Immunoglobulin-binding polypeptide

Abstract

The purpose of the present invention is to provide a polypeptide, which has excellent alkali stability and allows the elution of immunoglobulin or a fragment of the same, which has been bound thereto, in a weakly acidic region, by modifying the amino acid sequence in an immunoglobulin-binding domain of Protein A. A polypeptide, which has a binding activity to immunoglobulin or a polypeptide having the Fc region of the same and which has excellent alkali stability and allows efficient elution of immunoglobulin or the polypeptide having the Fc region of the same, which has been bound thereto, under weakly acidic mild conditions, can be obtained by substituting serine at the 41st position in each immunoglobulin-binding domain of Protein A with an amino acid having a hydrophobic side chain, tyrosine or histidine.

Description

Technical Field

[0001] The present invention relates to a polypeptide that binds to a polypeptide containing an immunoglobulin or its Fc region. More specifically, the present invention relates to a polypeptide having binding activity to a polypeptide containing an immunoglobulin or its Fc region, excellent stability under alkaline conditions (alkaline stability), and capable of efficiently eluting the once-bound immunoglobulin or its fragment in a weakly acidic region. Furthermore, the present invention relates to a method for producing the polypeptide, an immobilized product of the polypeptide, and a method for separating an immunoglobulin or its fragment using the polypeptide, etc.

Background Art

[0002] Conventionally, immunoglobulin (also referred to as an antibody) has high binding specificity to a target substance and is widely used as a research reagent or a clinical test reagent. In recent years, with the progress of gene recombination technology, the production technology of human antibodies and humanized antibodies has been established, and immunoglobulin has come to be put into practical use in medical fields such as rheumatism and cancer as an antibody drug.

[0003] Immunoglobulin is mainly produced by animal cell culture, and affinity chromatography using a ligand having an immunoglobulin-binding ability is widely used for its purification. In the affinity chromatography used for the purification of immunoglobulin, polypeptides such as protein A, protein L, protein G, or their immunoglobulin-binding domains are used as ligands that specifically bind to immunoglobulin.

[0004] On the other hand, in recent years, there has been a demand for improving the stability of the ligand used for the purification of immunoglobulin. In particular, in the purification of immunoglobulin, an alkaline solution is used for inactivating viruses, etc., and washing the carrier to which the ligand is immobilized, etc., and high stability against alkali is one important factor for the ligand used for the purification of immunoglobulin.

[0005] Conventionally, techniques for improving the alkaline stability of protein A have been actively investigated. For example, Patent Document 1 reports that by substituting a specific amino acid in the C domain of protein A, or a specific amino acid residue in the Z domain of protein A, excellent immunoglobulin binding ability and alkaline stability can be achieved. Furthermore, Patent Document 2 reports that by substituting an asparagine residue in protein A with another amino acid, alkaline stability can be achieved.

[0006] On the other hand, when eluting protein A or immunoglobulins bound to its immunoglobulin-binding domain, strongly acidic eluents (pH 3 or lower) are typically used. Using such strongly acidic eluents can lead to immunoglobulin denaturation and subsequent aggregate formation. Therefore, there is a need for the development of immunoglobulin-binding polypeptides that can elute bound immunoglobulins under mild, weakly acidic conditions.

[0007] As mentioned above, although various techniques for conferring alkaline stability to protein A and its immunoglobulin-binding domain have been reported, techniques for efficiently eluting the immunoglobulin or its fragments once bound in a weakly acidic environment have not been sufficiently investigated. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] International Publication Number 2007 / 097361 [Patent Document 2] International Publication No. 2000 / 023580 [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] The object of the present invention is to provide a polypeptide that exhibits excellent alkali stability and can elute immunoglobulin or a polypeptide containing its Fc region, once bound, in a weakly acidic region by modifying the amino acid sequence of the immunoglobulin-binding domain of protein A. [Means for solving the problem]

[0010] The inventors of the present invention conducted diligent studies to solve the aforementioned problems and found that by substituting the serine at position 41 in each immunoglobulin-binding domain of protein A with an amino acid, tyrosine, or histidine having a hydrophobic side chain, it is possible to obtain a polypeptide that has binding activity to immunoglobulins or polypeptides containing their Fc region, exhibits excellent alkali stability, and can efficiently elute the immunoglobulin or polypeptide containing its Fc region once bound under mild, weakly acidic conditions. Furthermore, in addition to the substitution at position 41, the inventors found that by substituting the glutamine at position 9 with an amino acid or histidine having a hydrophobic aliphatic side chain, or by substituting glutamic acid at position 15 with alanine or histidine and glutamic acid or alanine at position 24 with glutamine, alkali stability and elution in the weakly acidic region can be further improved. The present invention was completed by further studies based on these findings.

