, 33, 40, 53; positions 21, 5, 33, 42, 53; positions 21, 24, 33, 42, 53; or positions 21, 2, 33, 42, 53.

[0154] In some embodiments, the polypeptide contains an amino acid substitution mutation as shown in any of the following, compared to the amino acid sequence shown in SEQ ID NO:110:

[0155] (1) N21H; S33K;

[0156] (2) N21H; D53A;

[0157] (3) N21H; N28Q;

[0158] (4) N21H; E8A;

[0159] (5) N21H; N28A;

[0160] (6) N21H; E11D;

[0161] (7) N21H; D36E;

[0162] (8) N21H; K7R;

[0163] (9) N21H; E15K;

[0164] The mutated amino acid sites are the natural sequence sites relative to the amino acid sequence shown in SEQ ID NO:110.

[0165] As used herein, the mutation “N21H;S33K;” means that the amino acid residue N at the position corresponding to position 21 of SEQ ID NO:110 in the polypeptide is replaced with H, and the amino acid residue E at the position corresponding to position 11 of SEQ ID NO:110 is replaced with D.

[0166] Unless otherwise specified or there is a clear contradiction between the preceding and following statements, the meaning of the remaining similar expressions in this document shall be defined in a manner similar to that described above.

[0167] In some embodiments, the polypeptide contains an amino acid substitution mutation as shown in any of the following, compared to the amino acid sequence shown in SEQ ID NO:110:

[0168] (1) N21H; E15K; D53A;

[0169] (2) N21H; E15K; N28A;

[0170] (3) N21H; F13L; E15K;

[0171] (4) N21H; E11D; E15K;

[0172] (5) N21H; S33K; D53A;

[0173] (6) N21H; E15K; K49Q;

[0174] (7) N21H; N28Q; S33K;

[0175] (8) N21H; N28Q; D53A;

[0176] (9) N21H; E15A; N28Q;

[0177] (10) N21H; F13L; S33K;

[0178] (11) N21H; F13L; D53A;

[0179] (12) N21H; F13L; N28Q;

[0180] (13)E15K;H18K;S33K

[0181] (14)E15K; N28Q; S33K

[0182] (15)E15K; N28Q; D53A

[0183] The mutated amino acid sites are the natural sequence sites relative to the amino acid sequence shown in SEQ ID NO:110.

[0184] In some embodiments, the polypeptide contains an amino acid substitution mutation as shown in any of the following, compared to the amino acid sequence shown in SEQ ID NO:110:

[0185] (1) N21H; F5L; E15K; D53A

[0186] (2) N21H; E11D; E15K; D53A

[0187] (3) N21H; E15K; S33K; K49Q

[0188] (4) N21H; F13L; E15K; N28Q

[0189] (5) N21H; E15K; V40A; K49Q

[0190] (6) N21H; K7R; E15K; S33K

[0191] (7) N21H; E15K; H18K; D53A

[0192] (8) N21H; E15K; N28Q; K42A

[0193] (9) N21H; F13L; E15K; S33K

[0194] (10) N21H; F13L; E15K; D53A

[0195] (11) N21H; E15K; E25A; D53A

[0196] (12) N21H; E15K; V40K; D53A

[0197] (13) N21H; E11D; F13L; E15K

[0198] (14) N21H; F13L; N28Q; D53A

[0199] (15) N21H; E15K; N28Q; S33K

[0200] (16) N21H; E15K; N28Q; D53A

[0201] (17) N21H; E15K; S33K; D53A

[0202] (18) N21H; N28Q; S33K; D53A

[0203] (19) N21H; S33K; K49Q; D53A

[0204] (20) N21H; S33K; D53A; P57K

[0205] (21) N21H; S33K; V40K; D53A

[0206] The mutated amino acid sites are the natural sequence sites relative to the amino acid sequence shown in SEQ ID NO:110.

[0207] In some embodiments, the polypeptide contains an amino acid substitution mutation as shown in any of the following, compared to the amino acid sequence shown in SEQ ID NO:110:

[0208] (1)N21H; E15K; N28Q; S33K; D53A;

[0209] (2)N21H; Q10K; E15K; S33K; D53A;

[0210] (3)N21H; E15K; S33K; K42A; D53A;

[0211] (4)N21H; F5L; E15K; S33K; D53A;

[0212] (5)N21H; E15K; E24D; S33K; D53A;

[0213] (6)N21H; D2E; E15K; S33K; D53A;

[0214] (7)N21H; E15K; L17M; S33K; D53A;

[0215] (8)N21H; E15K; S33K; K49Q; D53A;

[0216] (9)N21H; E15K; S33K; V40A; D53A;

[0217] (10)N21H; Q10K; E15K; V40A; D53A;

[0218] (11)N21H; E15K; S33K; D53A; P57K;

[0219] (12)N21H; E15K; S33K; V40K; D53A;

[0220] (13)N21H; E15K; I31L; S33K; D53A;

[0221] (14)N21H; K7E; E15K; S33K; D53A;

[0222] (15)N21H; F5L; S33K; K42A; D53A;

[0223] (16)N21H; E24D; S33K; K42A; D53A;

[0224] (17)N21H; D2E; S33K; K42A; D53A;

[0225] The mutated amino acid sites are the natural sequence sites relative to the amino acid sequence shown in SEQ ID NO:110.

[0226] In some embodiments, the polypeptide contains an amino acid substitution mutation as shown in any of the following, compared to the amino acid sequence shown in SEQ ID NO:110:

[0227] (1)N21H; Q10K; E15K; S33K; K42A; D53A;

[0228] (2)N21H; F5L; Q10K; E15K; S33K; D53A;

[0229] (3)N21H; F5L; E15K; S33K; K42A; D53A;

[0230] (4)N21H; Q10K; E15K; E24D; S33K; D53A;

[0231] (5)N21H; Q10K; E15K; S33K; V40A; D53A;

[0232] (6)N21H; Q10K; E15K; S33K; K49Q; D53A;

[0233] (7)N21H; E15K; E24D; S33K; K42A; D53A;

[0234] (8)N21H; F5L; E15K; E24D; S33K; D53A;

[0235] (9)N21H; D2E; E15K; S33K; K42A; D53A;

[0236] (10)N21H; E15K; S33K; K42A; K49Q; D53A;

[0237] The mutated amino acid sites are the natural sequence sites relative to the amino acid sequence shown in SEQ ID NO:110.

[0238] In some embodiments, the polypeptide contains an amino acid substitution mutation as shown in any of the following, compared to the amino acid sequence shown in SEQ ID NO:110:

[0239] (1)N21H; F5L; Q10K; E15K; S33K; K42A; D53A;

[0240] (2)N21H; Q10K; E15K; E24D; S33K; K42A; D53A;

[0241] (3)N21H; F5L; Q10K; E15K; E24D; S33K; D53A;

[0242] (4)N21H; F5L; Q10K; E15K; S33K; K49Q; D53A;

[0243] (5)N21H; Q10K; E15K; S33K; V40A; K49Q; D53A;

[0244] (6)N21H; Q10K; E15K; E24D; S33K; V40A; D53A;

[0245] (7)N21H; Q10K; E15K; E24D; S33K; K49Q; D53A;

[0246] The mutated amino acid sites are the natural sequence sites relative to the amino acid sequence shown in SEQ ID NO:110.

[0247] The mutated amino acid sites are the amino acid sites in the polypeptide that correspond to the corresponding amino acid sites in SEQ ID NO:110. For example, the mutation "N21H" means that the amino acid residue N at the position corresponding to the 21st position in SEQ ID NO:110 is replaced with H.

[0248] In some embodiments, the polypeptide comprises an amino acid sequence as shown in any one of SEQ ID NOs:1-109, or an amino acid sequence having at least 90%, for example at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with an amino acid sequence shown in any one of SEQ ID NOs:1-109.

[0249] In a second aspect, the present invention provides a polymer comprising an amino acid sequence formed by the fusion of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, or 9) polypeptides described in the first aspect;

[0250] Wherein, the one or more polypeptides (e.g., 2, 3, 4, 5, 6, 7, 8, 9) are completely identical, partially identical, or completely different;

[0251] The polypeptides are optionally linked together by peptide linkers.

