WO2023143525A1 - 蛋白a的b结构域和z结构域突变体及其应用 - Google Patents

蛋白a的b结构域和z结构域突变体及其应用 Download PDF

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WO2023143525A1
WO2023143525A1 PCT/CN2023/073587 CN2023073587W WO2023143525A1 WO 2023143525 A1 WO2023143525 A1 WO 2023143525A1 CN 2023073587 W CN2023073587 W CN 2023073587W WO 2023143525 A1 WO2023143525 A1 WO 2023143525A1
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substitution mutation
seq
polypeptide
positions
protein
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PCT/CN2023/073587
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English (en)
French (fr)
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张洪
石海涛
张艳
胡慧霞
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博格隆(浙江)生物技术有限公司
博格隆(上海)生物技术有限公司
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Publication of WO2023143525A1 publication Critical patent/WO2023143525A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
    • B01D15/3804—Affinity chromatography
    • B01D15/3809—Affinity chromatography of the antigen-antibody type, e.g. protein A, G, L chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to B domain and Z domain mutants of protein A and applications thereof.
  • Protein A affinity chromatography medium has become the most widely used method for purifying monoclonal antibodies due to its special adsorption capacity for the unique Fc segment of antibodies.
  • Protein A affinity chromatography media may have problems such as ligand shedding and low binding capacity during antibody purification.
  • Protein A is composed of highly homologous immunoglobulin binding domains E, D, A, B, and C, among which domain B has a greater advantage in binding specificity to immunoglobulin.
  • Domain Z is obtained by replacing alanine at position 1 with valine and glycine at position 29 with alanine in Protein A domain B. This mutation endows domain Z with more prominent chemical stability, and similarly, domain Z through domain amino acid mutations also has certain stability under alkaline conditions. However, it is still unstable at higher pH conditions and cannot meet the requirements of the CIP washing step in the antibody purification process.
  • the existing protein A chromatography medium with B domain or Z domain mutation has certain alkaline stability.
  • CN101522278A mutates glycine at position 29 on the B domain to a Amino acid, thus improving alkali resistance.
  • CN1642976A discloses that at least one asparagine residue on the Z domain is mutated to an amino acid other than alanine, threonine or aspartic acid.
  • CN105377881A discloses that at least 15 glutamic acid residues in the B domain or Z domain are mutated to amino acids other than asparagine or glutamine. These mutants have higher alkali resistance under alkaline conditions compared to the natural ligands.
  • the chromatography medium prepared by using the above ligands can withstand 0.1-0.5M sodium hydroxide washing during the antibody purification process, but there is still a certain gap from the standard CIP (0.5-1.0M sodium hydroxide).
  • the first aspect of the present invention provides an isolated polypeptide selected from the group consisting of:
  • polypeptide Compared with the natural B domain of protein A shown in SEQ ID NO: 1 or the Z domain of protein A shown in SEQ ID NO: 2, the polypeptide is selected from positions 3, 6, Substitution mutations at one or more positions at positions 9, 15 and 23;
  • the substitution mutation at the 3rd position is that asparagine is substituted with leucine, isoleucine, valine or tyrosine;
  • the substitution mutation at the 6th position is that asparagine is substituted is glutamic acid, leucine, isoleucine, valine, serine or threonine;
  • the substitution mutation at position 9 is that glutamine is replaced by isoleucine, leucine, valine amino acid, phenylalanine or methionine;
  • the substitution mutation at position 15 is that glutamic acid is replaced by threonine, tryptophan, leucine, valine, isoleucine, Phenylalanine, serine, tyrosine or aspartic acid;
  • the substitution mutation at position 23 is asparagine mutation to valine, isoleucine, leucine or tyrosine;
  • the substitution mutation at position 3 is asparagine replaced by leucine, valine or tyrosine.
  • the substitution mutation at position 6 is asparagine replaced by glutamic acid, leucine or serine.
  • the substitution mutation at position 9 is glutamine replaced by isoleucine, phenylalanine or methionine.
  • the substitution mutation at position 15 is that glutamic acid is substituted by Threonine, Tryptophan, Leucine, Isoleucine, Valine, Phenylalanine, Serine, or Tyrosine.
  • the substitution mutation at position 23 is asparagine to valine, isoleucine or tyrosine.
  • the polypeptide has the substitution mutation at least at position 3, and at least one position selected from position 15, position 6, position 9 and position 23, Said substitution mutations are present in at least 2 positions, at least 3 positions or all 4 positions.
  • the polypeptide has the substitution mutation at least at position 6, and at least one position selected from position 3, position 15, position 9 and position 23, Said substitution mutations are present in at least 2 positions, at least 3 positions or all 4 positions.
  • the polypeptide has the substitution mutation at least at position 9, and at least one position selected from position 3, position 6, position 15 and position 23, Said substitution mutations are present in at least 2 positions, at least 3 positions or all 4 positions.
  • the polypeptide has the substitution mutation at least at the 15th position, and at least one position selected from the 3rd, 6th, 9th and 23rd positions, Said substitution mutations are present in at least 2 positions, at least 3 positions or all 4 positions.
  • said polypeptide has said substitution mutation at least at position 23, and at least one position selected from said position 3, position 6, position 9 and position 15, Said substitution mutations are present in at least 2 positions, at least 3 positions or all 4 positions.
  • the substitution mutation at position 15 is E15I, E15L or E15V
  • the substitution mutation at position 3 is N3L or N3V
  • the substitution mutation at position 6 is N6E or N6L
  • the mutation at position 9 is N6E or N6L.
  • the substitution mutation is Q9I or Q9F
  • the substitution mutation at position 23 is N23I, N23Y or N23V.
  • the polypeptide has the substitution mutation at position 3, position 9 and position 15, and the substitution mutation at position 6 and/or position 23; preferably, The substitution mutation at the 3rd position is N3L or N3V, the substitution mutation at the 6th position is N6L or N6E, the substitution mutation at the 9th position is Q9I or Q9F, and the substitution mutation at the 15th position is E15L, E15I or E15V, the substitution mutation at position 23 is N23V, N23I or N23Y.
  • said polypeptide has said substitution mutations at positions 3, 6, 9 and 15.
  • the substitution mutation at position 3 is N3L or N3V, preferably N3V
  • the substitution mutation at the 6th position is N6L or N6E, preferably N6E
  • the substitution mutation at the 9th position is Q9I or Q9F, preferably Q9I
  • the substitution mutation at the 15th position is E15L, E15I or E15V, preferably E15L.
  • the amino acid sequence of the polypeptide is as shown in any of SEQ ID NO: 3-71 and 118-124.
  • the second aspect of the present invention provides an isolated polypeptide consisting of the following (i), (ii) and (iii): (i) the polypeptide described in any embodiment of the first aspect of the present invention, (ii) One or more coupling elements at (i) the C-terminus or N-terminus of the amino acid sequence of the polypeptide, and optionally (iii) residues from the signal transduction sequence which are excised.
  • the coupling element is selected from the group consisting of: a cysteine residue, a plurality of lysine residues and a plurality of histidine residues; the cleaved signal transduction sequence The residue is AQ.
  • the third aspect of the present invention provides a fusion protein, which is characterized in that the fusion protein comprises an amino acid sequence formed by fusion of 2-8 polypeptides according to any embodiment of the first aspect of the present invention, wherein the 2-8 The polypeptides are different, partially or completely identical to each other, and a linker sequence is optionally present between the polypeptides.
  • At least one polypeptide has the substitution mutation at the 15th position, optionally at the 3rd position, the 6th position A polypeptide having said substitution mutation at least one position, at least two positions, at least three positions or all four positions of position, position 9 and position 23; preferably, at least one polypeptide is at position 3, A polypeptide having said substitution mutation at position 9 and 15, and having said substitution mutation at position 6 and/or 23.
  • the polypeptide in the fusion protein, is selected from the group consisting of: SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 60, SEQ ID NO: 71, EQ ID NO: 64, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 68, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO One or more of: 121, SEQ ID NO: 122, SEQ ID NO: 123 and SEQ ID NO: 124.
  • the C-terminal or N-terminal of the fusion protein also includes one or more coupling elements and/or residues from excised signal transduction sequences; preferably, said coupling elements are selected from the group consisting of cysteine residues, lysine residues and histidine residues acid residues.
  • the amino acid sequence of the fusion protein is as shown in any of SEQ ID NO: 73-109 and 111-117.
  • the fourth aspect of the present invention provides a recombinant protein A, and the B domain of the recombinant protein A is the polypeptide described in any embodiment of the first aspect of the present invention.
  • the fifth aspect of the present invention provides an isolated nucleic acid molecule, the polynucleotide sequence of the nucleic acid molecule is selected from: (1) a polynucleotide encoding the polypeptide, fusion protein or recombinant protein A described in any embodiment of the present invention sequence; (2) the complementary sequence of the polynucleotide sequence of (1).
  • the sixth aspect of the present invention provides a nucleic acid construct, which contains the nucleic acid molecule according to any embodiment of the present invention; preferably, the nucleic acid construct is an expression cassette; more preferably, the nucleic acid construct
  • the object is an expression vector or a cloning vector.
  • the seventh aspect of the present invention provides an expression system, which contains the nucleic acid construct according to any embodiment of the present invention; preferably, the expression system is a host cell.
  • the eighth aspect of the present invention provides a separation matrix, which includes the polypeptide, fusion protein and/or recombinant protein A of any embodiment of the present invention coupled to a solid support.
  • the polypeptide, fusion protein or recombinant protein A is coupled to the solid support through a thioether bond.
  • the solid support is selected from: polyhydroxy-containing polymers, preferably polysaccharides, more preferably selected from: dextran, starch, cellulose, pullulan, agar and Agarose; a synthetic polymer preferably selected from the group consisting of: polyvinyl alcohol, polystyrene, polystyrene divinylbenzene, polyhydroxyalkyl acrylates, polyhydroxyalkyl methacrylates, polyacrylamides and polymethylmethacrylates acrylamide; and a support of inorganic nature, preferably selected from silica and zirconia.
  • polyhydroxy-containing polymers preferably polysaccharides, more preferably selected from: dextran, starch, cellulose, pullulan, agar and Agarose
  • a synthetic polymer preferably selected from the group consisting of: polyvinyl alcohol, polystyrene, polystyrene divinylbenzene, polyhydroxyalkyl acrylates, polyhydroxyalkyl methacrylates, poly
  • the ninth aspect of the present invention provides a chromatographic column, which contains the separation matrix according to any embodiment of the present invention.
  • the tenth aspect of the present invention provides a method for isolating Fc-containing proteins, the method comprising combining an immunoglobulin-containing sample with the polypeptide, fusion protein, recombinant protein A, separation matrix or chromatographic The step of column contacting; preferably, the Fc-containing protein is immunoglobulin.
  • the method comprises: (1) contacting a sample containing an Fc-containing protein with the separation matrix; (2) washing the separation matrix; (3) eluting the separation matrix from the separation matrix. Fc-containing protein; (4) cleaning the separation matrix.
  • the separation matrix is washed with a 0.1-2.0M or 0.5-1.0M NaOH or KOH solution.
  • the eleventh aspect of the present invention provides the application of the polypeptide, fusion protein or recombinant protein A described in any embodiment of the present invention in the separation of Fc-containing proteins, or in the preparation of separation matrices or chromatographic columns for the separation of Fc-containing proteins in the application.
  • Figure 1 (B domain) 4 gene result verification analysis diagram.
  • Fig. 2 Analysis diagram of mutant B(N3V)4 gene result verification.
  • Figure 3 Analysis diagram of mutant B(N6E)4 gene result validation.
  • Figure 4 Analysis diagram of the result verification of mutant B(Q9I)4 gene.
  • Figure 5 Analysis diagram of the result verification of mutant B(Q9F)4 gene.
  • Fig. 6 Analysis diagram of mutant B(E15L) 4 gene result verification.
  • Fig. 7 Analysis diagram of mutant B(E15I)4 gene result verification.