[0011] In other words, the present invention provides inventions in the following embodiments. Item 1. A polypeptide comprising at least one immunoglobulin-binding domain as shown in any of (A) to (C) below. (A) An immunoglobulin-binding domain containing an amino acid sequence in which any of the amino acid sequences shown in Sequence ID No. 1 to 15 has been modified to satisfy the following conditions (i) to (iv): (i) The serine at position 41 is replaced with an amino acid, tyrosine, or histidine having a hydrophobic side chain. (ii) The glutamine at position 9 is unsubstituted, or substituted with an amino acid or histidine having a hydrophobic aliphatic side chain. (iii) The glutamic acid or glutamine at position 15 is unsubstituted or substituted with alanine, histidine, tyrosine, or leucine. (iv) The glutamic acid or alanine at position 24 is unsubstituted, substituted with glutamine, histidine, or alanine in the case of SEQ ID NOs. 1-12, or substituted with glutamine or histidine in the case of SEQ ID NOs. 13-15. (B) An immunoglobulin-binding domain having an amino acid sequence shown in any of Sequence IDs 1 to 15, in which a modification satisfying the conditions of (i) to (iv) above has been introduced, and one or more amino acids in the unmodified site have been substituted, added, inserted and / or deleted, and which has alkaline stability equal to or better than that of a polypeptide consisting of the corresponding unmodified amino acid sequence, and high IgG elution ability in the weakly acidic region. (C) An immunoglobulin-binding domain in which a modification satisfying the conditions of (i) to (iv) above has been introduced in any of the amino acid sequences shown in Sequence ID No. 1 to 15, the sequence identity of the unmodified region with respect to the corresponding unmodified amino acid sequence is 80% or more, and the alkali stability is equal to or better than that of a polypeptide consisting of the corresponding unmodified amino acid sequence, and the IgG elution ability in the weakly acidic region is high. Item 2. The polypeptide described in Item 1, which is a single-domain peptide containing one immunoglobulin-binding domain selected from among the immunoglobulin-binding domains shown in (A) to (C) above. Item 3. The polypeptide according to Item 1, which is a polydomain peptide comprising two or more immunoglobulin-binding domains selected from the immunoglobulin-binding domains shown in (A) to (C) above. Item 4. The polypeptide according to any one of items 1 to 3, wherein the glutamine at position 9 in the amino acid sequence is substituted with an amino acid having a hydrophobic aliphatic side chain or histidine. Item 5. In the above amino acid sequence, glutamic acid or glutamine at position 15 is substituted with alanine or histidine, and The polypeptide described in any of items 1 to 4, wherein in the case of SEQ ID NOs. 1 to 12, the glutamic acid at position 24 is replaced with glutamine, or in the case of SEQ ID NOs. 13 to 15, the alanine at position 24 is replaced with glutamine. Item 6. The polypeptide according to any one of items 1 to 5, wherein the serine at position 41 in the amino acid sequence is substituted with alanine, valine, leucine, phenylalanine, tyrosine, or histidine. Item 7. The polypeptide according to any one of items 1 to 6, wherein the glutamine at position 9 in the amino acid sequence is substituted with alanine, valine, leucine, isoleucine, or histidine. Item 8. DNA encoding a polypeptide as described in any of items 1-7. Item 9. A recombinant vector containing the DNA described in Item 8. Item 10. A transformant obtained by transforming a host using the recombinant vector described in Item 9. Item 11. A method for producing a polypeptide according to any one of items 1 to 7, comprising the step of culturing the transformant described in item 10. Item 12. An immunoglobulin-binding carrier comprising a polypeptide described in any of Items 1 to 7 immobilized on an insoluble carrier. Item 13. A method for separating an immunoglobulin or a fragment thereof, comprising separating an immunoglobulin or a polypeptide containing its Fc region using an immunoglobulin-binding carrier as described in Item 12. Item 14. The separation method according to Item 13, wherein an immunoglobulin or a polypeptide containing its Fc region is first bound to the immunoglobulin-binding carrier, and then the immunoglobulin or the polypeptide containing its Fc region is eluted under pH conditions of 3 to 5. [Effects of the Invention]