[0252] As used herein, the term "peptide linker" refers to an element in a polymer that links two polypeptide monomers or domains together. A peptide linker can be a linker conventionally used in the art, the presence of which does not affect the activity of the polypeptide. Exemplary peptide linkers comprise one or more glycine (G) and / or serine (S) peptide linkers, for example, flexible peptides comprising (G4S)n, where n is an integer not less than 0, such as 1, 2, 3, or 4. Other alternative peptide linkers include: APKVDAKFDKE, APKVDNKFNKE, APKADNKFNKE, APKVFDKE, APAKFDKE, AKFDKE, APKVDA, VDAKFDKE, APKKFDKE, APK, APKYEDGVDAKFDKE, and YEDG.

[0253] In some embodiments, the polymer is a dimer, trimer, tetramer, pentamer, hexamer, heptametamer, octamer, or nonamer; preferably, the polymer is a tetramer.

[0254] In some embodiments, the polymer comprises an amino acid sequence as shown in any one of SEQ ID NOs: 112-220, or an amino acid sequence having at least 90%, for example at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 112-220.

[0255] In some embodiments, the polypeptide described in the first aspect or the polymer described in the second aspect is further linked to one or more coupling elements at its C-terminus or N-terminus;

[0256] Preferably, the coupling element is selected from one or more cysteine ​​residues, multiple lysine residues, and multiple histidine residues.

[0257] As used herein, suitable "coupling elements" are well known in the art and include, but are not limited to, one or more cysteine ​​residues (e.g., 3-15 or 5-10), multiple lysine residues (e.g., 3-15 or 5-10), and multiple histidine residues (e.g., 3-15 or 5-10). The coupling element may be a single cysteine ​​residue located at the C-terminus of the peptide, thereby allowing the peptide to be coupled to the solid support by reacting the thiol group of the cysteine ​​residue with an electrophilic group on the solid support. The coupling element may be directly attached to the peptide or connected to the N- or C-terminus of the peptide via a linker.

[0258] In a third aspect, the present invention also provides an isolated polypeptide construct, the polypeptide construct comprising:

[0259] (1) The polypeptide described in the first aspect or the polymer described in the second aspect;

[0260] (2) One or more coupling elements located at the C-terminus of the polypeptide or polymer described in (1); and

[0261] (3) The leader sequence located at the N-terminus of the polypeptide or polymer described in (1).

[0262] Preferably, the coupling element is selected from one or more cysteine ​​residues, multiple lysine residues, and multiple histidine residues.

[0263] Preferably, the leader sequence is, for example, an amino acid residue derived from the cloning process, or an amino acid residue constituting a signal sequence from cleavage, and the number of amino acid residues may be, for example, 20 or less, such as 15 or less, such as 10 or less, or 5 or less. As a specific example, the N-terminal leader sequence comprises the sequences GAQGT, MGAQGT, AQ, AQGT, VDAKFDKE, AQVDAKFDKE, or AQGTVDAKFDKE.

[0264] In some embodiments, one or more coupling elements at the C-terminus are a single cysteine ​​residue, and the N-terminal leader sequence is GAQGT; optionally, the leader sequence contains amino acid modifications, such as a modification on the first amino acid G (e.g., glucose acetylation).

[0265] In some embodiments, the construct of the present invention comprises, from the N-terminus to the C-terminus, (1) GAQGT; (2) an amino acid sequence as shown in any one of SEQ ID NOs: 1-220; and (3) a single cysteine ​​(C).

[0266] The coupling element or leader sequence of the present invention should be sufficiently stable in an alkaline environment and should not impair the properties of the mutant protein (such as activity, alkaline stability, etc.).

[0267] In a fourth aspect, the present invention also provides isolated nucleic acid molecules that encode the polypeptides described in the first aspect, the polymers described in the second aspect, or the polypeptide constructs described in the third aspect.

[0268] In a fifth aspect, the present invention also provides a carrier comprising the isolated nucleic acid molecules of the fourth aspect.

[0269] In a sixth aspect, the present invention also provides a host cell comprising the isolated nucleic acid molecule of the fourth aspect or the carrier of the fifth aspect.

[0270] In a seventh aspect, the present invention also provides a method for preparing a polypeptide of the first aspect, a polymer of the second aspect, or a polypeptide construct of the third aspect, comprising culturing a host cell of the sixth aspect under conditions allowing expression of the polypeptide, the polymer, or the polypeptide construct, and recovering the polypeptide, the polymer, or the polypeptide construct from the cultured host cell culture.

[0271] In an eighth aspect, the present invention also provides a separation matrix comprising the polypeptide of the first aspect, the polymer of the second aspect, or the polypeptide construct of the third aspect coupled to a solid support.

[0272] Preferably, the polypeptide, polymer, or polypeptide construct is coupled to the solid support via a thioether bond;

[0273] Preferably, the solid support is selected from polysaccharides, such as dextran, starch, cellulose, pullulan, agar, or agarose.

[0274] The separation matrix of this invention can be used to separate immunoglobulins or other Fc-containing proteins, and due to the improved alkaline stability of the polypeptide / multimer / polypeptide construct, the separation matrix will withstand highly alkaline conditions during cleaning, which is crucial for long-term repeated use in biological treatment separation devices. The alkaline stability of the matrix can be evaluated by measuring immunoglobulin-binding capacity using polyclonal human IgG, typically before and after incubation in an alkaline solution at a specific temperature, such as 22 ± 2 °C. Such evaluation methods are well known in the art.

[0275] As those skilled in the art should understand, expressed peptides, polymers, or peptide constructs should be purified to an appropriate degree before being immobilized onto a solid support. Such purification methods are well known in the art, and immobilization of protein-based ligands onto solid supports is readily performed using standard methods.

[0276] The solid support for the separation matrix disclosed in this invention can be any suitable and well-known type. Conventional affinity separation matrices typically have organic properties and are based on polymers whose hydrophilic surfaces are exposed to the aqueous medium used, i.e., hydroxyl (-OH), carboxyl (-COOH), carboxylamino (-CONH2, possibly in N-substituted form), amino (-NH2, possibly in substituted form), oligo- or polyoxyethylene groups are exposed on their outer surfaces and (if present) also on their inner surfaces. The solid support can be suitable to be porous. Porosity can be expressed as a Kav or Kd value (the fraction of pore volume available for probe molecules of a specific size), which is measured by reversed-phase size exclusion chromatography, for example according to the method described in *Gel Filtration Principles and Methods*, Pharmacia LKB Biotechnology 1991, pp. 6-13.

[0277] Peptides, polymers, or peptide constructs can be attached to solid supports via conventional coupling techniques, utilizing groups such as thiol, amino, and / or carboxyl groups present in the ligand. Bisepoxides, epichlorohydrin, CNBr, and N-hydroxysuccinimide (NHS) are well-known coupling agents. A molecule called a spacer region can be introduced between the solid support and the peptide / polymer / peptide construct, which improves the availability of the peptide / polymer / peptide construct and facilitates its chemical coupling to the solid support. Depending on the nature of the peptide / polymer / peptide construct and the coupling conditions, coupling can be multi-point coupling (e.g., via multiple lysine residues) or single-point coupling (e.g., via a single cysteine ​​residue). Alternatively, peptide / polymer / peptide constructs can be attached to solid supports via non-covalent bonding, such as physical adsorption or biospecific adsorption.

[0278] The term "spacer region" in this article refers to the element that connects a polypeptide, polypeptide multimer, or polypeptide construct to a solid support.

[0279] In some embodiments, the solid support comprises a polyhydroxy polymer, such as a polysaccharide. Examples of polysaccharides include, for example, dextran, starch, cellulose, amylopectin, agar, agarose, etc. Polysaccharides are inherently hydrophilic, have a low degree of nonspecific interactions, provide a high content of reactive (activatable) hydroxyl groups, and are generally stable to alkaline cleaning solutions used for biological treatment.

[0280] In a ninth aspect, the present invention also provides the use of the polypeptide of the first aspect, the polymer of the second aspect, or the polypeptide construct of the third aspect in the preparation of the separation matrix of the eighth aspect.

[0281] In a tenth aspect, the present invention also provides a method for isolating a protein containing Fc, the method comprising the step of contacting the protein to be purified with the separation matrix described in the eighth aspect;

[0282] Preferably, the protein containing Fc is an immunoglobulin, such as IgG;

[0283] Preferably, the IgG is selected from IgG1, IgG2, IgG3, and IgG4.