  • Fig. 8 Analysis diagram for verification of 4 gene results of mutant B (N3V, N6E, Q9I, E15L).
  • Figure 10 Analysis diagram of the result verification of the mutant Z(Q9I)4 gene.
  • Figure 11 Analysis diagram of the result verification of mutant Z(E15L)4 gene.
  • Fig. 12 Analysis diagram of the result verification of mutant Z(E15I)4 gene.
  • Figure 13 Gene result verification analysis diagram of mutant Z(N3V, N6E, Q9I, E15L)4.
  • Figure 14 Alkaline-resistant stability curves of Z domain mutants shown in SEQ ID NO: 72-91.
  • Figure 15 Alkali-resistant stability curves of Z domains shown in SEQ ID NO: 72, 98-102, 104, 105 and 107-109.
  • Figure 16 Alkali-resistant stability curves of B domain mutants shown in SEQ ID NO: 110-117.
  • antibody and “immunoglobulin” are used interchangeably and have meanings known in the art.
  • Antibodies or immunoglobulins as described herein also include fragments of antibodies, and fusion proteins or conjugates containing antibody fragments, so long as such fragments, fusion proteins or conjugates contain the Fc portion of the antibody and can be expressed by binding to Protein A. Combined to achieve separation and purification. Fragments of an antibody may be functionally active fragments thereof.
  • amino acid residues are also described with the following abbreviations: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D ), Cysteine (Cys or C), Glutamine (Gln or Q), Glutamic Acid (Glu or E), Glycine (Gly or G), Histidine (His or H), Isoleucine (Ile or I), Leucine (Leu or L), Lysine (Lys or K), Methionine (Met or M), Phenylalanine (Phe or F), Proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), valine (Val or V), and any amino acid residue (Xaa or X).
  • N-terminus amino terminus
  • C-terminus carboxy-terminus
  • Protein A is widely used as an affinity media ligand for the purification of immunoglobulins.
  • Natural protein A has five domains that engage immunoglobulins, especially IgG, which are E domain, D domain, A domain, B domain and C domain from N-terminus to C-terminus.
  • Domain Z is obtained by replacing the alanine at position 1 in domain B of protein A with valine, and the glycine at position 29 with alanine. This mutation confers more prominent chemical stability on domain Z.
  • immunoglobulin purification after immunoglobulin is combined with protein A, it needs to be eluted with alkali. This process will cause protein A to fall off from the affinity chromatography medium and cause contamination to the immunoglobulin product.
  • the present invention finds that the natural B domain of protein A or at least one of the 3rd, 6th, 9th, 15th and 23rd positions of the Z domain are substituted and mutated, and the mutants of the B or Z domain obtained are relatively Based on the natural B domain or the Z domain before mutation, it has significantly improved alkaline stability during alkaline cleaning.
  • the concentration of lye used can be increased from 0.1-0.5M to 0.5-2.0M, such as 0.5-1.0M, complete this invention.
  • the present invention provides a polypeptide, compared with the natural B domain shown in SEQ ID NO: 1, at least one position, at least two positions selected from positions 3, 6, 9, 15 and 23 Position, at least three positions, at least four positions, or all five positions have substitution mutations, or compared with the Z domain shown in SEQ ID NO: 2, at positions selected from positions 3, 6, 9, 15 and 23 There are substitution mutations at at least one, at least two, at least three, at least four, or all five of the positions.
  • the substitution mutation at position 3 may be asparagine replaced by leucine, isoleucine, valine or tyrosine. In some embodiments, the substitution mutation at position 3 is that asparagine is replaced by leucine, valine or tyrosine, preferably by leucine or valine. In some embodiments, the substitution mutation at position 3 is asparagine replaced by valine.
  • the polypeptide described herein has a substitution mutation at position 3, preferably, the amino acid sequence of the polypeptide is shown in SEQ ID NO: 3, 4, 5 or 118.
  • the substitution mutation at position 6 may be asparagine replaced by glutamic acid, leucine, isoleucine, valine, serine or threonine. In some embodiments, the substitution mutation at position 6 is asparagine replaced by glutamic acid, leucine or serine. In some embodiments, the substitution mutation at position 6 is asparagine replaced by leucine. In some embodiments, the polypeptide described herein has a substitution mutation at position 6, preferably, the amino acid sequence of the polypeptide is shown in SEQ ID NO: 6, 7, 8 or 119.
  • the substitution mutation at position 9 may be a substitution of glutamine for isoleucine, leucine, valine, phenylalanine, or methionine. In some embodiments, the substitution mutation at position 9 is glutamine replaced by isoleucine, phenylalanine or methionine.
  • the polypeptide described herein has a substitution mutation at position 9, preferably, the amino acid sequence of the polypeptide is shown in SEQ ID NO: 9, 10, 11, 120 or 121.
  • the substitution mutation at position 15 may be a glutamic acid mutation replaced by threonine, tryptophan, leucine, valine, isoleucine, phenylalanine acid, serine, tyrosine or aspartic acid. In some embodiments, the substitution mutation at position 15 may be a substitution of glutamic acid for threonine, tryptophan, leucine, valine, phenylalanine, serine or tyrosine. In some embodiments, the substitution mutation at position 15 is a substitution of glutamic acid for leucine or serine. In some embodiments, the polypeptide described herein has a substitution mutation at position 15, preferably, the amino acid sequence of the polypeptide is as shown in any one of SEQ ID NO: 12-20 and 122, 123.
  • the substitution mutation at position 23 may be asparagine replaced by valine, isoleucine, leucine or tyrosine. In some embodiments, the substitution mutation at position 23 is asparagine replaced by valine, isoleucine or tyrosine. In some embodiments, the substitution mutation at position 23 is asparagine replaced by valine or isoleucine. In some embodiments, the polypeptide described herein has a substitution mutation at position 23, preferably, the amino acid sequence of the polypeptide is as shown in any one of SEQ ID NO: 21-23.
  • the polypeptide of the present invention has the substitution mutation described in any embodiment herein at least at position 3, and optionally at one or more positions selected from positions 6, 9, 15 and 23. There are substitution mutations described in any embodiment herein at multiple positions, preferably optionally at one or more positions selected from positions 6, 9 and 15. In some other embodiments, the polypeptide of the present invention has at least the substitution mutation described in any embodiment herein at position 6, and optionally at one or more positions selected from positions 3, 9, 15 and 23 Positions, preferably optionally at one or more positions selected from positions 3, 9 and 15, have the substitution mutations described in any of the embodiments herein.
  • the polypeptide of the present invention has at least the substitution mutation described in any embodiment herein at position 9, and optionally at one or more positions selected from positions 3, 6, 15 and 23. Positions, preferably optionally at one or more positions selected from positions 3, 6 and 15, have the substitution mutations described in any of the embodiments herein. In some other embodiments, the polypeptide of the present invention has at least the substitution mutation described in any embodiment herein at position 15, and optionally at one or more positions selected from positions 3, 6, 9 and 23. Positions, preferably optionally at one or more positions selected from positions 3, 6 and 9, have the substitution mutations described in any of the embodiments herein.
  • the polypeptide of the present invention has at least the substitution mutation described in any embodiment herein at position 23, and at one or more positions selected from positions 3, 6, 9 and 15 Having a substitution mutation as described in any of the embodiments herein.
  • the substitution mutation at the 3rd position is N3L or N3V
  • the substitution mutation at the 6th position is N6L or N6E
  • the substitution mutation at the 9th position is Q9I or Q9F
  • the The substitution mutation at position 15 is E15L, E15I or E15V
  • the substitution mutation at position 23 is N23V, N23I or N23Y.
  • the polypeptide of the present invention has at least at position 15 any of the embodiments herein The substitution mutation described in the above formula, and at least 1 position, at least 2 positions, at least 3 positions or all 4 positions selected from the 3rd, 6th, 9th and 23rd positions Substitution mutations described in any of the embodiments herein.
  • the substitution mutation at position 15 is E15T, E15W, E15L, E15V, E15I, E15F, E15S, E15Y or E15T
  • the substitution mutation at position 3 is N3L, N3V or N3Y
  • the substitution mutation at the 6th position is N6S, N6L or N6E
  • the substitution mutation at the 9th position is Q9I or Q9F
  • the substitution mutation at the 23rd position is N23V, N23I or N23Y.
  • the substitution mutation at the 15th position is E15I or E15V
  • the substitution mutation at the 3rd position is N3L or N3V
  • the substitution mutation at the 6th position is N6E or N6L
  • the substitution mutation at the 9th position is Q9I or In Q9F
  • the substitution mutation at position 23 is N23I, N23Y or N23V.
  • the polypeptide of the present invention has the substitution mutation described in any of the embodiments herein at position 3, position 9 and position 15, and any of the mutations described herein at position 6 and/or 23.
  • the substitution mutation at position 15 is E15T, E15W, E15L, E15V, E15I, E15F, E15S, E15Y or E15T
  • the substitution mutation at position 3 is N3L, N3V or N3Y
  • the substitution mutation at the 6th position is N6S, N6L or N6E
  • the substitution mutation at the 9th position is Q9I or Q9F
  • the substitution mutation at the 23rd position is N23V, N23I or N23Y.
  • the substitution mutation at the 3rd position is N3L or N3V
  • the substitution mutation at the 6th position is N6L or N6E
  • the substitution mutation at the 9th position is Q9I or Q9F
  • the substitution mutation at the 15th position is E15L, E15I or E15V
  • the substitution mutation at position 23 is N23V, N23I or N23Y.
  • the amino acid sequence of the polypeptide of the present invention has an N3V mutation at position 3, or an N6E mutation at position 6, or an N6E mutation at position 9.
  • amino acid sequence of the polypeptide described herein having substitution mutations at least two positions selected from positions 3, 6, 9, 15 and 23 is as set forth in any of SEQ ID NO: 24-71 stated.
  • the polypeptide of the present invention can be directly used as a ligand for binding and separating immunoglobulins. Accordingly, in some embodiments, the polypeptide further comprises one or more coupling elements at its C- or N-terminus for coupling the polypeptide to a solid support. Suitable coupling elements are well known in the art, including but not Cysteine residues, multiple lysine residues (eg 3-15 or 5-10) and multiple histidine residues (eg 3-15 or 5-10).
  • the coupling element can be a cysteine residue, and it is located at the C-terminal of the polypeptide, so that the polypeptide can be coupled by using the thiol group in the cysteine residue to react with the electrophilic group on the solid support.
  • a coupling element can be attached directly to the polypeptide, or it can be attached to the N- or C-terminus of the polypeptide via a linker.
  • the linker may be a linker commonly used in the art, and its presence will not affect the binding activity of the polypeptide.
  • An exemplary linker is a linker sequence comprising or consisting of G and S, typically 2-20 amino acid residues in length. Exemplary linker sequences are (GS) n , (GSS) n , (GSSS) n and (GSSSS) n , n can be an integer ranging from 2 to 10, but the total length of the linker usually does not exceed 20 amino acid residues.
  • the polypeptide may also comprise a plurality (eg, within 15, within 10 or within 5) of amino acid residues at the N-terminus, derived from the cloning process or from residues from signal transduction sequences.
  • a polypeptide of the invention may comprise AQ at the N-terminus.
  • the present invention also provides a fusion protein, which is formed by fusion of 2-8 aforementioned polypeptides of the present invention.
  • the polypeptides forming the fusion protein may be all different, partly or all the same.
  • Each polypeptide can be connected directly through the peptide bonds at the C-terminal and N-terminal, or any two polypeptides can also be connected through a linker.
  • the linker may be a linker commonly used in the art, and its presence will not affect the binding activity of the polypeptide.
  • An exemplary linker is a linker sequence comprising or consisting of G and S, typically 2-20 amino acid residues in length.
  • Exemplary linker sequences are (GS) n and (GSS) n , etc., n can be an integer ranging from 2 to 10, but the total length of the linker usually does not exceed 20 amino acid residues.