[0012] The polypeptide of the present invention has the ability to bind to a polypeptide containing an immunoglobulin or its Fc region, and even when exposed to alkaline conditions, it can maintain the ability to bind to a polypeptide containing an immunoglobulin or its Fc region. Therefore, even if elution and washing with an alkaline solution are repeated, a decrease in the binding ability to a polypeptide containing an immunoglobulin or its Fc region can be suppressed. In addition, the polypeptide of the present invention can efficiently elute the once-bound immunoglobulin or its fragment under mild conditions of weak acidity (about pH 3 to 5), so that denaturation of the polypeptide containing the immunoglobulin or its Fc region to be separated can be suppressed.

Brief Description of the Drawings

[0013] [Figure 1] This is the result of measuring the IgG elution profile by pH gradient using each multi-domain type polypeptide of Example 56 (PN-621 variant) and Comparative Example 50 (PN-621). In FIG. 1, (A) is the result of Example 56 (PN-621 variant), and (B) is the result of Comparative Example 50 (PN-621).

Modes for Carrying Out the Invention

[0014] Hereinafter, the present invention will be described in detail. In addition to the sequence listing, the 20 types of amino acid residues in the amino acid sequence may be represented by one-letter abbreviations. That is, glycine (Gly) is G, alanine (Ala) is A, valine (Val) is V, leucine (Leu) is L, isoleucine (Ile) is I, phenylalanine (Phe) is F, tyrosine (Tyr) is Y, tryptophan (Trp) is W, serine (Ser) is S, threonine (Thr) is T, cysteine (Cys) is C, methionine (Met) is M, aspartic acid (Asp) is D, glutamic acid (Glu) is E, asparagine (Asn) is N, glutamine (Gln) is Q, lysine (K) is K, arginine (Arg) is R, histidine (His) is H, and proline (Pro) is P.

[0015] In this specification, expressions such as "Q9V" and "Q9V / S41V" are notations for amino acid substitutions. For example, "Q9V" means that glutamine at the 9th position from the N-terminal side in a specific amino acid sequence is substituted with valine. Also, for example, "Q9V / S41V" means that glutamine at the 9th position from the N-terminal side in a specific amino acid sequence is substituted with valine, and serine at the 41st position from the N-terminal side is substituted with valine.

[0016] In this specification, "amino acids having hydrophobic side chains" include alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, and tryptophan. Also, in this specification, "amino acids having hydrophobic aliphatic side chains" include alanine, valine, leucine, isoleucine, and methionine.

[0017] 1. Polypeptide As one aspect of the polypeptide of the present invention, there is a polypeptide containing at least one immunoglobulin-binding domain shown in (A) below. (A) An immunoglobulin-binding domain containing an amino acid sequence in which modifications satisfying the following conditions (i) to (iv) are introduced in the amino acid sequences shown in any of SEQ ID NOs: 1 to 15: (i) Serine at the 41st position is substituted with an amino acid having a hydrophobic side chain, tyrosine, or histidine. (ii) Glutamine at the 9th position is unsubstituted, or substituted with an amino acid having a hydrophobic aliphatic side chain or histidine. (iii) Glutamic acid or glutamine at the 15th position is unsubstituted, or substituted with alanine, histidine, tyrosine, or leucine. (iv) Glutamic acid or alanine at the 24th position is unsubstituted, substituted with glutamine, histidine, or alanine in the case of SEQ ID NOs: 1 to 12, or substituted with glutamine or histidine in the case of SEQ ID NOs: 13 to 15.

[0018] The amino acid sequences shown in Sequence IDs 1-15 are those of the wild-type immunoglobulin-binding domains (A, B, C, D, E) of protein A derived from Staphylococcus aureus, and their variants.