[0284] In an eleventh aspect, the present invention also provides the use of the polypeptide of the first aspect, the polymer of the second aspect, the polypeptide construct of the third aspect, or the separation matrix of the eighth aspect in the separation of proteins containing Fc.

[0285] Terminology Definition

[0286] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the virological, biochemical, and immunological laboratory procedures used herein are all standard procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0287] As used herein, the terms "antibody" and "immunoglobulin" are used interchangeably and have the meanings known in the art. The antibodies or immunoglobulins described herein also include antibody fragments, fusion proteins containing antibody fragments, and conjugates containing antibodies or antibody fragments, provided that such fragments, fusion proteins, or conjugates contain the Fc moiety of an antibody and can be classified and purified by binding to protein A. Antibody fragments may be functionally active fragments.

[0288] As used herein, the terms “Fc-binding polypeptide” and “Fc-binding protein” refer to polypeptides or proteins capable of binding to the crystallizable portion (Fc) of an antibody, including, for example, protein A and protein G, or any fragment or fusion protein thereof that retains the binding property.

[0289] When the terms “for example,” “such as,” “like,” “including,” “contains,” or variations thereof are used herein, these terms will not be considered restrictive terms but will be interpreted as meaning “but not limited to” or “not limited to.”

[0290] As used herein, the term “contains only” will be interpreted as meaning “limited to” but not including any scope beyond that.

[0291] Unless otherwise specified herein or clearly contradicted by the context, the terms “an” and “a kind” as well as “the” and similar designations shall be interpreted to cover both the singular and the plural in the context of describing the invention (especially in the context of the following claims).

[0292] As used herein, the term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. To determine the percentage identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., a gap may be introduced in the first amino acid sequence or nucleic acid sequence to best align with the second amino acid sequence or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecule is identical at that position. The percentage identity between two sequences is a function of the number of identity positions shared by the sequences (i.e., percentage identity = number of identical overlapping positions / total number of positions × 100%). In some embodiments, the two sequences are of the same length.

[0293] The determination of percentage identity between two sequences can also be achieved using mathematical algorithms. A non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, as an improvement upon that in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. Such algorithms are integrated into the NBLAST and XBLAST procedures of Altschul et al., 1990, J. Mol. Biol. 215:403.

[0294] The twenty common amino acids mentioned in this article are written in accordance with conventional usage. See, for example, Immunology-ASynthesis (2nd Edition, E.S. Golub and D.G. Ren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this invention, the terms “polypeptide” and “protein” have the same meaning and are used interchangeably. Furthermore, in this 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.

[0295] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0296] 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.

[0297] Beneficial effects of the invention

[0298] The present invention provides a novel B domain construct of protein A, which, compared to the B domain of wild-type protein A, has one or more improved properties, such as: (1) alkali resistance; (2) high binding capacity to IgG, thereby improving the efficiency of IgG production and reducing costs. Attached Figure Description

[0299] Figure 1: Schematic diagram of the protein A tetramer sequence structure.

[0300] Figure 2: SDS-PAGE strip images of some variants with improved alkali resistance. Detailed Implementation

[0301] The invention will now be described with reference to the following embodiments, which are intended to illustrate the invention (and not limit it). Those skilled in the art will appreciate that the embodiments are described by way of example and are not intended to limit the scope of protection claimed by the invention.

[0302] Example 1: Design and preparation of domain B variant of protein A

[0303] Through protein design and multiple rounds of single- or multi-site mutations, this invention generates a series of variants of domain B of protein A. From these variants, high-performance variants are selected, and their mutation sites and sequences are shown in Table 1.

[0304] The wild-type sequence is derived from the B domain of Staphylococcus aureus protein A, and its monomeric amino acid sequence is shown in SEQ ID NO:111. The reference sequence monomer is shown in SEQ ID NO:110. Compared with the wild-type Staphylococcus aureus protein A B domain monomeric amino acid sequence SEQ ID NO:111, the reference sequence (Sequence 11 in patent CN109311949B) monomer SEQ ID NO:110 is mutated to: A1V; N3A; N6D; Q9A; N11E; N23T; G29A; Q40V; A42K; N43A; L44I.

[0305] Variants 1 to 30 were the molecules selected by the first round of single-point mutation. Among them, variant 3 performed the best in the alkali resistance and elution performance tests. Further multi-round multi-site mutations were carried out based on variant 3, and more preferred variant molecules 31 to 109 were obtained through screening.

[0306] The mutated sequence was artificially synthesized and constructed for transforming *E. coli* host cells to express the target protein. To increase the binding capacity to the antibody, the monomer sequence was constructed into a tetramer, as shown in Table 5. To improve the stability of protein expression and facilitate matrix coupling, the tetramer protein was actually expressed as a tandem of four repeating domains, containing an N-terminal GAQGT leader sequence and a single C-terminal cysteine ​​residue; in the following examples, it is referred to as a protein tetramer or tetramer.

[0307] The variant's gene was constructed into plasmid pET16b, which was artificially synthesized (GenScript). The correctly sequenced recombinant plasmid was then transformed into *E. coli* BL21(DE3) competent cells (from Weidi Biotechnology). The cells were cultured at 37°C until the OD reached 0.6, then induced at 25°C. IPTG (from Solarbio) was added at a final concentration of 1 mM. After 18 hours of induction, the cells were harvested. The cells were resuspended in PBS buffer, sonicated on ice, and the supernatant was collected by centrifugation. The supernatant was then heat-treated and centrifuged again. The heat-treated supernatant was precipitated with ammonium sulfate and concentrated to 10 mg / ml by ultrafiltration with PBS.

[0308] Table 1: Variant sequences and their mutations relative to the reference sequence

[0309] Example 2: Performance evaluation of protein A B domain variants

[0310] 2.1 Evaluation of Alkali Resistance of Optimized Molecules in One Round

[0311] Alkali resistance: Due to their specific binding to target proteins, affinity media capture these proteins during the crude purification stage of the process. The impurity level in this stage is high, and repeated use affects the lifespan of the affinity media. Alkali washing can effectively clean the media and extend its lifespan. Therefore, improving the alkali resistance of the ligand protein and its affinity media is particularly important.

[0312] The mutant protein and the reference sequence protein tetramer were coupled to agarose-based spheres under similar ligand density conditions to form an affinity medium. The dynamic binding capacity (DBC) of protein A in the affinity medium for the model protein was determined using the model protein (normal human IgG - concentration 5 mg / ml - Biotech). This capacity was the initial binding capacity. The binding capacity of the coupled affinity medium for the model protein was measured every 24 hours in a 0.5 M NaOH environment at laboratory temperature (22±2℃) for a cumulative incubation of 120 hours, and the final remaining binding capacity was calculated. The results are shown in Table 2.1.

[0313] As shown in Table 2.1, variant 3 showed better residual binding capacity to the model protein than the reference sequence after alkali treatment for 24, 48, 72, 96, and 120 hours.

[0314] Table 2.1: Evaluation of Alkali Resistance of Optimized Molecules in One Round

[0315] 2.2 Evaluation of the Elution Performance of Optimized Molecules in One Round

[0316] Elution performance: Protein A affinity medium has standard performance. Low pH conditions, such as pH < 4.0, are used as elution conditions to elute the target protein. Higher or higher pH conditions, such as pH > 4.5, are used as elution conditions, mainly to remove non-specifically adsorbed impurities. In this case, the amount of target protein eluted should be low or there should be no target protein elution.

[0317] The mutant protein and the reference sequence protein tetramer were coupled to agarose-based spheres under similar ligand density conditions to form an affinity medium. To ensure no protein flow-through during the loading process of the coupled affinity medium, the loading capacity was set to the initial loading capacity * 0.8 safety factor. The chromatography process was set to elute at pH 5.5 and pH 3.4. The yield of the target protein under different pH conditions was investigated to verify the elution performance of the medium. The results are shown in Table 2.2.

[0318] As shown in Table 2.2, the yield of variant 3 under rinsing conditions was lower than that of the reference sequence, while the yield under elution conditions was higher than that of the reference sequence. Variant 3 exhibits superior elution performance compared to the reference sequence.

[0319] Table 2.2: Evaluation of the Elution Performance of Optimized Molecules in One Round

[0320] 2.3 The binding ability of the constructed variant to IgG was detected using the ELISA method.