  • the fusion protein can be used as a ligand for binding and separating immunoglobulins. Accordingly, in some embodiments, the fusion protein further comprises one or more coupling elements at its C-terminus or N-terminus for coupling the fusion protein to a solid support.
  • Suitable coupling elements are well known in the art and include, but are not limited to, cysteine residues, multiple lysine residues (such as 3-15 or 5-10), and multiple histidine residues (such as 3-15 or 5-10).
  • the coupling element can be a cysteine residue, and it is located at the C-terminus of the fusion protein, so that the fusion Proteins are coupled to solid supports.
  • a coupling element can be attached directly to the polypeptide, or it can be attached to the N- or C-terminus of the polypeptide via a linker.
  • the linker may be a linker commonly used in the art, and its presence will not affect the binding activity of the polypeptide.
  • An exemplary linker is a linker sequence comprising or consisting of G and S, typically 2-20 amino acid residues in length.
  • Exemplary linker sequences such as (GS) n and (GSS) n , etc., n can be an integer of 2-10, but the total length of the linker usually does not exceed 20 amino acid residues.
  • the fusion protein may also comprise a plurality (eg, within 15, within 10, or within 5) of amino acid residues at the N-terminus, derived from the cloning process or from residues from signal transduction sequences.
  • the fusion protein may comprise AQ at its N-terminus.
  • At least one polypeptide has the substitution mutation described in any embodiment herein at the 15th position, optionally at the 3rd position A polypeptide having a substitution mutation according to any embodiment herein at least 1 position, at least 2 positions, at least 3 positions or all 4 positions of position 6, position 9 and position 23.
  • at least one polypeptide has the substitution mutation described in any embodiment herein at position 3, position 9 and position 15, and at position 6 and/or A polypeptide having a substitution mutation as described in any of the embodiments herein at position 23.
  • the substitution mutation at the 3rd position is N3L or N3V
  • the substitution mutation at the 6th position is N6L or N6E
  • the substitution mutation at the 9th position is Q9I or Q9F
  • the substitution mutation at the 15th position is E15L, E15I or E15V
  • the substitution mutation at position 23 is N23V, N23I or N23Y.
  • the polypeptide of the present invention that forms the melting protein of the present invention includes at least one or more of the following substitution mutations: SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 60, SEQ ID NO: 71, EQ ID NO: 64, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 68, SEQ ID NO: 118, SEQ ID NO: 119.
  • SEQ ID NO: 120 SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, and SEQ ID NO: 124.
  • the amino acid sequence of the exemplary fusion protein of the present invention is shown in any one of SEQ ID NO: 73-109 and 111-117.
  • the present invention also provides a recombinant protein A whose B domain is the Z domain mutant or B domain mutant described in any embodiment of the present invention.
  • the remaining structural domains of the recombinant protein A may be E structural domains, D structural domains, A structural domains and C structural domains known in the art, including natural E structural domains, D structural domains, A structural domains and B structural domains and existing E domain, D domain, A domain and/or C domain with known mutations but retaining biological activity. Except that the polypeptide of the present invention is used in the B domain, mutations or modifications to the rest of the protein A known in the art can be used in the recombinant protein of the present invention. White A.
  • polypeptide having at least 85% sequence identity with the polypeptide, fusion protein or recombinant protein A described in any of the embodiments herein, and selected from the sequence corresponding to SEQ ID NO: 1 or 2, 3rd, 6th, 9th, 15th Polypeptides, fusion proteins and recombinant protein A having substitution mutations described in any embodiment herein at one or more amino acid positions at position 23 and 23.
  • the "at least 85% identity" related to the amino acid sequence and the nucleotide sequence refers to an identity of more than 85%, preferably more than 90%, more preferably more than 95%, and even more preferably The identity is 97% or more, more preferably 98% or more, and still more preferably 99% or more.
  • the identity between two aligned sequences can be calculated using tools well known in the art, including BLASTP for amino acid sequence alignment, and the like.
  • the present invention includes variants of any of the amino acid sequences shown in SEQ ID NO: 3-71, 118-124 and 73-109, 111-117, SEQ ID NO: 3-71, Each variant of 118-124 and 73-109, 111-117 has at least 85%, preferably at least 90%, more preferably at least 97%, more preferably at least 99% sequence identity compared to the corresponding parental sequence and its The amino acid residues at the 3rd, 6th, 9th, 15th and 23rd positions of the corresponding parents still remain as the corresponding parental amino acid residues.
  • the present invention also includes nucleic acid molecules encoding the aforementioned polypeptides, fusion proteins and recombinant protein A and their complementary sequences.
  • the invention includes all forms of the nucleic acid molecules of the invention, including RNA and DNA. It should be understood that the complementary sequence described herein refers to a complementary sequence whose length is substantially the same as that of the nucleic acid molecule.
  • nucleic acid constructs comprising said nucleic acid molecule or its complement are also included within the scope of the present invention.
  • the nucleic acid construct may be an expression cassette comprising a promoter, the nucleic acid molecule and a transcription termination sequence. Promoters and transcription termination sequences are well known in the art, and can be appropriately selected by those skilled in the art according to the host cell selected for expressing the polypeptide, fusion protein or recombinant protein A.
  • nucleic acid constructs are vectors, including expression vectors and cloning vectors.
  • Expression vectors are vectors suitable for expressing foreign genes such as nucleic acid molecules encoding polypeptides, fusion proteins or recombinant protein A of the present invention in host cells, including prokaryotic expression vectors and eukaryotic expression vectors.
  • Eukaryotic expression systems include yeast expression systems, mammalian cell expression systems and insect cell expression systems.
  • the cloning vector is used to amplify the target gene (such as the nucleic acid molecule encoding the polypeptide, fusion protein or protein A of the present invention) in the host cell.
  • Cloning vectors include plasmid vectors, phage vectors, viral vectors, and vectors that are combined with each other or with other genomic DNA. The most commonly used host in molecular cloning The main cell is Escherichia coli.
  • the present invention also provides an expression system comprising the nucleic acid molecule, nucleic acid construct or vector disclosed herein.
  • the expression system can be, for example, a Gram-positive or Gram-negative prokaryotic host cell system, such as E. coli or Bacillus, modified to express a polypeptide, fusion protein or recombinant protein A of the invention.
  • the expression system is a eukaryotic host cell system, such as a yeast, such as Pichia pastoris or Saccharomyces cerevisiae.
  • the present invention also provides a separation matrix.
  • the separation matrix of the present invention can be used to separate immunoglobulins or Fc-containing proteins.
  • the separation matrix of the present invention contains the polypeptide, fusion protein and/or recombinant protein A described in any embodiment of the present invention coupled to a solid support. Due to the improved alkaline stability of the polypeptide, fusion protein and recombinant protein A of the present invention, the separation matrix of the present invention can withstand highly alkaline conditions (0.5-2.0M NaOH) during washing.
  • the solid support in the separation matrix of the present invention may be a solid support known in the art and used in the separation of immunoglobulin or Fc-containing protein.
  • the shape of the solid phase support may be any shape such as particle, film, plate, tube, needle, and fiber.
  • the solid support of the present invention may be a porous material or a non-porous material.
  • the solid support is in the form of a porous or non-porous bead or particle.
  • Substrates in beaded or granular form can be used as packed beds or in suspension.
  • Suspension forms include expanded beds in which the particles or beads move freely.
  • the particle size is preferably 20-200 ⁇ m.
  • the particle size is preferably 20-100 ⁇ m, preferably 30-80 ⁇ m.
  • the particle size is preferably 50-200 ⁇ m, more preferably 60-150 ⁇ m.
  • the "particle diameter" in this specification refers to the volume average particle diameter measured by the laser diffraction method in accordance with ISO 13320 and JIS Z 8825-1.
  • solid supports include, but are not limited to, polymers containing polyhydroxyl groups, such as polysaccharides.
  • Polysaccharides include dextran, starch, cellulose, pullulan, agar and agarose, etc.
  • the solid support comprises agar or agarose.
  • the solid supports of the invention are synthetic polymers with clear surfaces.
  • such polymers can be treated by hydrophilization on the outer surface (and, where present, also Inner surface) polymers with hydroxyl, carboxyl, aminocarbonyl, amino or oligovinyloxy or polyethyleneoxy, preferably such as multifunctional (meth)acrylate, divinylbenzene and other polyfunctional monomers Synthetic polymers of cross-linked copolymers.
  • Exemplary synthetic polymers include polyvinyl alcohol, polystyrene, polystyrene divinylbenzene, polyhydroxyalkyl acrylates, polyhydroxyalkyl methacrylates, polyacrylamides, polymethacrylamides, and the like.
  • the surface can be hydrophilized to expose the hydrophilic groups to the surrounding aqueous liquid.
  • the solid supports of the present invention comprise supports of an inorganic nature, including, but not limited to, silica, zirconia, and the like.
  • a solid support of the invention is in the form of a surface, chip, capillary or filter, eg, a membrane or the like.
  • the polypeptide, fusion protein and recombinant protein A of the present invention can be connected to the corresponding solid support through conventional coupling techniques through the thiol group, amino group and/or carboxyl group contained therein.
  • Commonly used coupling reagents include, but are not limited to, diepoxides, epichlorohydrin, CNBr, and N-hydroxysuccinimide.
  • a spacer can be introduced between the solid support and the polypeptide, fusion protein and recombinant protein A of the present invention, which can facilitate their chemical coupling with the solid support.
  • the polypeptide, fusion protein or recombinant protein A of the present invention can be coupled to a solid support through a thioether bond.
  • the polypeptide, fusion protein or recombinant protein A of the present invention is coupled through its C-terminal cysteine, wherein the cysteine thiol group is coupled to the electrophilic group on the solid support Such as epoxy group, halohydrin group, etc. are effectively coupled to form thioether bridge coupling.
  • the concentration of the binding partner coupled to the solid support is usually 5-20 mg/ml, such as 5-15 mg/ml. ml.
  • the amount of the binding partner coupled can be controlled by adjusting the concentration of the polypeptide, fusion protein and/or recombinant protein A used in the coupling process, the coupling conditions used and/or the pore structure of the solid support used .
  • the present invention also provides a chromatographic column containing the separation matrix of the present invention.
  • the separation matrix of the present invention is packed in a chromatographic column.
  • the present invention also provides a method for isolating an Fc-containing protein (such as immunoglobulin), the method comprising combining the polypeptide, fusion protein and/or recombinant protein A described in any embodiment herein with a sample containing an Fc-containing protein contact steps.
  • the method includes combining the sample with the The step of contacting the separation matrix or chromatographic column described in any embodiment.
  • the method includes:
  • the samples may be various samples containing Fc-containing proteins (especially immunoglobulins).
  • the immunoglobulin referred to herein refers to IgG.
  • the washing solution for washing the separation matrix used in the method, the eluent for eluting the protein and the cleaning solution for cleaning the separation matrix are all routinely used in the art (especially in the field of protein A chromatography) Washes, eluents, and washes.
  • the wash solution can be PBS buffer.
  • the eluent can be a solution or buffer with pH ⁇ 5, preferably, the pH of the eluent is 2.5-5 or 3-5.
  • the pH of the eluent is 11 or higher, eg, the pH of the eluent is 11-14 or 11-13.
  • elution is performed using a citrate solution, such as a sodium citrate solution.
  • Cleaning solutions are usually alkaline and can have a pH of 13-14.
  • the cleaning solution is sodium hydroxide solution or potassium hydroxide solution, the concentration of which is 0.1-2.0M, such as 0.5-2.0M or 0.5-1.0M.
  • the methods described herein further comprise the step of recovering the eluate and further separating and purifying it.
  • further separation and purification can be carried out by anion or cation exchange chromatography, complex ion exchange chromatography and/or hydrophobic interaction chromatography and the like.
  • the present invention also provides the use of the polypeptide, fusion protein and protein A described in any embodiment herein in the separation and purification of Fc-containing proteins, or in the preparation of separation matrices for the separation and purification of Fc-containing proteins.