[0019] Specifically, Sequence ID 1 is the wild-type C domain, Sequence IDs 2 and 3 are variants of the C domain, Sequence ID 4 is the wild-type A domain, Sequence IDs 5 and 6 are variants of the A domain, Sequence ID 7 is the wild-type B domain, Sequence IDs 8 and 9 are variants of the B domain, Sequence ID 10 is the wild-type D domain, Sequence IDs 11 and 12 are variants of the D domain, Sequence ID 13 is the wild-type E domain, and Sequence IDs 14 and 15 are variants of the E domain.

[0020] The amino acid sequence shown in Sequence ID No. 2 is the same as the amino acid sequence of the wild-type C domain (Sequence ID No. 1), but with the following substitutions: lysine at position 4 is replaced by alanine, lysine at position 7 is replaced by threonine, lysine at position 35 is replaced by arginine, valine at position 40 is replaced by lysine, glutamic acid at position 43 is replaced by lysine, alanine at position 46 is replaced by lysine, and aspartic acid at position 53 is replaced by lysine. In the amino acid sequence shown in Sequence ID No. 2, the introduction of lysine at positions 40, 43, 46, and 53 makes the sequence from position 40 onwards lysine-rich, making it easier to immobilize on a carrier.

[0021] The amino acid sequence shown in Sequence ID No. 3 is the same as the amino acid sequence of the wild-type C domain (Sequence ID No. 1), but with the following substitutions: lysine at position 4 is replaced by alanine, lysine at position 7 is replaced by threonine, lysine at position 35 is replaced by arginine, lysine at position 42 is replaced by alanine, lysine at position 49 is replaced by arginine, lysine at position 50 is replaced by arginine, and lysine at position 58 is replaced by arginine.

[0022] The amino acid sequence shown in Sequence ID No. 5 is the same as the amino acid sequence of the wild-type A domain (Sequence ID No. 4), but with the following substitutions: asparagine at position 4 is replaced with alanine, lysine at position 7 is replaced with threonine, lysine at position 35 is replaced with arginine, glutamine at position 40 is replaced with lysine, asparagine at position 43 is replaced with lysine, serine at position 46 is replaced with lysine, and glutamic acid at position 53 is replaced with lysine. In the amino acid sequence shown in Sequence ID No. 5, the introduction of lysine at positions 40, 43, 46, and 53 makes the sequence from position 40 onwards lysine-rich, making it easier to immobilize on a carrier.

[0023] The amino acid sequence shown in Sequence ID No. 6 is the same as the amino acid sequence of the wild-type A domain (Sequence ID No. 4), but with the following substitutions: asparagine at position 4 is replaced with alanine, lysine at position 7 is replaced with threonine, lysine at position 35 is replaced with arginine, lysine at position 49 is replaced with arginine, lysine at position 50 is replaced with arginine, and lysine at position 58 is replaced with arginine.

[0024] The amino acid sequence shown in Sequence ID No. 8 is the same as the amino acid sequence of the wild-type B domain (Sequence ID No. 7), but with the following substitutions: lysine at position 4 is replaced by alanine, lysine at position 7 is replaced by threonine, lysine at position 35 is replaced by arginine, glutamine at position 40 is replaced by lysine, asparagine at position 43 is replaced by lysine, alanine at position 46 is replaced by lysine, and aspartic acid at position 53 is replaced by lysine. In the amino acid sequence shown in Sequence ID No. 8, the introduction of lysine at positions 40, 43, 46, and 53 makes the sequence from position 40 onwards lysine-rich, making it easier to immobilize on a carrier.

[0025] The amino acid sequence shown in Sequence ID No. 9 is the same as the amino acid sequence of the wild-type B domain (Sequence ID No. 7), but with the following substitutions: lysine at position 4 is replaced by alanine, lysine at position 7 is replaced by threonine, lysine at position 35 is replaced by arginine, lysine at position 49 is replaced by arginine, lysine at position 50 is replaced by arginine, and lysine at position 58 is replaced by arginine.