[0321] Experimental principle: The B domain of protein A is attached to an ELISA plate, where it binds to human IgG antibody to form a solid-phase antigen-antibody complex. Horseradish peroxidase-labeled goat anti-human IgG (HRP) then binds to the antibody in the solid-phase immune complex, forming a solid-phase antigen-antibody-ELISA-labeled secondary antibody complex. Finally, the degree of color development after substrate addition is quantitatively measured using an ELISA instrument (Agilent Synergy) to determine the human IgG antibody content, which is used to represent the affinity of the B domain of protein A for IgG.

[0322] Experimental Methods: Eleven gradients of 40 μg / ml protein A B-domain variant tetramer were coated onto 96-well plates, 50 μl / well, with three replicates per concentration, and coated overnight. The plates were blocked with 100 μl of PBS containing 5% skim milk at 37°C for 1 h, followed by washing. Human IgG (purchased from Yisheng Biotechnology) was diluted to 0.5 mg / ml with PBS containing 2% skim milk, 50 μl / well, and incubated at 37°C for 1.5 hours, followed by washing. Horseradish peroxidase-conjugated goat anti-human IgG (HRP) (from Beyotime) diluted 1:250 with 2% skim milk was added, 50 μl / well, and incubated at 37°C for 1 hour, followed by washing, color development, and termination. The plates were shaken to mix, and OD values ​​were read using a microplate reader. 450 The absorbance value was calculated. The EC50 (half-maximum concentration) was obtained using GraphPad Prism 8 software with a four-parameter method.

[0323] The experimental results are shown in Table 2.3. The B domain of the protein A obtained by the present invention can bind IgG, showing excellent binding ability.

[0324] 2.4 Alkali tolerance of protein A by SDS-PAGE detection

[0325] Strong alkaline environments disrupt protein hydrogen bonds and hydrolyze peptide bonds, leading to protein degradation. SDS molecules are used to convert proteins to a linear conformation. Under an electric field, degraded and intact proteins are separated, and the proportion of undegraded protein is calculated based on band density to determine the alkali resistance of protein A. SDS-PAGE analysis of band density allows for rapid screening of variants resistant to high concentrations of NaOH.

[0326] Wild-type protein A's B domain or its variant tetramer was treated with alkali-resistant solution in 1M NaOH and placed in a 25℃ incubator. Samples were taken at 24h, 48h, and 72h of alkali treatment, neutralized with 1M HCl, and then analyzed by SDS-PAGE using a 15% high-resolution precast gel (from Yisheng Biotechnology). Samples were diluted with ultrapure water, and 5× protein buffer was added at a 4:1 ratio. The gel was heated at 95℃ for 10 minutes, followed by electrophoresis at 150V for 60 minutes, staining, and destaining. The average density of the samples was analyzed using Tanon Image software. The protein loading amount was 1μg, and the average density of the bands in samples treated with 0h alkali was taken as 100%. The relative average density of samples treated for different alkali times was calculated. Figure 2 shows the SDS-PAGE band images of some representative variants to demonstrate alkali resistance.

[0327] According to the test results, the B domain of wild-type protein A was completely degraded after treatment with 1M NaOH at 25°C for 72 hours, and the residual average density of the protein bands detected by SDS-PAGE was 0%. The residual average density of the reference sequence under the same treatment conditions was 29.8%, while the experimental results of the variants of this invention are shown in Table 2.3. The residual average density of the bands of the variants shown in Table 2.3 is greater than that of the reference sequence. Particularly noteworthy are the variants 3, 14, 43, 44, 57, 58, 64, 68, 71, 73, 75, 77, 80, 81, 82, 83, 84, 87, 88, 89, 90, 92, 93, 97, 99, 100, 101, 102, 104, 106, 107, and 108, which exhibited an alkali resistance greater than 70% after 72 hours, significantly higher than the reference sequence. This indicates that the alkali resistance of the variant protein molecules screened by this invention is significantly superior to that of the reference sequence.

[0328] Table 2.3: SDS-PAGE band density and affinity determination of variants with improved alkali resistance

[0329] The effect of the 2.5N-terminal GAQGT leader sequence and the terminal C-cysteine ​​residue on the affinity and alkali resistance of variant IgG.

[0330] A variant tetramer without the N-terminal GAQGT leader sequence and a single C-terminal cysteine ​​residue was constructed and named the truncated group. A variant tetramer containing the N-terminal leader sequence and a single C-terminal cysteine ​​residue was named the original sequence group. The EC50 value and 72-hour residual mean density percentage of the truncated group were detected using the ELISA method in section 2.3 and the SDS-PAGE method in section 2.4. The effects of the N-terminal GAQGT leader sequence and the single C-terminal cysteine ​​residue on the binding affinity and alkali resistance of the constructed variant IgG were evaluated.

[0331] The experimental results are shown in Table 2.4. Compared with the original sequence group, there was no significant difference in the EC50 value and the percentage of the average remaining density after 72 hours in the truncated group. Therefore, removing the N-terminal GAQGT leader sequence and the single cysteine ​​residue at the C-terminus does not affect the ability of the variant tetramer to bind IgG and its alkali resistance.

[0332] Table 2.4: Results of determination of affinity and alkali resistance of truncated tetramer IgG variants

[0333] Example 3: SPR technology for detecting the alkali resistance of protein A B domain variants

[0334] In Example 2, a subset of protein variants with high residual average density of protein bands in SDS-PAGE analysis after alkali treatment were selected, and their tolerance to high concentrations of NaOH in the mobile phase was further tested and verified using SPR technology.

[0335] The alkali treatment process is a coupling surface contact treatment, the concentration of sodium hydroxide in the alkali solution is 0.5M, the treatment temperature is constant at 20℃, and the treatment time is 24h-32h.

[0336] The instrument used in this assay was a Biacore 8K, with a CM5 chip (from Cytiva). Wild-type protein A's B domain or its variant tetramer was used as the stationary phase, and human immunoglobulin G (Human IgG, from Yisheng Biotechnology) was used as the mobile phase. The affinity of wild-type protein A's B domain or its variant for human IgG was detected. Enrichment tests were performed on these proteins at different pH values ​​(pH 4.0, pH 4.5, pH 5.0, pH 5.5) in 10mM sodium acetate buffer to determine the optimal coupling pH for each protein.

[0337] The alkali resistance of the B domain of wild-type protein A or its variants is characterized by binding signals. Wild-type or variant molecules are immobilized on the sensor surface, and the surface of the immobilized wild-type or variant molecules is treated with an alkaline solution. The change in the antibody binding signal before and after alkali treatment is detected, thus reflecting the alkali resistance of the wild-type or variant molecules. Specifically, 1 mg / ml of human IgG is injected before and after alkali treatment, and the binding signal is recorded. The percentage of remaining binding signal after alkali treatment is calculated with the binding signal at 0 h as 100%. The binding capacity of the variant relative to the reference sequence with IgG after 24 h is calculated as: (Percentage of remaining binding signal of the variant with IgG after 24 h) / (Percentage of remaining binding signal of the reference sequence with IgG after 24 h).

[0338] The experimental results are shown in Table 3. The B domain variant of protein A tested and verified showed that its remaining binding capacity after alkaline treatment was comparable to or better than that of the reference sequence.

[0339] Table 3: Residual binding capacity of alkali-resistant variants as detected by Biacore

[0340] * Experimental data from a reference sequence were used as the standard for comparison; the reference sequence value was 1.0.

[0341] Example 4: Alkali resistance of B-domain variants of protein A

[0342] The alkali resistance of the affinity media formed by coupling some variants obtained from multiple rounds of screening with agarose-based spheres was evaluated according to the alkali resistance evaluation method described in 2.1.

[0343] Tetramers of mutant proteins and reference sequence proteins were coupled to agarose-based spheres under similar ligand density conditions to form affinity media. The dynamic binding capacity (DBC) of protein A in the affinity media for the model protein was determined using the model protein (normal human IgG - concentration 5 mg / ml - Biotech). This capacity was the initial binding capacity. The binding capacity of the coupled affinity media for the model protein was measured every 24 hours in a 0.5 M NaOH environment at laboratory temperature (22±2℃) for a cumulative incubation of 120 hours, and the final remaining binding capacity was calculated. The results are shown in Table 4.