  • a single recombinant plasmid is used to transform into Escherichia coli, and LB liquid medium is used for fermentation and expression induction. After fermentation, the cells were collected and the cell wall was destroyed by thermal lysis to release the expression product and centrifuged. Purify the separation liquid through the IgG affinity medium, load the separation liquid through the affinity medium, wash the medium with 10mM phosphate buffer, collect the target protein with a buffer solution of pH 3.8, adjust to a neutral environment, and store it for later use .
  • an affinity chromatography medium was prepared by a conventional method.
  • An exemplary preparation process specifically includes the following steps:
  • Table 1 shows the kinetic analysis results of Z domain mutants and B domain mutants using Biacore.
  • Buffer Buffer A (PBS, pH 7.6); Buffer B (0.1M Sodium Citrate, pH 3.0); Buffer C (1M NaOH).
  • Sample prepare a 1mg/ml IgG sample with the pure product (the solution for diluting the pure product is buffer A).
  • step d Continue to load the sample until the UV absorption peak reaches the volume corresponding to the UV absorption peak in step d, which is 10% dynamic loading.
  • Control room temperature 23 ⁇ 0.5°C; update the sample load in the program according to the decrease of the load, and keep other programs consistent.
  • the initial IgG binding abilities of the mutants in the table are all above 130mg/ml, which has good affinity.
  • the IgG binding ability of most of the mutants was better than that of the Z domain and the B domain, except for a small number of mutants that showed slightly worse IgG binding ability than the Z domain.
  • the mutant Z(E15S)4 still has 43.57% remaining IgG capacity after 24 hours of alkali treatment, these data indicate that the mutation site selected in the present invention can improve the alkaline stability of the Z domain and the B domain.
  • Example 2 was repeated with the affinity chromatography medium prepared from the Z domain mutants in Table 3, and the results are shown in Table 3 and FIG. 15 .
  • Table 3 shows the IgG capacity (in terms of dynamic binding capacity, mg/ml) of each sample at 10% breakthrough (Qb10%).
  • Table 3 shows the alkali resistance of the mutants with mutations at multiple sites on the Z domain. After 24 hours of 1.0M NaOH treatment, the remaining IgG binding ability of the mutants was improved to a certain extent compared with the Z domain, indicating that the above mutations Improved alkali resistance. Among them, mutant Z (N3L, N6L, Q9F, E15I, N23V) 6 still had 63.93% IgG ability after 24 hours of alkaline treatment.

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Abstract

一种蛋白A的B结构域和Z结构域突变体及其应用。具体而言，提供一种分离的多肽，所述多肽选自：(1)与SEQ ID NO：1所示的蛋白A的天然B结构域或SEQ ID NO：2所示的蛋白A的Z结构域相比，该多肽在选自第3位、第6位、第9位、第15位和第23位的一个或多个位置上具有取代突变；和(2)与(1)所述的多肽具有至少85％序列相同性并保留了所述的第3位、第6位、第9位、第15位和第23位中一个或多个位置上的取代突变的多肽。还提供含有多肽的融合蛋白或蛋白A以及分离基质。含所述多肽的融合蛋白或蛋白A在碱清洗过程中具有明显提升的碱性稳定性。

Description

蛋白A的B结构域和Z结构域突变体及其应用 技术领域
本发明涉及蛋白A的B结构域和Z结构域突变体及其应用。
背景技术
近年来抗体药物以其高特异性成为全球药品市场上的热品，其中单克隆抗体已成为生物制药产业增长最快的领域之一。单克隆抗体的纯度越高，其效应性越好，效率越高，关于单克隆抗体等各类抗体药物的纯化研究一直是目前领域内研究的重点内容。Protein A亲和层析介质凭借其对抗体Fc独特段具有特殊吸附能力，成为运用最广泛的纯化单抗的方式。然而，Protein A亲和层析介质在抗体的纯化过程中会出现配基脱落、结合载量较低等问题。另外，纯化程序中的在位清洗(CIP)过程会使用到0.5-1.0M NaOH溶液，高浓度的NaOH会破坏ProteinA亲和层析介质，大大降低其使用寿命。因此，科研人员对如何提升ProteinA的耐碱性进行了大量研究，例如Susanne Gilich等人提出可以通过蛋白质工程来提高蛋白配体的结构域在碱性条件下的稳定性。为此，本领域技术人员对蛋白配体各个结构域中的氨基酸进行突变研究，找寻能提高蛋白配体耐碱性的氨基酸。
ProteinA是由高度同源的免疫球蛋白结合域E、D、A、B、C，其中结构域B与免疫球蛋白结合特异性具有较大的优势。结构域Z是将Protein A结构域B中1位的丙氨酸替换为缬氨酸，29位的甘氨酸替换为丙氨酸所得。该突变赋予了结构域Z更加突出的化学稳定性，同样地，对通过结构域氨基酸突变的结构域Z在碱性条件下也具有一定的稳定性。但是，其在更高的pH条件下仍然不稳定，无法满足抗体纯化过程中的CIP清洗步骤的要求。目前已有的B结构域或Z结构域突变的Protein A层析介质相较于天然Protein A层析介质有一定的碱性稳定性。例如CN101522278A将B结构域上29位甘氨酸突变为丙 氨酸，从而提高了耐碱性。CN1642976A公开了Z结构域上至少一个天冬酰胺残基突变为除丙氨酸、苏氨酸或天冬氨酸之外的氨基酸。另外，CN105377881A公开了B结构域或Z结构域中至少15位的谷氨酸残基突变为天冬酰胺或谷氨酰胺以外的氨基酸。这些突变体与天然配体相比在碱性条件下具有更高的耐碱性。使用上述配体制备的层析介质在抗体纯化过程中可以耐受0.1～0.5M氢氧化钠清洗，但距标准CIP(0.5～1.0M氢氧化钠)仍有一定差距。
发明内容
本发明第一方面提供一种分离的多肽，所述多肽选自：
(1)与SEQ ID NO：1所示的蛋白A的天然B结构域或SEQ ID NO：2所示的蛋白A的Z结构域相比，该多肽在选自第3位、第6位、第9位、第15位和第23位的一个或多个位置上具有取代突变；
其中，所述第3位上的取代突变为天冬酰胺被取代为亮氨酸、异亮氨酸、缬氨酸或酪氨酸；所述第6位上的取代突变为天冬酰胺被取代为谷氨酸、亮氨酸、异亮氨酸、缬氨酸、丝氨酸或苏氨酸；所述第9位上的取代突变为谷氨酰胺被取代为异亮氨酸、亮氨酸、缬氨酸、苯丙氨酸或甲硫氨酸；所述第15位上的取代突变为谷氨酸被取代为苏氨酸、色氨酸、亮氨酸、缬氨酸、异亮氨酸、苯丙氨酸、丝氨酸、酪氨酸或天冬氨酸；所述第23位上的取代突变为天冬酰胺突变为缬氨酸、异亮氨酸、亮氨酸或酪氨酸；
(2)与(1)所述的多肽具有至少85％序列相同性并保留了所述的第3位、第6位、第9位、第15位和第23位中一个或多个位置上的取代突变的多肽。
在一个或多个实施方案中，所述第3位上的取代突变为天冬酰胺被取代为亮氨酸、缬氨酸或酪氨酸。
在一个或多个实施方案中，所述第6位上的取代突变为天冬酰胺被取代为谷氨酸、亮氨酸或丝氨酸。
在一个或多个实施方案中，所述第9位上的取代突变为谷氨酰胺被取代为异亮氨酸、苯丙氨酸或甲硫氨酸。
在一个或多个实施方案中，所述第15位上的取代突变为谷氨酸被取代为 苏氨酸、色氨酸、亮氨酸、异亮氨酸、缬氨酸、苯丙氨酸、丝氨酸或酪氨酸。
在一个或多个实施方案中，所述第23位上的取代突变为天冬酰胺突变为缬氨酸、异亮氨酸或酪氨酸。
在一个或多个实施方案中，所述多肽至少在第3位具有所述取代突变，并在选自所述第15位、第6位、第9位和第23位的至少1个位置、至少2个位置、至少3个位置或全部4个位置上具有所述取代突变。
在一个或多个实施方案中，所述多肽至少在第6位具有所述取代突变，并在选自所述第3位、第15位、第9位和第23位的至少1个位置、至少2个位置、至少3个位置或全部4个位置上具有所述取代突变。
在一个或多个实施方案中，所述多肽至少在第9位具有所述取代突变，并在选自所述第3位、第6位、第15位和第23位的至少1个位置、至少2个位置、至少3个位置或全部4个位置上具有所述取代突变。
在一个或多个实施方案中，所述多肽至少在第15位具有所述取代突变，并在选自所述第3位、第6位、第9位和第23位的至少1个位置、至少2个位置、至少3个位置或全部4个位置上具有所述取代突变。