[0026] The amino acid sequence shown in Sequence ID No. 11 is the same as the amino acid sequence of the wild-type D domain (Sequence ID No. 10), but with the following substitutions: asparagine at position 4 is replaced by alanine, lysine at position 7 is replaced by threonine, lysine at position 35 is replaced by arginine, glutamine at position 40 is replaced by lysine, asparagine at position 43 is replaced by lysine, glycine at position 46 is replaced by lysine, and glutamic acid at position 53 is replaced by lysine. In the amino acid sequence shown in Sequence ID No. 11, the introduction of lysine at positions 40, 43, 46, and 53 makes the sequence from position 40 onwards lysine-rich, making it easier to immobilize on a carrier.

[0027] The amino acid sequence shown in Sequence ID No. 12 is the same as the amino acid sequence of the wild-type D domain (Sequence ID No. 10), but with the following substitutions: asparagine at position 4 is replaced by alanine, lysine at position 7 is replaced by threonine, lysine at position 35 is replaced by arginine, threonine at position 42 is replaced by alanine, lysine at position 49 is replaced by arginine, lysine at position 50 is replaced by arginine, and lysine at position 58 is replaced by arginine.

[0028] The amino acid sequence shown in Sequence ID No. 14 is the same as the amino acid sequence of the wild-type E domain (Sequence ID No. 13), but with the following substitutions: glutamine at position 4 is replaced by alanine, glutamic acid at position 7 is replaced by threonine, lysine at position 35 is replaced by arginine, glutamine at position 40 is replaced by lysine, asparagine at position 43 is replaced by lysine, glycine at position 46 is replaced by lysine, and aspartic acid at position 53 is replaced by lysine. In the amino acid sequence shown in Sequence ID No. 14, the introduction of lysine at positions 40, 43, 46, and 53 makes the sequence from position 40 onwards lysine-rich, making it easier to immobilize on a carrier.

[0029] The amino acid sequence shown in Sequence ID No. 15 is the same as the amino acid sequence of the wild-type E domain (Sequence ID No. 13), but with the following substitutions: glutamine at position 4 is replaced by alanine, glutamic acid at position 7 is replaced by threonine, lysine at position 35 is replaced by arginine, glutamine at position 49 is replaced by arginine, lysine at position 50 is replaced by arginine, and lysine at position 58 is replaced by arginine.

[0030] The immunoglobulin-binding domain of protein A contains three α-helices from the N-terminus: the first α-helix, the second α-helix, and the third α-helix. The serine at position 41 in each amino acid sequence shown in SEQ ID NOs. 1-15 is conserved across all immunoglobulin-binding domains (A-E). It is located in the third α-helix, forming a linkage with the second α-helix and also interacting with the first α-helix. By substituting this serine at position 41 with an amino acid having a hydrophobic side chain, tyrosine, or histidine, the hydrophobic interactions between amino acids within the immunoglobulin-binding domain can be enhanced, improving alkaline stability. Furthermore, the serine at position 41 is located on the reverse side of the α-helix structures of glutamine at position 9, glutamine at position 10, and asparagine at position 11, which form the immunoglobulin binding sites within the first α-helix. It is thought that substituting this serine at position 41 with an amino acid having a hydrophobic side chain, such as tyrosine or histidine, would affect the IgG binding structure formed by these amino acids.

[0031] The amino acid that replaces the serine at position 41 in each amino acid sequence shown in Sequence IDs 1 to 15 may be an amino acid having a hydrophobic side chain, tyrosine, or histidine, but preferably alanine, valine, leucine, phenylalanine, tyrosine, or histidine.

[0032] The glutamine at position 9 in each amino acid sequence shown in SEQ ID NOs: 1-15 is one of the IgG binding sites and contributes to binding to IgG by forming a hydrogen bond with serine at position 254 of IgG. The glutamine at position 9 in each amino acid sequence shown in SEQ ID NOs: 1-15 may be unsubstituted, or it may be substituted with an amino acid or histidine having a hydrophobic aliphatic side chain. When the glutamine at position 9 in each amino acid sequence shown in SEQ ID NOs: 1-15 is substituted with an amino acid or histidine having a hydrophobic aliphatic side chain, the alkali stability can be further improved while enhancing the elution properties in the weakly acidic range. While we do not wish for a restrictive interpretation, in each amino acid sequence shown in Sequence IDs 1-15, the amino acids at positions 41 and 9 are spatially close. Therefore, by analogy, substituting the glutamine at position 9 in each amino acid sequence shown in Sequence IDs 1-15 with an amino acid or histidine having a hydrophobic aliphatic side chain could further improve alkaline stability while reducing mutual binding with IgG, thereby decreasing binding activity in the weakly acidic region through interaction between the hydrophobic amino acid, tyrosine, or histidine at position 41 and the hydrophobic amino acid or histidine at position 9.