[0344] As shown in Table 4, the remaining binding load of the test variants on the model protein was better than that of the reference sequence after 120 hours of alkali treatment.

[0345] Table 4: Evaluation of Alkali Resistance of Multiple Optimized Molecules

[0346] Table 5 Sequence Information

[0347] The sequences involved in this application are described in the table below.

Claims

1. An Fc-binding polypeptide, wherein, compared with the amino acid sequence shown in SEQ ID NO:110, the polypeptide contains amino acid substitution mutations selected from one or more of the following positions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10): position 1, position 2, position 4, position 5, position 6, position 7, position 8, position 10, position 11, position 13, position 15, position 16, position 17, position 18, position 21, position 24, position 25, position 28, position 31, position 33, position 36, position 40, position 42, position 49, position 53, position 57; Preferably, the polypeptide comprises amino acid substitution mutations selected from one or more of the following positions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10): (i) The amino acid at position 1 is mutated and replaced with leucine (1L); (ii) The amino acid at position 2 is mutated to glutamic acid (2E); (iii) The amino acid at position 4 is mutated to glutamic acid (4E); (iv) The amino acid at position 5 is mutated and replaced with leucine (5L); (v) The amino acid at position 6 is mutated to threonine (6T); (vi) The amino acid at position 7 is mutated to glutamic acid (7E) or arginine (7R); (vii) The amino acid at position 8 is mutated and replaced with alanine (8A); (viii) The amino acid at position 10 is mutated and replaced with lysine (10K); (ix) The amino acid at position 11 is mutated to aspartic acid (11D); (x) The amino acid at position 13 is mutated and replaced with leucine (13L); (xi) The amino acid at position 15 is substituted and mutated to lysine (15K) or alanine (15A); (xii) The amino acid at position 16 is mutated and replaced with leucine (16L); (xiii) The amino acid at position 17 is mutated and replaced with methionine (17M); (xiv) describes the amino acid at position 18 being mutated to lysine (18K); (xv) The amino acid at position 21 is mutated and replaced with histidine (21H); (xvi) The amino acid at position 24 is mutated to aspartic acid (24D); (xvii) The amino acid at position 25 is mutated to alanine (25A); (xviii) The amino acid at position 28 is mutated to alanine (28A) or glutamine (28Q); (xix) The amino acid at position 31 is mutated and replaced with leucine (31L); (xx) The amino acid at position 33 is substituted and mutated to lysine (33K) or threonine (33T); (xxi) The amino acid at position 36 is mutated to glutamic acid (36E); (xxii) The amino acid at position 40 is substituted and mutated to lysine (40K) or alanine (40A); (xxiii) The amino acid at position 42 is mutated and replaced with alanine (42A); (xxiv) The amino acid at position 49 was mutated to glutamine (49Q); The amino acid at position 53 in (xxv) is mutated to alanine (53A); The amino acid at position 57 in (xxvi) is mutated and replaced with lysine (57K); wherein Each mutated amino acid site is a natural sequence site relative to the amino acid sequence shown in SEQ ID NO:

110.

2. The polypeptide of claim 1, wherein, Compared with the amino acid sequence shown in SEQ ID NO:110, the polypeptide is selected from any of the following: (1) The polypeptide contains an amino acid substitution mutation selected from any of the following positions: position 1, position 2, position 4, position 5, position 6, position 7, position 8, position 10, position 11, position 13, position 15, position 16, position 17, position 18, position 21, position 24, position 25, position 28, position 33, position 36, position 40, position 42, position 49, position 53, position 57; Preferably, the polypeptide contains an amino acid substitution mutation selected from any of the following positions: 1L, 2E, 4E, 5L, 6T, 7E or 7R, 8A, 10K, 11D, 13L, 15K or 15A, 16L, 17M, 18K, 21H, 24D, 25A, 28A or 28Q, 33K or 33T, 36E, 40K or 40A, 42A, 49Q, 53A, 57K; (2) The polypeptide contains an amino acid substitution mutation selected from any two of the following positions: position 7, position 8, position 11, position 15, position 21, position 28, position 33, position 36, position 53; Preferably, the polypeptide contains an amino acid substitution mutation selected from any two of the following positions: 7R, 8A, 11D, 15K, 21H, 28A or 28Q, 33K, 36E, 53A; (3) The polypeptide contains an amino acid substitution mutation selected from any three of the following positions: position 11, position 13, position 15, position 18, position 21, position 28, position 33, position 49, and position 53; Preferably, the polypeptide contains an amino acid substitution mutation selected from any three of the following positions: 11D, 13L, 15K or 15A, 18K, 21H, 28A or 28Q, 33K, 49Q, 53A; (4) The polypeptide contains amino acid substitution mutations selected from any four of the following positions: position 5, position 7, position 11, position 13, position 15, position 18, position 21, position 25, position 28, position 33, position 40, position 42, position 49, position 53, and position 57. Preferably, the polypeptide contains an amino acid substitution mutation selected from any four of the following positions: 5L, 7R, 11D, 13L, 15K, 18K, 21H, 25A, 28Q, 33K, 40K or 40A, 42A, 49Q, 53A, 57K. (5) The polypeptide contains an amino acid substitution mutation selected from any five of the following positions: position 2, position 5, position 7, position 10, position 15, position 17, position 21, position 24, position 28, position 31, position 33, position 40, position 42, position 49, position 53, and position 57. Preferably, the polypeptide contains an amino acid substitution mutation selected from any five of the following positions: 2E, 5L, 7E, 10K, 15K, 17M, 21H, 24D, 28Q, 31L, 33K, 40K or 40A, 42A, 49Q, 53A, 57K. (6) The polypeptide contains an amino acid substitution mutation selected from any six of the following positions: position 2, position 5, position 10, position 15, position 21, position 24, position 33, position 40, position 42, position 49, and position 53. Preferably, the polypeptide contains amino acid substitution mutations selected from any six of the following positions: 2E, 5L, 10K, 15K, 21H, 24D, 33K, 40A, 42A, 49Q, 53A; (7) The polypeptide contains an amino acid substitution mutation selected from any seven of the following positions: position 5, position 10, position 15, position 21, position 24, position 33, position 40, position 42, position 49, and position 53; Preferably, the polypeptide comprises an amino acid substitution mutation selected from any seven of the following positions: 5L, 10K, 15K, 21H, 24D, 33K, 40A, 42A, 49Q, 53A; The mutated amino acid sites are the natural sequence sites relative to the amino acid sequence shown in SEQ ID NO:

110.

3. The polypeptide of claim 1 or 2, wherein, Compared to the amino acid sequence shown in SEQ ID NO:110, the polypeptide comprises: (1) There is an amino acid substitution mutation at least at position 21; preferably, the amino acid at position 21 is substituted with histidine (21H); And optional location, (2) The polypeptide further comprises amino acid substitution mutations selected from one or more of the following positions (e.g., 1, 2, 3, 4, 5 or 6): position 2, position 5, position 7, position 8, position 10, position 11, position 13, position 15, position 17, position 18, position 24, position 25, position 28, position 31, position 33, position 36, position 40, position 42, position 49, position 53, position 57; Preferably, the polypeptide further comprises an amino acid substitution mutation selected from one or more of the following positions (e.g., 1, 2, 3, 4, 5 or 6): 2E, 5L, 7E or 7R, 8A, 10K, 11D, 13L, 15K or 15A, 17M, 18K, 24D, 25A, 28A or 28Q, 31L, 33K, 36E, 40K or 40A, 42A, 49Q, 53A, 57K; The mutated amino acid sites are the natural sequence sites relative to the amino acid sequence shown in SEQ ID NO:

110.