在一个或多个实施方案中，所述多肽至少在第23位具有所述取代突变，并在选自所述第3位、第6位、第9位和第15位的至少1个位置、至少2个位置、至少3个位置或全部4个位置上具有所述取代突变。
在一个或多个实施方案中，所述第15位的取代突变为E15I、E15L或E15V，第3位的取代突变为N3L或N3V，第6位的取代突变为N6E或N6L，第9位的取代突变为Q9I或Q9F，第23位的取代突变为N23I、N23Y或N23V。
在一个或多个实施方案中，所述多肽在第3位、第9位和第15位具有所述取代突变，并在第6位和/或第23位具有所述取代突变；优选地，所述第3位的取代突变为N3L或N3V，所述第6位的取代突变为N6L或N6E，所述第9位的取代突变为Q9I或Q9F，所述第15位的取代突变为E15L、E15I或E15V，所述第23位的取代突变为N23V、N23I或N23Y。
在一个或多个实施方案中，所述多肽在第3位、第6位、第9位和第15位具有所述取代突变。优选地，所述第3位的取代突变为N3L或N3V、优选 N3V，所述第6位的取代突变为N6L或N6E、优选N6E，所述第9位的取代突变为Q9I或Q9F、优选Q9I，所述第15位的取代突变为E15L、E15I或E15V、优选E15L。
在一个或多个实施方案中，所述多肽的氨基酸序列如SEQ ID NO：3-71和118-124中任一所示。
本发明第二方面提供一种分离的多肽，所述多肽由以下(i)、(ii)和(iii)组成：(i)本发明第一方面任一实施方案所述的多肽，(ii)在(i)所述多肽的氨基酸序列的C端或N端的一个或多个偶联元件，和任选的(iii)来自被切下的信号转导序列的残基。
在一个或多个实施方案中，所述偶联元件选自：半胱氨酸残基、多个赖氨酸残基和多个组氨酸残基；所述被切下的信号转导序列的残基为AQ。
本发明第三方面提供一种融合蛋白，其特征在于，该融合蛋白包含由2-8条本发明第一方面任一实施方案所述的多肽融合形成的氨基酸序列，其中，所述2-8条多肽互不相同、部分相同或完全相同，且所述多肽之间任选地存在接头序列。
在一个或多个实施方案中，所述融合蛋白所含的2-8条多肽中，至少一条多肽为在第15位具有所述取代突变、任选在选自所述第3位、第6位、第9位和第23位的至少1个位置、至少2个位置、至少3个位置或全部4个位置上具有所述取代突变的多肽；优选地，至少一条多肽为在第3位、第9位和第15位具有所述取代突变、并在第6位和/或第23位具有所述取代突变的多肽。
在一个或多个实施方案中，所述融合蛋白中，所述多肽选自：SEQ ID NO：4、SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：10、SEQ ID NO：11、SEQ ID NO：14、SEQ ID NO：16、SEQ ID NO：18、SEQ ID NO：21、SEQ ID NO：22、SEQ ID NO：51、SEQ ID NO：53、SEQ ID NO：60、SEQ ID NO：71、EQ ID NO：64、SEQ ID NO：69、SEQ ID NO：70、SEQ ID NO：68、SEQ ID NO：118、SEQ ID NO：119、SEQ ID NO：120、SEQ ID NO：121、SEQ ID NO：122、SEQ ID NO：123和SEQ ID NO：124中的一条或多条。
在一个或多个实施方案中，所述融合蛋白的C端或N端还包括一个或多 个偶联元件和/或来自被切下的信号转导序列的残基；优选地，所述偶联元件选自：半胱氨酸残基、多个赖氨酸残基和多个组氨酸残基。
在一个或多个实施方案中，所述融合蛋白的氨基酸序列如SEQ ID NO：73-109和111-117中任一所示。
本发明第四方面提供一种重组蛋白A，该重组蛋白A的B结构域为本发明第一方面任一实施方案所述的多肽。
本发明第五方面提供一种分离的核酸分子，所述核酸分子的多核苷酸序列选自：(1)编码本发明任一实施方案所述的多肽、融合蛋白或重组蛋白A的多核苷酸序列；(2)(1)所述多核苷酸序列的互补序列。
本发明第六方面提供一种核酸构建物，所述核酸构建物含有本发明任一实施方案所述的核酸分子；优选地，所述核酸构建物为表达盒；更优选地，所述核酸构建物为表达载体或克隆载体。
本发明第七方面提供一种表达系统，其含有本发明任一实施方案所述的核酸构建物；优选地，所述表达系统为宿主细胞。
本发明第八方面提供一种分离基质，所述分离基质包括与固体支持物偶联的本发明任一实施方案所述的多肽、融合蛋白和/或重组蛋白A。
在一个或多个实施方案中，所述多肽、融合蛋白或重组蛋白A通过硫醚键与所述固体支持物偶联。
在一个或多个实施方案中，所述固体支持物选自：含多羟基的多聚体，优选为多糖，更优选选自：葡聚糖、淀粉、纤维素、普鲁兰多糖、琼脂和琼脂糖；合成的聚合物，优选选自：聚乙烯醇、聚苯乙烯、聚苯乙烯二乙烯苯、多羟基烷基丙烯酸酯、多羟基烷基甲基丙烯酸酯、聚丙烯酰胺和聚甲基丙烯酰胺；和无机性质的支持物，优选选自二氧化硅和氧化锆。
本发明第九方面提供一种色谱柱，所述色谱柱含有本发明任一实施方案所述的分离基质。
本发明第十方面提供一种分离含Fc的蛋白质的方法，所述方法包括使含免疫球蛋白的样品与本发明任一实施方案所述的多肽、融合蛋白、重组蛋白A、分离基质或色谱柱接触的步骤；优选地，所述含Fc的蛋白质为免疫球蛋白。
在一个或多个实施方案中，所述方法包括：(1)使含有含Fc的蛋白质的样品与所述分离基质接触；(2)洗涤分离基质；(3)从分离基质上洗脱所述含Fc的蛋白质；(4)清洗分离基质。
在一个或多个实施方案中，使用0.1-2.0M或0.5-1.0M的NaOH或KOH溶液清洗分离基质。
本发明第十一方面提供本发明任一实施方案所述的多肽、融合蛋白或重组蛋白A在分离含Fc的蛋白质中的应用，或在制备用于分离含Fc的蛋白质的分离基质或色谱柱中的应用。
附图说明
图1：(B结构域)4基因结果验证分析图。
图2：突变体B(N3V)4基因结果验证分析图。
图3：突变体B(N6E)4基因结果验证分析图。
图4：突变体B(Q9I)4基因结果验证分析图。
图5：突变体B(Q9F)4基因结果验证分析图。
图6：突变体B(E15L)4基因结果验证分析图。
图7：突变体B(E15I)4基因结果验证分析图。
图8：突变体B(N3V，N6E，Q9I，E15L)4基因结果验证分析图。
图9：(Z结构域)4基因结果验证分析图。
图10：突变体Z(Q9I)4基因结果验证分析图。
图11：突变体Z(E15L)4基因结果验证分析图。
图12：突变体Z(E15I)4基因结果验证分析图。
图13：突变体Z(N3V，N6E，Q9I，E15L)4的基因结果验证分析图。
图14：SEQ ID NO：72-91所示的Z结构域突变体的耐碱稳定性曲线图。
图15：SEQ ID NO：72、98-102、104、105和107-109所示的Z结构域的耐碱稳定性曲线图。
图16：SEQ ID NO：110-117所示的B结构域突变体的耐碱稳定性曲线图。
具体实施方式
应理解，在本发明范围中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成优选的技术方案。
本文中，“抗体”和“免疫球蛋白”可互换使用，其具有本领域周知含义。本文所述的抗体或免疫球蛋白也包括抗体的片段，以及含有抗体片段的融合蛋白或辍合物，只要这类片段、融合蛋白或辍合物含有抗体的Fc部分并可通过与蛋白A的结合而实现分离纯化即可。抗体的片段可以是其功能活性片段。
在本说明书中，氨基酸残基也以下列简写符号记载：丙氨酸(Ala或A)，精氨酸(Arg或R)，天冬酰胺(Asn或N)，天冬氨酸(Asp或D)，半胱氨酸(Cys或C)，谷氨酰胺(Gln或Q)，谷氨酸(Glu或E)，甘氨酸(Gly或G)，组氨酸(His或H)，异亮氨酸(Ile或I)，亮氨酸(Leu或L)，赖氨酸(Lys或K)，甲硫氨酸(Met或M)，苯丙氨酸(Phe或F)，脯氨酸(Pro或P)，丝氨酸(Ser或S)，苏氨酸(Thr或T)，色氨酸(Trp或W)，酪氨酸(Tyr或Y)，缬氨酸(Val或V)，以及任意的氨基酸残基(Xaa或X)。此外，在本说明书中，肽的氨基酸序列是依常法以氨基末端(以下称为N末端)位于左侧，羧基末端(以下称为C末端)位于右侧的方式加以记载。
蛋白A被广泛用作纯化免疫球蛋白的亲和介质配基。天然的蛋白A具有5个接合免疫球蛋白尤其是IgG的结构域，从N端到C端依次为E结构域、D结构域、A结构域、B结构域以及C结构域。结构域Z是将蛋白A结构域B中1位的丙氨酸替换为缬氨酸，29位的甘氨酸替换为丙氨酸所得。该突变赋予了结构域Z更加突出的化学稳定性。在免疫球蛋白纯化过程中，免疫球蛋白与蛋白A结合后需要用碱洗脱，此过程会造成蛋白A从亲和层析介质上脱落，对免疫球蛋白产品造成污染。
本发明发现，对蛋白A的天然B结构域或对Z结构域的第3、6、9、15和23位中的至少一位进行取代突变，得到的该B或Z结构域的突变体相对于天然B结构域或突变前的Z结构域，在碱清洗过程中具有明显提升的碱性稳定性，所用碱液浓度可从0.1-0.5M提高到0.5-2.0M，如0.5-1.0M，由此完成本 发明。
因此，本发明提供一种多肽，该多肽与SEQ ID NO：1所示的天然B结构域相比，在选自第3、6、9、15和23位中的至少一个位置、至少两个位置、至少三个位置、至少四个位置或全部五个位置上具有取代突变，或与SEQ ID NO：2所示的Z结构域相比，在选自第3、6、9、15和23位中的至少一个位置、至少两个位置、至少三个位置、至少四个位置或全部五个位置上具有取代突变。
在一个或多个实施方案中，所述3位上的取代突变可以是天冬酰胺被取代为亮氨酸、异亮氨酸、缬氨酸或酪氨酸。在一些实施方案中，所述3位上的取代突变为天冬酰胺被取代为亮氨酸、缬氨酸或酪氨酸，优选被取代为亮氨酸或缬氨酸。在一些实施方案中，所述3位上的取代突变为天冬酰胺被取代为缬氨酸。在一些实施方案中，本文所述的多肽在第3位具有取代突变，优选地，所述多肽的氨基酸序列如SEQ ID NO：3、4、5或118所示。
在一个或多个实施方案中，所述6位上的取代突变可以是天冬酰胺被取代为谷氨酸、亮氨酸、异亮氨酸、缬氨酸、丝氨酸或苏氨酸。在一些实施方案中，所述6位上的取代突变为天冬酰胺被取代为谷氨酸、亮氨酸或丝氨酸。在一些实施方案中，所述6位上的取代突变为天冬酰胺被取代为亮氨酸。在一些实施方案中，本文所述的多肽在第6位具有取代突变，优选地，所述多肽的氨基酸序列如SEQ ID NO：6、7、8或119所示。
在一个或多个实施方案中，所述9位上的取代突变可以是谷氨酰胺被取代为异亮氨酸、亮氨酸、缬氨酸、苯丙氨酸或甲硫氨酸。在一些实施方案中，所述9位上的取代突变为谷氨酰胺被取代为异亮氨酸、苯丙氨酸或甲硫氨酸。在一些实施方案中，本文所述的多肽在第9位具有取代突变，优选地，所述多肽的氨基酸序列如SEQ ID NO：9、10、11、120或121所示。
在一个或多个实施方案中，所述15位上的取代突变可以是谷氨酸突变被取代为苏氨酸、色氨酸、亮氨酸、缬氨酸、异亮氨酸、苯丙氨酸、丝氨酸、酪氨酸或天冬氨酸。在一些实施方案中，所述15位上的取代突变可以是谷氨酸被取代为苏氨酸、色氨酸、亮氨酸、缬氨酸、苯丙氨酸、丝氨酸或酪氨酸。在一些实施方案中，所述15位上的取代突变为谷氨酸被取代为亮氨酸或丝氨酸。 在一些实施方案中，本文所述的多肽在第15位具有取代突变，优选地，所述多肽的氨基酸序列如SEQ ID NO：12-20和122、123中任一所示。
在一个或多个实施方案中，所述23位上的取代突变可以是天冬酰胺被取代为缬氨酸、异亮氨酸、亮氨酸或酪氨酸。在一些实施方案中，所述23位上的取代突变为天冬酰胺被取代为缬氨酸、异亮氨酸或酪氨酸。在一些实施方案中，所述23位上的取代突变为天冬酰胺被取代为缬氨酸或异亮氨酸。在一些实施方案中，本文所述的多肽在第23位具有取代突变，优选地，所述多肽的氨基酸序列如SEQ ID NO：21-23中任一所示。