[0033] The amino acids to which the glutamine at position 9 in each amino acid sequence shown in Sequence ID No. 1 to 15 is substituted are preferably alanine, valine, leucine, isoleucine, or histidine, and more preferably alanine, valine, leucine, or histidine.

[0034] In each amino acid sequence shown in SEQ ID NOs: 1-15, the glutamic acid or glutamine at position 15 (glutamic acid in SEQ ID NOs: 1-12, and glutamine in SEQ ID NOs: 13-15) may be unsubstituted, or it may be substituted with alanine, histidine, tyrosine, or leucine. Furthermore, the glutamic acid at position 24 in each amino acid sequence shown in SEQ ID NOs: 1-12 may be unsubstituted, or it may be substituted with glutamine, histidine, or alanine. Similarly, the alanine at position 24 in each amino acid sequence shown in SEQ ID NOs: 13-15 may be unsubstituted, or it may be substituted with glutamine or histidine. In particular, when the glutamic acid or glutamine at position 15 in each amino acid sequence shown in SEQ ID NOs: 1-15 is substituted with alanine or histidine, and the glutamic acid or alanine at position 24 is substituted with glutamine, alkaline stability and elution in the weakly acidic range can be further improved.

[0035] A preferred example of the immunoglobulin-binding domain of (A) is one having the amino acid sequence shown in (1) to (5) below. (1) Amino acid sequences in which positions 9, 15, and 24 are unsubstituted, and position 41 is substituted with valine, phenylalanine, tyrosine, or histidine, as shown in SEQ ID NOs. (2) Amino acid sequences in which, in each of the amino acid sequences shown in Sequence IDs 1 to 15, positions 15 and 24 are unsubstituted, position 9 is substituted with leucine, and position 41 is substituted with valine, leucine, phenylalanine, alanine, tyrosine, or histidine. (3) Amino acid sequences in which, in each of the amino acid sequences shown in Sequence IDs 1 to 15, positions 15 and 24 are unsubstituted, position 9 is substituted with valine, and position 41 is substituted with leucine, valine, phenylalanine, tyrosine, or histidine. (4) Amino acid sequences in which, in each of the amino acid sequences shown in Sequence IDs 1 to 15, positions 15 and 24 are unsubstituted, position 9 is substituted with histidine, and position 41 is substituted with valine. (5) Amino acid sequences in which, in each of the amino acid sequences shown in Sequence IDs 1 to 15, positions 15 and 24 are unsubstituted, position 9 is substituted with alanine, and position 41 is substituted with phenylalanine, valine, tyrosine, or histidine. (6) Amino acid sequences in which, in each of the amino acid sequences shown in Sequence IDs 1 to 15, positions 15 and 24 are unsubstituted, position 9 is substituted with isoleucine, and serine at position 41 is substituted with valine or alanine. (7) Amino acid sequences in which, in each of the amino acid sequences shown in Sequence IDs 1 to 15, the 24th position is unsubstituted, the 9th position is substituted with valine, the 15th position is substituted with alanine, and the 41st position is substituted with valine. (8) In each of the amino acid sequences shown in Sequence IDs 1 to 15, the 24th position is unsubstituted, the 9th position is substituted with leucine or valine, the 15th position is substituted with histidine, and the 41st position is substituted with valine. (9) Amino acid sequences in which, in each of the amino acid sequences shown in Sequence IDs 1 to 15, position 24 is unsubstituted, position 9 is substituted with valine, position 15 is substituted with tyrosine, and position 41 is substituted with valine. (10) Amino acid sequences in which, in each of the amino acid sequences shown in Sequence IDs 1 to 15, position 24 is unsubstituted, position 9 is unsubstituted or substituted with leucine, position 15 is substituted with leucine, and position 41 is substituted with valine. (10) Amino acid sequences in which, in each of the amino acid sequences shown in Sequence IDs 1 to 15, position 9 is unsubstituted, position 15 is substituted with alanine or histidine, position 24 is substituted with glutamine, and position 41 is substituted with valine or histidine. (11) Amino acid sequences in which, in each of the amino acid sequences shown in Sequence IDs 1 to 15, the 9th position is replaced with leucine, the 15th position with stidine, the 24th position with glutamine, and the 41st position with valine. (12) Amino acid sequences in which, in each of the amino acid sequences shown in Sequence IDs 1 to 15, the 9th position is replaced with leucine, the 15th position is replaced with histidine or leucine, the 24th position is replaced with glutamine, and the 41st position is replaced with valine. (13) Amino acid sequences in which, in each of the amino acid sequences shown in Sequence IDs 1 to 15, position 15 is unsubstituted, position 9 is substituted with leucine, position 24 is substituted with histidine, and position 41 is substituted with valine. (14) Amino acid sequences in which, in each of the amino acid sequences shown in Sequence IDs 1 to 15, the 9th position is replaced with leucine, the 15th position is replaced with histidine or leucine, the 24th position is replaced with histidine, and the 41st position is replaced with valine. (15) Amino acid sequences in which, in each of the amino acid sequences shown in Sequence IDs 1 to 15, the 9th position is replaced with valine, the 15th position is replaced with histidine or leucine, the 24th position is replaced with alanine, and the 41st position is replaced with valine.