4. The polypeptide of claim 3, wherein, Compared to the amino acid sequence shown in SEQ ID NO:110, the polypeptide comprises: (1) It has an amino acid substitution mutation at at least position 21; preferably, the amino acid at position 21 is substituted with histidine (21H); and (2) The polypeptide further comprises any one of the following: (i) The polypeptide further comprises an amino acid substitution mutation selected from any of the following positions: position 7, position 8, position 11, position 15, position 28, position 33, position 36, position 53; preferably, the polypeptide further comprises an amino acid substitution mutation selected from any of the following positions: 7R, 8A, 11D, 15K, 28A or 28Q, 33K, 36E, 53A; (ii) The polypeptide further comprises an amino acid substitution mutation selected from any two of the following positions: position 11, position 13, position 15, position 28, position 33, position 49, position 53; preferably, the polypeptide further comprises an amino acid substitution mutation selected from any two of the following positions: 11D, 13L, 15K or 15A, 28A or 28Q, 33K, 49Q, 53A; (iii) The polypeptide further comprises an amino acid substitution mutation selected from any three of the following positions: position 5, position 7, position 11, position 13, position 15, position 18, position 25, position 28, position 33, position 40, position 42, position 49, position 53, and position 57; preferably, the polypeptide further comprises an amino acid substitution mutation selected from any three of the following positions: 5L, 7R, 11D, 13L, 15K, 18K, 25A, 28Q, 33K, 40K or 40A, 42A, 49Q, 53A, and 57K; (iv) The polypeptide further comprises an amino acid substitution mutation selected from any four of the following positions: position 2, position 5, position 7, position 10, position 15, position 17, position 24, position 28, position 31, position 33, position 40, position 42, position 49, position 53, and position 57; preferably, the polypeptide further comprises an amino acid substitution mutation selected from any four of the following positions: 2E, 5L, 7E, 10K, 15K, 17M, 24D, 28Q, 31L, 33K, 40K or 40A, 42A, 49Q, 53A, and 57K; (v) The polypeptide further comprises an amino acid substitution mutation selected from any five of the following positions: position 2, position 5, position 10, position 15, position 24, position 33, position 40, position 42, position 49, and position 53; preferably, the polypeptide further comprises an amino acid substitution mutation selected from any five of the following positions: 2E, 5L, 10K, 15K, 24D, 33K, 40A, 42A, 49Q, and 53A; (vi) The polypeptide further comprises an amino acid substitution mutation selected from any six of the following positions: position 5, position 10, position 15, position 24, position 33, position 40, position 42, position 49, and position 53; preferably, the polypeptide further comprises an amino acid substitution mutation selected from any six of the following positions: 5L, 10K, 15K, 24D, 33K, 40A, 42A, 49Q, and 53A; The mutated amino acid sites are the natural sequence sites relative to the amino acid sequence shown in SEQ ID NO:

110.

5. The polypeptide of any one of claims 1-4, wherein, Compared to the amino acid sequence shown in SEQ ID NO:110, the polypeptide comprises: (1) It has an amino acid substitution mutation at at least position 21; preferably, the amino acid at position 21 is substituted with histidine (21H); and (2) There is an amino acid substitution mutation at least at position 15; preferably, the amino acid at position 15 is substituted with lysine (15K); And optional location, (3) The polypeptide further comprises amino acid substitution mutations selected from one or more of the following positions (e.g., 1, 2, 3, 4 or 5): position 2, position 5, position 7, position 10, position 11, position 13, position 15, position 17, position 18, position 24, position 25, position 28, position 31, position 33, position 40, position 42, position 49, position 53, position 57; Preferably, the polypeptide further comprises amino acid substitution mutations selected from one or more of the following positions (e.g., 2, 3, 4 or 5): 2E, 5L, 7E or 7R, 10K, 11D, 13L, 15K, 17M, 18K, 24D, 25A, 28Q or 28A, 31L, 33K, 40K or 40A, 42A, 49Q, 53A, 57K; The mutated amino acid sites are the natural sequence sites relative to the amino acid sequence shown in SEQ ID NO:

110.

6. The polypeptide of claim 5, wherein, Compared to the amino acid sequence shown in SEQ ID NO:110, the polypeptide comprises: (1) There is an amino acid substitution mutation at least at position 21; preferably, the amino acid at position 21 is substituted with histidine (21H); (2) It has an amino acid substitution mutation at at least position 15; preferably, the amino acid at position 15 is substituted with lysine (15K); and (3) The polypeptide further comprises any one of the following: (i) The polypeptide further comprises an amino acid substitution mutation selected from any of the following positions: position 11, position 13, position 28, position 49, position 53; preferably, the polypeptide further comprises an amino acid substitution mutation selected from any of the following positions: 11D, 13L, 28A, 49Q, 53A; (ii) The polypeptide further comprises an amino acid substitution mutation selected from any two of the following positions: position 5, position 7, position 11, position 13, position 18, position 25, position 28, position 33, position 40, position 42, position 49, position 53; preferably, the polypeptide further comprises an amino acid substitution mutation selected from any two of the following positions: 5L, 7R, 11D, 13L, 18K, 25A, 28Q, 33K, 40K or 40A, 42A, 49Q, 53A; (iii) The polypeptide further comprises an amino acid substitution mutation selected from any three of the following positions: position 2, position 5, position 7, position 10, position 17, position 24, position 28, position 31, position 33, position 40, position 42, position 49, position 53, and position 57; preferably, the polypeptide further comprises an amino acid substitution mutation selected from any three of the following positions: 2E, 5L, 7E, 10K, 17M, 24D, 28Q, 31L, 33K, 40K or 40A, 42A, 49Q, 53A, and 57K; (iv) The polypeptide further comprises an amino acid substitution mutation selected from any four of the following positions: position 2, position 5, position 10, position 24, position 33, position 40, position 42, position 49, and position 53; preferably, the polypeptide further comprises an amino acid substitution mutation selected from any four of the following positions: 2E, 5L, 10K, 24D, 33K, 40A, 42A, 49Q, and 53A; (iv) The polypeptide further comprises an amino acid substitution mutation selected from any five of the following positions: position 5, position 10, position 24, position 33, position 40, position 42, position 49, and position 53; preferably, the polypeptide further comprises an amino acid substitution mutation selected from any four of the following positions: 5L, 10K, 24D, 33K, 40A, 42A, 49Q, and 53A; The mutated amino acid sites are the natural sequence sites relative to the amino acid sequence shown in SEQ ID NO:

110.

7. The polypeptide of any one of claims 1-6, wherein, Compared to the amino acid sequence shown in SEQ ID NO:110, the polypeptide comprises: (1) There is an amino acid substitution mutation at least at position 21; preferably, the amino acid at position 21 is substituted with histidine (21H); (2) It has an amino acid substitution mutation at at least position 15; preferably, the amino acid at position 15 is substituted with lysine (15K); and (3) There is an amino acid substitution mutation at least at position 49; preferably, the amino acid at position 49 is substituted with glutamine (49Q); And optional location, (4) The polypeptide further comprises an amino acid substitution mutation selected from one or more of the following positions (e.g., 1, 2, 3 or 4): position 5, position 10, position 24, position 33, position 40, position 42, position 53; Preferably, the polypeptide further comprises amino acid substitution mutations selected from one or more of the following positions (e.g., 2, 3 or 4): 5L, 10K, 24D, 33K, 40A, 42A, 53A; The mutated amino acid sites are the natural sequence sites relative to the amino acid sequence shown in SEQ ID NO:

110.

8. The polypeptide of claim 7, wherein, Compared to the amino acid sequence shown in SEQ ID NO:110, the polypeptide comprises: (1) There is an amino acid substitution mutation at least at position 21; preferably, the amino acid at position 21 is substituted with histidine (21H); (2) There is an amino acid substitution mutation at least at position 15; preferably, the amino acid at position 15 is substituted with lysine (15K); (3) It has an amino acid substitution mutation at least at position 49; preferably, the amino acid at position 49 is mutated to glutamine (49Q); and (4) The polypeptide further comprises any one of the following: (i) The polypeptide further comprises an amino acid substitution mutation selected from any of the following positions: position 33, position 40; preferably, the polypeptide further comprises an amino acid substitution mutation selected from any of the following positions: 33K, 40A; (ii) The polypeptide further comprises an amino acid substitution mutation selected from any two of the following positions: position 33 and position 53; preferably, the polypeptide further comprises an amino acid substitution mutation selected from any two of the following positions: 33K and 53A; (iii) The polypeptide further comprises an amino acid substitution mutation selected from any three of the following positions: position 10, position 33, position 42, and position 53; preferably, the polypeptide further comprises an amino acid substitution mutation selected from any three of the following positions: 10K, 33K, 42A, and 53A. (iv) The polypeptide further comprises an amino acid substitution mutation selected from any four of the following positions: position 5, position 10, position 24, position 33, position 40, and position 53; preferably, the polypeptide further comprises an amino acid substitution mutation selected from any four of the following positions: 5L, 10K, 24D, 33K, 40A, and 53A. The mutated amino acid sites are the natural sequence sites relative to the amino acid sequence shown in SEQ ID NO:

110.