在一个或多个实施方案中，本发明所述多肽至少在第3位具有本文任一实施方案所述的取代突变，并任选在选自第6、9、15和23位中的一个或多个位置上、优选任选在选自第6、9和15位中的一个或多个位置上具有本文任一实施方案所述的取代突变。在另外一些实施方案中，本发明所述多肽至少在第6位具有本文任一实施方案所述的取代突变，并任选在选自第3、9、15和23位中的一个或多个位置上、优选任选在选自第3、9和15位中的一个或多个位置上具有本文任一实施方案所述的取代突变。在另外一些实施方案中，本发明所述多肽至少在第9位具有本文任一实施方案所述的取代突变，并任选在选自第3、6、15和23位中的一个或多个位置上、优选任选在选自第3、6和15位中的一个或多个位置上具有本文任一实施方案所述的取代突变。在另外一些实施方案中，本发明所述多肽至少在第15位具有本文任一实施方案所述的取代突变，并任选在选自第3、6、9和23位中的一个或多个位置上、优选任选在选自第3、6和9位中的一个或多个位置上具有本文任一实施方案所述的取代突变。在另外一些实施方案中，本发明所述多肽至少在第23位具有本文任一实施方案所述的取代突变，并在选自第3、6、9和15位中的一个或多个位置上具有本文任一实施方案所述的取代突变。在一些实施方案中，所述第3位上的取代突变为N3L或N3V，所述第6位上的取代突变为N6L或N6E，所述第9位上的取代突变为Q9I或Q9F，所述第15位上的取代突变为E15L、E15I或E15V，所述第23位上的取代突变为N23V、N23I或N23Y。
在一些实施方案中，本发明所述多肽至少在第15位具有本文任一实施方 案所述的取代突变，并在选自所述第3位、第6位、第9位和第23位的至少1个位置、至少2个位置、至少3个位置或全部4个位置上具有本文任一实施方案所述的取代突变。在一些实施方案中，所述第15位的取代突变为E15T、E15W、E15L、E15V、E15I、E15F、E15S、E15Y或E15T，所述第3位上的取代突变为N3L、N3V或N3Y，所述第6位上的取代突变为N6S、N6L或N6E，所述第9位上的取代突变为Q9I或Q9F，所述第23位上的取代突变为N23V、N23I或N23Y。优选地，所述第15位的取代突变为E15I或E15V，第3位上的取代突变为N3L或N3V，第6位上的取代突变为N6E或N6L，第9位上的取代突变为Q9I或Q9F，第23位上的取代突变为N23I、N23Y或N23V。
在一些实施方案中，本发明所述多肽在第3位、第9位和第15位具有本文任一实施方案所述的取代突变，并在第6位和/或第23位具有本文任一实施方案所述的取代突变。在一些实施方案中，所述第15位的取代突变为E15T、E15W、E15L、E15V、E15I、E15F、E15S、E15Y或E15T，所述第3位上的取代突变为N3L、N3V或N3Y，所述第6位上的取代突变为N6S、N6L或N6E，所述第9位上的取代突变为Q9I或Q9F，所述第23位上的取代突变为N23V、N23I或N23Y。优选地，所述第3位上的取代突变为N3L或N3V，所述第6位上的取代突变为N6L或N6E，所述第9位上的取代突变为Q9I或Q9F，所述第15位上的取代突变为E15L、E15I或E15V，所述第23位上的取代突变为N23V、N23I或N23Y。
在一些实施方案中，本发明所述多肽的氨基酸序列与SEQ ID NO：1或2相比，在第3位上具有N3V突变，或在第6位上具有N6E突变，或在第9位上具有Q9I或Q9F突变，或在第15位上具有E15L或E15I突变，或在3、6、9和15位上具有N3V、N6E、Q9I或Q9F以及E15L或E15I突变。
在一些实施方案中，本文所述的在选自第3、6、9、15和23位的至少两个位置上具有取代突变的多肽的氨基酸序列如SEQ ID NO：24-71中任一所述。
本发明所述多肽可直接作为配体，用于结合并分离免疫球蛋白。因此，在一些实施方案中，所述多肽还在其C端或N端包含一个或多个用于将该多肽偶联到固体支持物上的偶联元件。合适的偶联元件为本领域所周知，包括但不 先于半胱氨酸残基、多个赖氨酸残基(如3-15个或5-10个)和多个组氨酸残基(如3-15个或5-10个)。偶联元件可以是1个半胱氨酸残基，且位于多肽的C端，从而可利用半胱氨酸残基中的硫醇基与固体支持物上的亲电子基团反应而将多肽偶联到固体支持物上。偶联元件可直接与多肽连接，或者可通过接头与多肽的N或C端连接。接头可以是本领域常规使用的接头，其存在不影响到多肽的结合活性。示例性的接头为含有G和S或由G和S组成的接头序列，通常长2-20个氨基酸残基。示例性的接头序列如(GS)n、(GSS)n、(GSSS)n以及(GSSSS)n，n可为2-10的整数，但接头总长度通常不超过20个氨基酸残基。所述多肽在N端还可包含多个(如15个以内，10个以内或5以内)氨基酸残基，其源自克隆过程或来自信号转导序列的残基。作为具体实例，本发明的多肽可在N端包含AQ。
本发明还提供一种融合蛋白，该融合蛋白是一种由2-8条本发明前述多肽融合而形成。形成融合蛋白的各条多肽可全部不同、部分相同或全部相同。各多肽之间可通过C端和N端的肽键各自直接连接，或者任意两条多肽之间也可通过接头连接。接头可以是本领域常规使用的接头，其存在不影响到多肽的结合活性。示例性的接头为含有G和S或由G和S组成的接头序列，通常长2-20个氨基酸残基。示例性的接头序列如(GS)n和(GSS)n等，n可为2-10的整数，但接头总长度通常不超过20个氨基酸残基。
该融合蛋白可作为配体，用于结合并分离免疫球蛋白。因此，在一些实施方案中，该融合蛋白还在其C端或N端包含一个或多个用于将该融合蛋白偶联到固体支持物上的偶联元件。合适的偶联元件为本领域所周知，包括但不限于半胱氨酸残基、多个赖氨酸残基(如3-15个或5-10个)和多个组氨酸残基(如3-15个或5-10个)。偶联元件可以是1个半胱氨酸残基，且位于融合蛋白的C端，从而可利用半胱氨酸残基中的硫醇基与固体支持物上的前电子基团反应而将融合蛋白偶联到固体支持物上。偶联元件可直接与多肽连接，或者可通过接头与多肽的N或C端连接。接头可以是本领域常规使用的接头，其存在不影响到多肽的结合活性。示例性的接头为含有G和S或由G和S组成的接头序列，通常长2-20个氨基酸残基。示例性的接头序列如(GS)n和(GSS) n等，n可为2-10的整数，但接头总长度通常不超过20个氨基酸残基。所述融合蛋白在N端还可包含多个(如15个以内，10个以内或5以内)氨基酸残基，其源自克隆过程或来自信号转导序列的残基。作为具体实例，该融合蛋白可在其N端包含AQ。
在一些实施方案中，本发明的融合蛋白所含的2-8条多肽中，至少一条多肽为在第15位具有本文任一实施方案所述取代突变、任选在选自所述第3位、第6位、第9位和第23位的至少1个位置、至少2个位置、至少3个位置或全部4个位置上具有本文任一实施方案所述取代突变的多肽。优选地，所述融合蛋白所含的多肽中，至少一条多肽为在第3位、第9位和第15位具有本文任一实施方案所述的取代突变、并在第6位和/或第23位具有本文任一实施方案所述取代突变的多肽。优选地，所述第3位上的取代突变为N3L或N3V，所述第6位上的取代突变为N6L或N6E，所述第9位上的取代突变为Q9I或Q9F，所述第15位上的取代突变为E15L、E15I或E15V，所述第23位上的取代突变为N23V、N23I或N23Y。
在一些实施方案中，形成本发明融化蛋白的本发明所述多肽至少包括具有以下取代突变的多肽中的一条或多条：SEQ ID NO：4、SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：10、SEQ ID NO：11、SEQ ID NO：14、SEQ ID NO：16、SEQ ID NO：18、SEQ ID NO：21、SEQ ID NO：22、SEQ ID NO：51、SEQ ID NO：53、SEQ ID NO：60、SEQ ID NO：71、EQ ID NO：64、SEQ ID NO：69、SEQ ID NO：70、SEQ ID NO：68、SEQ ID NO：118、SEQ ID NO：119、SEQ ID NO：120、SEQ ID NO：121、SEQ ID NO：122、SEQ ID NO：123和SEQ ID NO：124。本发明中示例性的融合蛋白的氨基酸序列如SEQ ID NO：73-109和111-117中任一所示。
本发明还提够一种重组蛋白A，其B结构域为本发明任意实施方案所述的Z结构域突变体或B结构域突变体。该重组蛋白A的其余结构域可以是本领域周知的E结构域、D结构域、A结构域和C结构域，包括天然的E结构域、D结构域、A结构域和B结构域以及已知的突变但保留了生物活性的E结构域、D结构域、A结构域和/或C结构域。除B结构域采用本发明的多肽外，本领域已知的对蛋白A其余部分做出的突变或修饰均可用于本发明的重组蛋 白A。
本文也包括与本文任一实施方案所述的多肽、融合蛋白或重组蛋白A具有至少85％的序列相同性，并在选自对应于SEQ ID NO：1或2第3、6、9、15和23位的一个或多个氨基酸位置上具有本文任一实施方案所述的取代突变的多肽、融合蛋白和重组蛋白A。本文中，涉及氨基酸序列和核苷酸序列的“至少85％相同性”，是指85％以上的相同性，优选为90％以上的相同性，更优选为95％以上的相同性，进一步优选为97％以上的相同性，进一步优选为98％以上的相同性，更进一步优选为99％以上的相同性。可采用本领域周知的工具计算比对的两条序列之间的相同性，这些工具包括用于氨基酸序列比对的BLASTP等。在优选的实施方案中，本发明包括与SEQ ID NO：3-71、118-124和73-109、111-117中任一所示的氨基酸序列的变体，SEQ ID NO：3-71、118-124和73-109、111-117各自的变体与对应的亲本序列相比具有至少85％、优选至少90％、更优选至少97％、更优选至少99％的序列相同性且其在对应的亲本第3、6、9、15和23位上的氨基酸残基仍保持为对应亲本的氨基酸残基。
本发明也包括编码前述多肽、融合蛋白以及重组蛋白A的核酸分子及其互补序列。本发明包括本发明核酸分子的所有形式，包括RNA和DNA。应理解，本文所述的互补序列指长度与该核酸分子基本一致的互补序列。
含有所述核酸分子或其互补序列的核酸构建物也包括在本发明的范围内。核酸构建物可以是一表达盒，其含有启动子、所述核酸分子以及转录终止序列。启动子和转录终止序列为本领域所周知，并可由本领域技术人员根据所选择的用于表达所述多肽、融合蛋白或重组蛋白A的宿主细胞而做出适当的选择。
在一些实施方案中，核酸构建物为载体，包括表达载体和克隆载体。表达载体是适合在宿主细胞中表达外源基因如编码本发明所述多肽、融合蛋白或重组蛋白A的核酸分子的载体，包括原核表达载体和真核表达载体。真核表达系统有酵母表达系统，哺乳细胞表达系统和昆虫细胞表达系统等。克隆载体用于在宿主细胞中进行目的基因(如编码本发明所述多肽、融合蛋白或蛋白A的核酸分子)的扩增。克隆载体包括质粒载体、噬菌体载体、病毒载体、以及由它们互相组合或者与其他基因组DNA组合而成的载体。分子克隆中最常用的宿 主细胞是大肠杆菌。
本发明还提供一种表达系统，其包含本文公开的核酸分子、核酸构建物或载体。表达系统可为例如革兰氏阳性或革兰氏阴性原核宿主细胞系统，例如经修饰以表达本发明的多肽、融合蛋白或重组蛋白A的大肠杆菌或芽孢杆菌。在一些实施方案中，表达系统是真核宿主细胞系统，例如酵母，例如巴斯德毕赤酵母或酿酒酵母。
本发明还提供一种分离基质。本发明的分离基质可用于分离免疫球蛋白或含Fc的蛋白质。本发明的分离基质含有与固体支持物偶联的本发明任一实施方案所述的多肽、融合蛋白和/或重组蛋白A。由于本发明的多肽、融合蛋白和重组蛋白A的碱稳定性改进，本发明的分离基质在清洗时可承受高碱性条件(0.5-2.0M的NaOH)。
本发明分离基质中的固体支持物可以是本领域已知的在免疫球蛋白或含Fc的蛋白质的分离中使用到的固体支持物。
本文中，固相支持物的形状可以为粒子、膜、板、管、针状、纤维状等任意的形状。本发明的固体支持物可以是多孔状的材料，或者为无孔的材料。在一些实施方案中，固体支持物为多孔或无孔的珠状形式或颗粒形式。呈珠状形式或颗粒形式的基质可作为填充床或以悬浮形式使用。悬浮形式包括膨胀床，其中颗粒或小珠自由移动。
在该固相支持物为粒子的情况下，粒径优选为20-200μm。例如，在固相支持物为合成聚合物的情况下，粒径优选为20-100μm，优选为30-80μm。在固相支持物为多糖的情况下，粒径优选为50-200μm，更优选为60-150μm。本说明书中的“粒径”是指以按照ISO 13320和JIS Z 8825-1的激光衍射法进行测定而得的体积平均粒径。
固体支持物的例子包括但不限于包含多羟基的多聚体，例如多糖。多糖包括葡聚糖、淀粉、纤维素、普鲁兰多糖、琼脂和琼脂糖等。在优选的实施方案中，固体支持物包含琼脂或琼脂糖。
在一些实施方案中，本发明的固体支持物为具有清水性表面的合成聚合物。例如，这类聚合物可以是通过亲水化处理而在外表面(以及在存在的情况下也 在内表面)具有羟基、羧基、氨基羰基、氨基或寡乙烯氧基或聚乙烯氧基的聚合物，优选为诸如由多官能(甲基)丙烯酸酯、二乙烯基苯等多官能单体进行交联而得的共聚物的合成聚合物。示例性的合成聚合物包括聚乙烯醇、聚苯乙烯、聚苯乙烯二乙烯苯、多羟基烷基丙烯酸酯、多羟基烷基甲基丙烯酸酯、聚丙烯酰胺和聚甲基丙烯酰胺等。在聚合物表面疏水的情况下，可对其表面进行被亲水化处理，以使亲水基团暴露于周围的水性液体中。
在一些实施方案中，本发明的固体支持物包含无机性质的支持物，包括但不限于二氧化硅和氧化锆等。
在一些实施方案中，本发明的固体支持物呈表面、芯片、毛细管或滤器的形式，例如膜等。
本发明中，本发明所述的多肽、融合蛋白和重组蛋白A可通过其所含有的硫醇基、氨基和/或羧基等通过常规偶联技术与相应的固体支持物连接。常用的偶联试剂包括但不限于双环氧化物、表氯醇、CNBr和N-羟基琥珀酰亚胺。在固体支持物和本发明所述多肽、融合蛋白和重组蛋白A之间可引入间隔基，其可促进它们与固体支持物的化学偶联。
在一些实施方案中，本发明所述的多肽、融合蛋白或重组蛋白A可通过硫醚键与固体支持物偶联。在一些实施方案中，本发明所述的多肽、融合蛋白或重组蛋白A通过其C端的半胱氨酸偶联，其中，该半胱氨酸硫醇基与固体支持物上的亲电子基团如环氧基、卤代醇基等有效偶联，形成硫醚桥偶联。
本发明的分离基质中，与固体支持物偶联的结合配体(如本发明所述的多肽、融合蛋白和/或重组蛋白A)的浓度通常为5-20mg/ml，例如5-15mg/ml。可通过调整偶联过程中所用的多肽、融合蛋白和/或重组蛋白A的浓度、所用的偶联条件和/或所用固体支持物的孔结构，来控制偶联的所述结合配体的量。
本发明还提供一种色谱柱，其含有本发明的分离基质。通常，将本发明的分离基质填充于色谱柱中。
本发明还提供一种分离含Fc的蛋白质(如免疫球蛋白)的方法，该方法包括使本文任一实施方案所述的多肽、融合蛋白和/或重组蛋白A与含有含Fc的蛋白质的样品接触的步骤。在一些实施方案中，所述方法包括使样品与本文 任一实施方案所述的分离基质或色谱柱接触的步骤。
更具体而言，在一些实施方案中，该方法包括：
(1)使含有含Fc的蛋白质的样品与本文任一实施方案所述的分离基质接触；
(2)洗涤分离基质；
(3)从分离基质上洗脱所述含Fc的蛋白质；
(4)清洗分离基质。
本文中，样品可以是各类含有含Fc的蛋白质(尤其是免疫球蛋白)的样品。优选地，本文所述的免疫球蛋白指IgG。
所述方法中使用到的用于洗涤分离基质的洗液、用于洗脱蛋白质的洗脱液以及用于清洗分离基质的清洗液均为本领域(尤其是蛋白A层析领域)常规使用的洗液、洗脱液以及清洗液。例如，洗液可以是PBS缓冲液。洗脱液可以是pH≤5的溶液或缓冲液，优选地，洗脱液的pH为2.5-5或3-5。在一些实施方案中，洗脱液的pH为11或更高，例如洗脱液的pH为11-14或11-13。在一些实施方案中，使用柠檬酸盐溶液，如柠檬酸钠溶液进行洗脱。清洗液通常是碱性的，其pH可为13-14。在一些实施方案中，清洗液为氢氧化钠溶液或氢氧化钾溶液，其浓度为0.1-2.0M，例如0.5-2.0M或0.5-1.0M。
在一些实施方案中，本文所述方法还包括回收洗脱物并对其进行进一步分离纯化的步骤。可根据所分离的蛋白，通过阴离子或阳离子交换层析法、复合型离子交换层析法和/或疏水相互作用层析法等进行进一步的分离纯化。
本发明还提供本文任一实施方案所述的多肽、融合蛋白以及蛋白A在分离纯化含Fc的蛋白质中的应用，或在制备用于分离纯化含Fc的蛋白质的分离基质中的应用。
本文所述的序列如下：



















下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
制备例1：Z结构域突变体和B结构域突变体的制备
设计好突变位点并委托生工生物工程(上海)股份有限公司(Sangon Biotech)全基因合成、表达和纯化。
表达和纯化时，利用单个重组质粒转化到大肠杆菌，并通过LB液体培养基进行发酵培养并进行诱导表达。发酵后，收集菌体并采用热裂解的方式破坏细胞壁将表达产物释放出来并离心分离。将分离液体经过IgG亲和介质进行纯化，分离液上样经过亲和介质，用10mM的磷酸盐缓冲液清洗介质，并用pH3.8的缓冲溶液收集目的蛋白，调节到中性环境，保存待用。
合成的部分突变体的基因结果验证分析图如图1-13所示。
制备例2：使用Z结构域突变体和B结构域突变体制备亲和层析介质
利用得到的Z结构域突变体和B结构域突变体，采用常规的方法制备亲和层析介质。示例性的制备过程具体包括以下步骤：
a.活化
取10g高刚性琼脂糖，经纯化水清洗并抽干。称取10g上述琼脂糖，加入20mL纯化水，0.2g氢氧化钠以及10mL环氧氯丙烷加入100mL烧瓶中，在27℃下恒温反应2小时，将活化的凝胶用1L水洗涤，得到活化的琼脂糖微球。
b.交联
将活化后的琼脂糖微球加入到100mL烧瓶中，再加入150mg NaHCO3，10mg Na2CO3，150mg NaCl和10mg EDTA，搅拌均匀后加入50mL突变体溶液，在34℃下恒温反应8小时，完成交联。
c.封闭
在交联产物中加入100mL封闭液(乙醇胺溶液)，用氢氧化钠调整pH为8.6，26℃下恒温反应2小时，完成对残留的环氧基团的封闭，减小影响。
实施例1
使用下面描述的方法对表1的Z结构域突变体和B结构域突变体进行动力学分析。
实验步骤如下：
1)用IgG交联Series S CM5芯片。
a.用10mM乙酸-NaOH(pH5.0)稀释IgG至5μg/ml、10μg/ml、15μg/ml、20μg/ml。
b.配置运行缓冲液(10mM乙酸-NaOH，pH5.0)、解吸液1(0.5％SDS)、解吸液2(50mM甘氨酸-NaOH，pH9.5)、封闭液(1M乙醇胺pH8.5)、活化剂(0.4M EDC、0.1MNHS)。
c.在Biacore-4000中运行固定化。
d.交联结果如下表所示：

2)动力学分析
a.用1XPBS(pH7.6)稀释突变体样品至0μg/ml、1.615μg/ml、2.4375μg/ml、3.25μg/ml、4.875μg/ml、6.5μg/ml、9.75μg/ml、13μg/ml、19.5μg/ml、26μg/ml。
b.配置跑胶缓冲液1XPBS(pH7.6)、再生溶液(50mM柠檬酸-NaOH pH3.0)。
c.在Biacore-4000中运行动力学和亲和学。
接触时间 流速 解离时间 再生时间
150s 30μl/min 250s 120s
实验结果如下表1所示。表1显示了利用Biacore对Z结构域突变体和B结构域突变体进行的动力学分析结果。
表1
突变体 SEQ ID NO Ka Kd KD
(Z结构域)4 72 1.340x105 2.971x10-4 2.217x10-9
Z(N3L)4 73 1.334x105 2.346x10-4 1.759x10-9
Z(N3V)4 74 1.121x105 4.509x10-4 4.022x10-9
Z(N6L)4 75 8.815x104 2.693x10-4 3.055x10-9
Z(N6E)4 76 1.726x105 2.752x10-4 1.594x10-9
Z(Q9I)4 77 1.340x105 2.971x10-4 1.340x105
Z(Q9F)4 78 1.296x105 2.250x10-4 1.736x10-9
Z(Q9M)4 79 1.050x105 2.584x10-4 2.462x10-9
Z(E15T)4 80 1.331x105 6.263x10-4 4.707x10-9
Z(E15W)4 81 1.068x105 1.646x10-4 1.542x10-9
Z(E15L)4 82 1.065x105 1.330x10-4 1.249x10-9
Z(E15V)4 83 1.345x105 3.573x10-4 2.656x10-9
Z(E15I)4 84 2.248x105 5.732x10-4 2.551x10-9
Z(E15F)4 85 1.112x105 4.440x10-4 3.994x10-9
Z(E15S)4 86 2.049x105 3.651x10-4 1.782x10-9
Z(E15Y)4 87 1.389x105 4.051x10-4 2.917x10-9
Z(E15D)4 88 2.098x105 2.367x10-4 1.128x10-9
Z(N23V)4 89 9.553x104 3.468x10-4 3.638x10-9
Z(N23I)4 90 7.875x104 4.614x10-4 5.859x10-9
Z(N23Y)4 91 1.718x105 2.544x10-4 1.481x10-9
Z(N3L，N6L，Q9I，E15L)4 95 1.689x105 3.535x10-4 2.093x10-9
Z(N3V，N6E，Q9I，E15L)4 97 1.380x105 3.752x10-4 2.720x10-9
Z(N3L，Q9I，E15L，N23V)4 98 1.271x105 5.610x10-4 4.414x10-9
Z(N6L，Q9F，E15I，N23V)4 99 1.135x105 4.541x10-4 3.977x10-9
Z(N3L，N6L，Q9I，E15L，N23V)4 100 1.322x105 3.083x10-4 2.331x10-9
Z(N3L，N6E，Q9I，E15I，N23I)4 101 6.995x104 1.796x10-4 2.567x10-9
Z(N3V，N6L，Q9F，E15V，N23Y)4 102 9.564x104 2.641x10-4 2.762x10-9
(B结构域)4 110 1.003x105 2.861x10-4 2.852x10-9
B(N3V)4 111 7.052x104 2.921x10-4 4.142x10-9
B(N6E)4 112 7.656x104 1.655x10-4 2.162x10-9
B(Q9I)4 113 1.802x105 5.513x10-4 3.306x10-9
B(Q9F)4 114 8.360x104 1.842x10-4 2.203x10-9
B(E15L)4 115 1.513x105 3.647x10-4 2.410x10-9
B(E15I)4 116 1.083x105 3.614x10-4 3.337x10-9
B(N3V，N6E，Q9I，E15L)4 117 1.583x105 2.101x10-4 1.328x10-9