9. The polypeptide of any one of claims 1-4, wherein, Compared to the amino acid sequence shown in SEQ ID NO:110, the polypeptide comprises: (1) There is an amino acid substitution mutation at least at position 21; preferably, the amino acid at position 21 is substituted with histidine (21H); (2) It has an amino acid substitution mutation at at least position 33; preferably, the amino acid at position 33 is substituted with lysine (33K); and (3) There is an amino acid substitution mutation at least at position 53; preferably, the amino acid at position 53 is substituted with alanine (53A); And optional location, (4) The polypeptide further comprises amino acid substitution mutations selected from one or more of the following positions (e.g., 1, 2, 3 or 4): position 2, position 5, position 7, position 10, position 15, position 17, position 24, position 28, position 31, position 33, position 40, position 42, position 49, position 53, position 57; Preferably, the polypeptide further comprises amino acid substitution mutations selected from one or more of the following positions (e.g., 2, 3 or 4): 2E, 5L, 7E, 10K, 15K, 17M, 24D, 28Q, 31L, 33K, 40K or 40A, 42A, 49Q, 53A, 57K. The mutated amino acid sites are the natural sequence sites relative to the amino acid sequence shown in SEQ ID NO:

110.

10. The polypeptide of claim 9, wherein, Compared to the amino acid sequence shown in SEQ ID NO:110, the polypeptide comprises: (1) There is an amino acid substitution mutation at least at position 21; preferably, the amino acid at position 21 is substituted with histidine (21H); (2) There is an amino acid substitution mutation at least at position 33; preferably, the amino acid at position 33 is substituted with lysine (33K); (3) It has an amino acid substitution mutation at at least position 53; preferably, the amino acid at position 53 is mutated to alanine (53A); and (4) The polypeptide further comprises any one of the following: (i) The polypeptide further comprises an amino acid substitution mutation selected from any of the following positions: position 15, position 28, position 40, position 49, position 57; preferably, the polypeptide further comprises an amino acid substitution mutation selected from any of the following positions: 15K, 28Q, 40K, 49Q, 57K; (ii) The polypeptide further comprises an amino acid substitution mutation selected from any two of the following positions: position 2, position 5, position 7, position 10, position 15, position 17, position 24, position 28, position 31, position 40, position 42, position 49, position 57; preferably, the polypeptide further comprises an amino acid substitution mutation selected from any two of the following positions: 2E, 5L, 7E, 10K, 15K, 17M, 24D, 28Q, 31L, 40K or 40A, 42A, 49Q, 57K; (iii) The polypeptide further comprises an amino acid substitution mutation selected from any three of the following positions: position 2, position 5, position 10, position 15, position 24, position 40, position 42, and position 49; preferably, the polypeptide further comprises an amino acid substitution mutation selected from any three of the following positions: 2E, 5L, 10K, 15K, 24D, 40A, 42A, and 49Q; (iv) The polypeptide further comprises an amino acid substitution mutation selected from any four of the following positions: position 5, position 10, position 15, position 24, position 40, position 42, and position 49; preferably, the polypeptide further comprises an amino acid substitution mutation selected from any four of the following positions: 5L, 10K, 15K, 24D, 40A, 42A, and 49Q; The mutated amino acid sites are the natural sequence sites relative to the amino acid sequence shown in SEQ ID NO:

110.

11. The polypeptide of any one of claims 1-6, 9-10, wherein, Compared to the amino acid sequence shown in SEQ ID NO:110, the polypeptide comprises: (1) There is an amino acid substitution mutation at least at position 21; preferably, the amino acid at position 21 is substituted with histidine (21H); (2) There is an amino acid substitution mutation at least at position 33; preferably, the amino acid at position 33 is substituted with lysine (33K); (3) It has an amino acid substitution mutation at at least position 53; preferably, the amino acid at position 53 is mutated to alanine (53A); and (4) There is an amino acid substitution mutation at least at position 15; preferably, the amino acid at position 15 is substituted with lysine (15K); And optional location, (5) The polypeptide further comprises amino acid substitution mutations selected from one or more of the following positions (e.g., 2 or 3): position 2, position 5, position 7, position 10, position 15, position 17, position 24, position 28, position 31, position 33, position 40, position 42, position 49, position 53, position 57; Preferably, the polypeptide further comprises an amino acid substitution mutation selected from one or more of the following positions (e.g., 2 or 3): 2E, 5L, 7E, 10K, 15K, 17M, 24D, 28Q, 31L, 33K, 40A or 40K, 42A, 49Q, 53A, 57K. The mutated amino acid sites are the natural sequence sites relative to the amino acid sequence shown in SEQ ID NO:

110.

12. The polypeptide of claim 11, wherein, Compared to the amino acid sequence shown in SEQ ID NO:110, the polypeptide comprises: (1) There is an amino acid substitution mutation at least at position 21; preferably, the amino acid at position 21 is substituted with histidine (21H); (2) There is an amino acid substitution mutation at least at position 33; preferably, the amino acid at position 33 is substituted with lysine (33K); (3) There is an amino acid substitution mutation at least at position 53; preferably, the amino acid at position 53 is substituted with alanine (53A); (4) It has an amino acid substitution mutation at at least position 15; preferably, the amino acid at position 15 is substituted with lysine (15K); and (5) The polypeptide further comprises any one of the following: (i) The polypeptide further comprises an amino acid substitution mutation selected from any of the following positions: position 2, position 5, position 7, position 10, position 17, position 24, position 28, position 31, position 40, position 42A, position 49Q, position 57; preferably, the polypeptide further comprises an amino acid substitution mutation selected from any of the following positions: 2E, 5L, 7E, 10K, 17M, 24D, 28Q, 31L, 40A or 40K, 42A, 49Q, 57K; (ii) The polypeptide further comprises an amino acid substitution mutation selected from any two of the following positions: position 2, position 5, position 10, position 24, position 40, position 42, position 49; preferably, the polypeptide further comprises an amino acid substitution mutation selected from any two of the following positions: 2E, 5L, 10K, 24D, 40A, 42A, 49Q; (iii) The polypeptide further comprises an amino acid substitution mutation selected from any three of the following positions: position 5, position 10, position 24, position 40, position 42, and position 49; preferably, the polypeptide further comprises an amino acid substitution mutation selected from any three of the following positions: 5L, 10K, 24D, 40A, 42A, and 49Q. The mutated amino acid sites are the natural sequence sites relative to the amino acid sequence shown in SEQ ID NO:

110.