结果表明，各突变体与相应的Z结构域或B结构域相比，其KD值基本都处于一个数量级，解离常数并没有明显变化，说明本发明对Z结构域和B结构域进行突变得到的突变体对于IgG的结合能力没有影响。
实施例2
使用下面描述的方法对表2的Z结构域突变体和B结构域突变体亲和层析介质的IgG结合能力和耐碱稳定性进行评价。
实验步骤如下：
1)取4.4mL Z结构域突变体或B结构域突变体的亲和层析介质，用20％乙醇，5ml/min的流速装入层析柱中。
缓冲液：缓冲液A(PBS，pH 7.6)；缓冲液B(0.1M柠檬酸钠，pH 3.0)；缓冲液C(1M NaOH)。
样品：用纯品配制1mg/ml的IgG样品(稀释纯品的溶液为缓冲液A)。
2)10％动态载量测定：
设备：AKTA Pure。
测定过程：
a.缓冲液A平衡层析柱，流速：1.8ml/min；
b.IGg样品不流经柱子时，检测其紫外吸收峰值，计为λmax，并据此计算10％λmax；
c.上样：流速：1.8ml/min，记录基础流穿的紫外吸收峰值；
d.二者相加得出10％动态载量对应的紫外吸收峰值；
e.继续上样至紫外吸收峰值达到步骤d中紫外吸收峰值时所对应的体积为10％动态载量。
3)耐碱循环：
控制室温：23±0.5℃；根据载量的下降更新程序中的上样量，其他程序保持一致。
测定过程：
a.PBS平衡：流速：1.8ml/min；体积：1cv；
b.上样：流速：1.8ml/min；200ml(后续程序根据载量的下降更新上样量)；
c.PBS清洗：流速：1.8ml/min；体积：5cv；
d.0.1M柠檬酸钠洗脱：流速：1.8ml/min；体积：3cv；
e.1M NaOH处理2h：先用1.8ml/min的流速过1cv使层析柱内充满1M NaOH，后改为流速：0.5ml/min；体积：60ml；时间：2h；
f.PBS平衡：流速：1.8ml/min；体积：10cv；
4)上述碱处理程序进行13次，记录对应载量，绘制图表，结果见表2和图14、15(Z(N3V，N6E，Q9I，E15L)4)和16。表2显示了各样品在10％穿透下(Qb10％)的IgG能力(以动态结合载量计，mg/ml)。
表2

结果表明，突变体的初始IgG结合能力与Z结构域相比相差不大，表中突变体的初始IgG结合能力均在130mg/ml以上，具有较好的亲和能力。而在1.0M NaOH碱处理24小时后，除小部分突变体显示出比Z结构域稍差的IgG结合能力外，大多数突变体的IgG结合能力均优于Z结构域和B结构域。其中，突变体Z(E15S)4在碱处理24小时后，仍具有43.57％的剩余IgG能力，这些数据表明本发明选择的突变位点可以改善Z结构域和B结构域的碱性稳定性。
实施例3
用表3中的Z结构域突变体制备得到的亲和层析介质重复实施例2，结果见表3和图15。表3显示了各样品在10％穿透下(Qb10％)的IgG能力(以动态结合载量计，mg/ml)。
表3

表3显示了Z结构域上多个位点突变的突变体的耐碱性，在1.0M NaOH处理24小时后，突变体的剩余IgG结合能力较Z结构域均有一定程度提高，表明上述突变提高了耐碱性。其中，突变体Z(N3L，N6L，Q9F，E15I，N23V)6在碱处理24小时后，仍有63.93％的IgG能力。

Claims (23)

  1. 一种分离的多肽，其特征在于，所述多肽选自：
    (1)与SEQ ID NO：1所示的蛋白A的天然B结构域或SEQ ID NO：2所示的蛋白A的Z结构域相比，该多肽在选自第3位、第6位、第9位、第15位和第23位的一个或多个位置上具有取代突变；
    其中，所述第3位上的取代突变为天冬酰胺被取代为亮氨酸、异亮氨酸、缬氨酸或酪氨酸；所述第6位上的取代突变为天冬酰胺被取代为谷氨酸、亮氨酸、异亮氨酸、缬氨酸、丝氨酸或苏氨酸；所述第9位上的取代突变为谷氨酰胺被取代为异亮氨酸、亮氨酸、缬氨酸、苯丙氨酸或甲硫氨酸；所述第15位上的取代突变为谷氨酸被取代为苏氨酸、色氨酸、亮氨酸、缬氨酸、异亮氨酸、苯丙氨酸、丝氨酸、酪氨酸或天冬氨酸；所述第23位上的取代突变为天冬酰胺突变为缬氨酸、异亮氨酸、亮氨酸或酪氨酸；
    (2)与(1)所述的多肽具有至少85％序列相同性并保留了所述的第3位、第6位、第9位、第15位和第23位中一个或多个位置上的取代突变的多肽。
  2. 如权利要求1所述的多肽，其特征在于，
    所述第3位上的取代突变为天冬酰胺被取代为亮氨酸、缬氨酸或酪氨酸；
    所述第6位上的取代突变为天冬酰胺被取代为谷氨酸、亮氨酸或丝氨酸；
    所述第9位上的取代突变为谷氨酰胺被取代为异亮氨酸、苯丙氨酸或甲硫氨酸；
    所述第15位上的取代突变为谷氨酸被取代为苏氨酸、色氨酸、亮氨酸、异亮氨酸、缬氨酸、苯丙氨酸、丝氨酸或酪氨酸；
    所述第23位上的取代突变为天冬酰胺突变为缬氨酸、异亮氨酸或酪氨酸。
  3. 如权利要求1或2所述的多肽，其特征在于，所述多肽：
    至少在第3位具有所述取代突变，并在选自所述第15位、第6位、第9位和第23位的至少1个位置、至少2个位置、至少3个位置或全部4个位置上具有所述取代突变；
    至少在第6位具有所述取代突变，并在选自所述第3位、第15位、第9 位和第23位的至少1个位置、至少2个位置、至少3个位置或全部4个位置上具有所述取代突变；
    至少在第9位具有所述取代突变，并在选自所述第3位、第6位、第15位和第23位的至少1个位置、至少2个位置、至少3个位置或全部4个位置上具有所述取代突变；
    至少在第15位具有所述取代突变，并在选自所述第3位、第6位、第9位和第23位的至少1个位置、至少2个位置、至少3个位置或全部4个位置上具有所述取代突变；或
    至少在第23位具有所述取代突变，并在选自所述第3位、第6位、第9位和第15位的至少1个位置、至少2个位置、至少3个位置或全部4个位置上具有所述取代突变；
    优选地，所述第15位的取代突变为E15I、或E15L或E15V，第3位的取代突变为N3L或N3V，第6位的取代突变为N6E或N6L，第9位的取代突变为Q9I或Q9F，第23位的取代突变为N23I、N23Y或N23V。
  4. 如权利要求1或2所述的多肽，其特征在于，所述多肽在第3位、第9位和第15位具有所述取代突变，并在第6位和/或第23位具有所述取代突变；
    优选地，所述第3位的取代突变为N3L或N3V，所述第6位的取代突变为N6L或N6E，所述第9位的取代突变为Q9I或Q9F，所述第15位的取代突变为E15L、E15I或E15V，所述第23位的取代突变为N23V、N23I或N23Y。
  5. 如权利要求1所述的多肽，其特征在于，所述多肽的氨基酸序列如SEQ ID NO：3-71和118-124中任一所示。
  6. 一种分离的多肽，其特征在于，所述多肽由以下(i)、(ii)和(iii)组成：(i)权利要求1-5中任一项所述的多肽，(ii)在(i)所述多肽的氨基酸序列的C端或N端的一个或多个偶联元件，和任选的(iii)来自被切下的信号转导序列的残基。
  7. 如权利要求6所述的多肽，其特征在于，所述偶联元件选自：半胱氨酸残基、多个赖氨酸残基和多个组氨酸残基；所述被切下的信号转导序列的残 基为AQ。
  8. 一种融合蛋白，其特征在于，该融合蛋白包含由2-8条权利要求1-5中任一项所述的多肽融合形成的氨基酸序列，其中，所述2-8条多肽互不相同、部分相同或完全相同，且所述多肽之间任选地存在接头序列。
  9. 如权利要求8所述的融合蛋白，其特征在于，所述融合蛋白所含的2-8条多肽中，至少一条多肽为在第15位具有所述取代突变、任选在选自所述第3位、第6位、第9位和第23位的至少1个位置、至少2个位置、至少3个位置或全部4个位置上具有所述取代突变的多肽；优选地，至少一条多肽为在第3位、第9位和第15位具有所述取代突变、并在第6位和/或第23位具有所述取代突变的多肽。
  10. 如权利要求8所述的融合蛋白，其特征在于，所述融合蛋白中，所述多肽选自：SEQ ID NO：4、SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：10、SEQ ID NO：11、SEQ ID NO：14、SEQ ID NO：16、SEQ ID NO：18、SEQ ID NO：21、SEQ ID NO：22、SEQ ID NO：51、SEQ ID NO：53、SEQ ID NO：60、SEQ ID NO：71、EQ ID NO：64、SEQ ID NO：69、SEQ ID NO：70、SEQ ID NO：68、SEQ ID NO：118、SEQ ID NO：119、SEQ ID NO：120、SEQ ID NO：121、SEQ ID NO：122、SEQ ID NO：123和SEQ ID NO：124中的一条或多条。
  11. 如权利要求8-10中任一项所述的融合蛋白，其特征在于，所述融合蛋白的C端或N端还包括一个或多个偶联元件和/或来自被切下的信号转导序列的残基；优选地，所述偶联元件选自：半胱氨酸残基、多个赖氨酸残基和多个组氨酸残基。
  12. 如权利要求8所述的融合蛋白，其特征在于，所述融合蛋白的氨基酸序列如SEQ ID NO：73-109和110-117中任一所示。
  13. 一种重组蛋白A，该重组蛋白A的B结构域为权利要求1-5中任一项所述的多肽。
  14. 一种分离的核酸分子，所述核酸分子的多核苷酸序列选自：
    (1)编码权利要求1-7中任一项所述的多肽、权利要求8-12中任一项所述的融合蛋白或权利要求13所述的重组蛋白A的多核苷酸序列；
    (2)(1)所述多核苷酸序列的互补序列。
  15. 一种核酸构建物，其特征在于，所述核酸构建物含有权利要求14所述的核酸分子；优选地，所述核酸构建物为表达盒；更优选地，所述核酸构建物为表达载体或克隆载体。
  16. 一种表达系统，其含有权利要求15所述的核酸构建物；优选地，所述表达系统为宿主细胞。
  17. 一种分离基质，其特征在于，所述分离基质包括与固体支持物偶联的权利要求1-7中任一项所述的多肽、权利要求8-12中任一项所述的融合蛋白和/或权利要求13所述的重组蛋白A。
  18. 如权利要求17所述的分离基质，其特征在于，所述多肽、融合蛋白或重组蛋白A通过硫醚键与所述固体支持物偶联。
  19. 如权利要求18所述的分离基质，其特征在于，所述固体支持物选自：
    含多羟基的多聚体，优选为多糖，更优选选自：葡聚糖、淀粉、纤维素、普鲁兰多糖、琼脂和琼脂糖；
    合成的聚合物，优选选自：聚乙烯醇、聚苯乙烯、聚苯乙烯二乙烯苯、多羟基烷基丙烯酸酯、多羟基烷基甲基丙烯酸酯、聚丙烯酰胺和聚甲基丙烯酰胺；和
    无机性质的支持物，优选选自二氧化硅和氧化锆。
  20. 一种色谱柱，其特征在于，所述色谱柱含有权利要求17-19中任一项所述的分离基质。
  21. 一种分离含Fc的蛋白质的方法，其特征在于，所述方法包括使含免疫球蛋白的样品与权利要求1-7中任一项所述的多肽、权利要求8-12中任一项所述的融合蛋白、权利要求13所述的重组蛋白A、权利要求17-19中任一项所述的分离基质或权利要求20所述的色谱柱接触的步骤；优选地，所述含Fc的蛋白质为免疫球蛋白。
  22. 如权利要求21所述的方法，其特征在于，所述方法包括：
    (1)使含有含Fc的蛋白质的样品与所述分离基质接触；
    (2)洗涤分离基质；
    (3)从分离基质上洗脱所述含Fc的蛋白质；
    (4)清洗分离基质；
    优选地，使用0.1-2.0M或0.5-1.0M的NaOH或KOH溶液清洗分离基质。
  23. 权利要求1-7中任一项所述的多肽、权利要求8-12中任一项所述的融合蛋白、权利要求13所述的重组蛋白A在分离含Fc的蛋白质中的应用，或在制备用于分离含Fc的蛋白质的分离基质或色谱柱中的应用。
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