13. The polypeptide of any one of claims 1-12, wherein, compared with the amino acid sequence shown in SEQ ID NO:110, the polypeptide comprises an amino acid substitution mutation selected from any combination of the following positions: 21, 33, 21, 53, 21, 28, 21, 8, 21, 11, 21, 36, 21, 7, 21, 15, 21, 15, 53, 15, 18, 33, 21, 15, 28, 15, 28, 33, 21, 13, 15, 21, 11, 15, 15, 28, 53, 21, 33, 53, 21, 15, 49, 21, 28, 33, 21, 28, 53, 21, 15, 28, 21, 13, 33, 21, 13, 53, 21, 13, 28, 21, 5, 15, 53, 21, 11, 15, 53, 21, 15, 33, 49, 21, 13, 15, 28, 21, 15, 40, 49, 21, 7, 15, 33, 21, 15, 18, 53, 21, 15, 28, 42, 21, 13, 15, 33, 21, 13, 15, 53, 21, 15, 25, 53, 21, 15, 40, 53, 21, 11, 13, 15, 21, 13, 28, 53, 21, 15, 28, 33, 21, 15, 28, 53, 21, 15, 33, 53, 21, 28, 33, 53, 21, 15, 28, 33, 53, 21, 10, 15, 33, 53, 21, 15, 33, 42, 53, 21, 5, 15, 33, 53, 21, 15, 24, 33, 53, 21, 2, 15, 33, 53, 21, 15, 17, 33, 53, 21, 15, 33, 49, 53, 21, 15, 33, 40, 53, 21, 10, 15, 40, 53, 21, 15, 33, 53, 57, 21, 15, 33, 40, 53, 21, 15, 31, 33, 53, 21, 7, 15, 33, 53, 21, 10, 15, 33, 42, 53, 21, 5, 10, 15, 33, 53, 21, 5, 15, 33, 42, 53, 21, 10, 15, 24, 33, 53, 21, 10, 15, 33,10, 15, 33, 40, 49, 53, 21, 10, 15, 24, 33, 40, 53, 21, 10, 15, 24, 33, 49, 53, 21, 33, 49, 53, 21, 33, 53, 57, 21, 33, 40, 53, 21, 5, 33, 42, 53, 21, 24, 33, 42, 53, or 21, 2, 33, 42, 53; Preferably, the polypeptide comprises an amino acid substitution mutation selected from any one of the following combinations compared to the amino acid sequence set forth in SEQ ID NO: 110: N21H; S33K, N21H; D53A, N21H; N28Q, N21H; E8A, N21H; N28A, N21H; E11D, N21H; D36E, N21H; K7R, N21H; E15K, N21H; E15K; D53A, E15K; H18K; S33K, N21H; E15K; N28A, E15K; N28Q; S33K, N21H; F13L; E15K, N21H; E11D; E15K, E15K; N28Q; D53A, N21H; S33K; D53A, N21H; E15K; K49Q, N21H; N28Q; S33K, N21H; N28Q; D53A, N21H; E15A; N28Q, N21H; F13L; S33K, N21H; F13L; D53A, N21H; F13L; N28Q, N21H; F5L; E15K; D53A, N21H; E11D; E15K; D53A, N21H; E15K; S33K; K49Q, N21H; F13L; E15K; N28Q, N21H; E15K; V40A; K49Q, N21H; K7R; E15K; S33K, N21H; E15K; H18K; D53A, N21H; E15K; N28Q; K42A, N21H; F13L; E15K; S33K, N21H; F13L; E15K; D53A, N21H; E15K; E25A; D53A, N21H; E15K; V40K; D53A, N21H; E11D; F13L; E15K, N21H; F13L; N28Q; D53A, N21H; E15K; N28Q; S33K, N21H; E15K; N28Q; D53A, N21H; E15K; S33K; D53A, N21H; N28Q; S33K; D53A, N21H; E15K; N28Q; S33K; D53A, N21H; Q10K; E15K; S33K; D53A, N21H; E15K; S33K; K42A; D53A, N21H; F5L; E15K; S33K; D53A, N21H; E15P57K, N21H; E15K; S33K; V40K; D53A, N21H; E15K; I31L; S33K; D53A, N21H; K7E; E15K; S33K; D53A, N21H; Q10K; E15K; S33K; K42A; D53A, N21H; F5L; Q10K; E15K; S33K; D53A, N21H; F5L; E15K; S33K; K42A; D53A, N21H; Q10K; E15K; E24D; S33K; D53A, N21H; Q10K; E15K; S33K; V40A; D53A, N21H; Q10K; E15K; S33K; K49Q; D53A, N21H; E15K; E24D; S33K; K42A; D53A, N21H; F5L; E15K; E24D; S33K; D53A, N21H; D2E; E15K; S33K; K42A; D53A, N21H; E15K; S33K; K42A; K49Q; D53A, N21H; F5L; Q10K; E15K; S33K; K42A; D53A, N21H; Q10K; E15K; E24D; S33K; K42A; D53A, N21H; F5L; Q10K; E15K; E24D; S33K; D53A, N21H; F5L; Q10K; E15K; S33K; K49Q; D53A, N21H; Q10K; E15K; S33K; V40A; K49Q; D53A, N21H; Q10K; E15K; E24D; S33K; V40A; D53A, N21H; Q10K; E15K; E24D; S33K; K49Q; D53A, N21H; S33K; K49Q; D53A, N21H; S33K; D53A; P57K, N21H; S33K; V40K; D53A, N21H; F5L; S33K; K42A; D53A, N21H; E24D; S33K; K42A; D53A, or N21H; D2E; S33K; K42A; D53A.

14. The polypeptide according to any one of claims 1-13, compared with the amino acid sequence shown in SEQ ID NO:110, contains only the mutation described in any one of claims 1-13.

15. The polypeptide of any one of claims 1-14, comprising an amino acid sequence as shown in any one of SEQ ID NOs:1-109, or an amino acid sequence having at least 90%, for example at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs:1-109.

16. A polymer comprising one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9) amino acid sequences formed by the fusion of any one of the polypeptides according to claims 1 to 15; wherein The one or more polypeptides (e.g., 2, 3, 4, 5, 6, 7, 8, 9) are completely identical, partially identical, or completely different from each other; The polypeptides are optionally linked together by peptide linkers.

17. The polymer of claim 16 is a dimer, trimer, tetramer, pentamer, hexamer, heptametamer, octamer, or nonamer; preferably, the polymer is a tetramer.

18. The polymer of claim 16 or 17, comprising an amino acid sequence as shown in any one of SEQ ID NOs: 112-220, or an amino acid sequence having at least 90%, for example at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 112-220.

19. The polypeptide of any one of claims 1-15 or the polymer of any one of claims 16-18, further comprising being linked to one or more coupling elements at a C-terminus or N-terminus; Preferably, the coupling element is selected from one or more cysteine ​​residues, multiple lysine residues, and multiple histidine residues.

20. An isolated polypeptide construct comprising, The polypeptide construct includes: (1) The polypeptide of any one of claims 1-15 or the polymer of any one of claims 16-18; (2) One or more coupling elements located at the C-terminus of the polypeptide or polymer described in (1); and (3) The leader sequence located at the N-terminus of the polypeptide or polymer described in (1); Preferably, the leader sequence is selected from one or more of the following fragments: GAQGT, MGAQGT, AQ, AQGT, MAQGT, VDAKFDKE, AQVDAKFDKE, or AQGTVDAKFDKE; Preferably, the coupling element is selected from: one or more cysteine ​​residues, multiple lysine residues, and multiple histidine residues; Preferably, one or more coupling elements at the C-terminus are a single cysteine ​​residue, and the N-terminal leader sequence is GAQGT; optionally, the leader sequence contains amino acid modifications, such as a modification on the first amino acid G (e.g., glucose acetylation).

21. The polypeptide construct of claim 20, wherein, The construct comprises, from N-terminus to C-terminus, (1) GAQGT; (2) an amino acid sequence as shown in any one of SEQ ID NOs: 1-220; and (3) a single cysteine ​​(C).

22. An isolated nucleic acid molecule encoding a polypeptide as described in any one of claims 1-15, a polymer as described in any one of claims 16-19, or a polypeptide construct as described in any one of claims 20-21.

23. A vector comprising the isolated nucleic acid molecule of claim 22.

24. A host cell comprising the isolated nucleic acid molecule of claim 22 or the vector of claim 23.

25. A method for preparing a polypeptide of any one of claims 1-15, a polymer of any one of claims 16-19, or a polypeptide construct of any one of claims 20-21, comprising culturing a host cell of claim 24 under conditions allowing expression of the polypeptide or the polymer, and recovering the polypeptide, the polymer, or the polypeptide construct from the cultured host cell culture.

26. A separation matrix comprising a polypeptide of any one of claims 1-15, a polymer of any one of claims 16-19, or a polypeptide construct of any one of claims 20-21 coupled to a solid support; Preferably, the polypeptide, polymer, or polypeptide construct is coupled to the solid support via a thioether bond; Preferably, the solid support is selected from polysaccharides, such as dextran, starch, cellulose, pullulan, agar, or agarose.

27. The use of the polypeptide of any one of claims 1-15, the polymer of any one of claims 16-19, or the polypeptide construct of any one of claims 20-21 in the preparation of the separation matrix of claim 26.

28. A method for isolating a protein containing Fc, the method comprising the steps of: contacting the protein to be purified with a polypeptide of any one of claims 1-15, a polymer of any one of claims 16-19, a polypeptide construct of any one of claims 20-21, or a separation matrix of claim 26; Preferably, the protein containing Fc is an immunoglobulin, such as IgG; Preferably, the IgG is selected from IgG1, IgG2, IgG3, and IgG4.

29. The use of the polypeptide of any one of claims 1-15, the polymer of any one of claims 16-19, the polypeptide construct of any one of claims 20-21, and the separation matrix of claim 26 in the separation of proteins containing Fc.

Images