[0036] Furthermore, one embodiment of the polypeptide of the present invention is a polypeptide comprising at least one immunoglobulin-binding domain corresponding to either (B) or (C) below. (B) An immunoglobulin-binding domain having an amino acid sequence shown in any of Sequence IDs 1 to 15, in which a modification satisfying the conditions of (i) to (iv) above has been introduced, and one or more amino acids in the unmodified site have been substituted, added, inserted and / or deleted, and which has alkaline stability equal to or better than that of a polypeptide consisting of the corresponding unmodified amino acid sequence, and high IgG elution ability in the weakly acidic region. (C) An immunoglobulin-binding domain in which a modification satisfying the conditions of (i) to (iv) above has been introduced in any of the amino acid sequences shown in Sequence ID No. 1 to 15, the sequence identity of the unmodified region with respect to the corresponding unmodified amino acid sequence is 80% or more, and the alkali stability is equal to or better than that of a polypeptide consisting of the corresponding unmodified amino acid sequence, and the IgG elution ability in the weakly acidic region is high.

[0037] The immunoglobulin-binding domains of (B) and (C) are modified versions of the immunoglobulin-binding domain of (A), and the manner of amino acid substitutions introduced at positions 9, 15, 24, and 41 in the amino acid sequences shown in Sequence ID No. 1 to 15, as well as preferred amino acid substitution sites, are the same as in the case of the immunoglobulin-binding domain of (A).

[0038] In the immunoglobulin-binding domain of (B) above, "amino acids at the site where the modification has not been made" refers to amino acids other than those substituted by the modification that satisfies the conditions of (i) to (iv) above in the amino acid sequences shown in Sequence ID No. 1 to 15. For example, if the modification that satisfies the conditions of (i) to (iv) above substituted only the 41st position, and the 9th, 15th, and 24th positions were not substituted, then the amino acids other than the 41st position would be considered "amino acids at the site where the modification has not been made". Also, for example, if the modification that satisfies the conditions of (i) to (iv) above substituted the 41st and 9th positions, and the 15th and 24th positions were not substituted, then the amino acids other than the 41st and 9th positions would be considered "amino acids at the site where the modification has not been made". Also, for example, if the modification that satisfies the conditions of (i) to (iv) above substituted the 41st, 15th, and 24th positions, and the 9th position was not substituted, then the amino acids other than the 41st, 15th, and 24th positions would be considered "amino acids at the site where the modification has not been made".

[0039] In the immunoglobulin-binding domain of (B) above, the modification of the amino acids introduced may include only one type of modification (e.g., substitution) from among substitution, addition, insertion, and deletion, or it may include two or more types of modifications (e.g., substitution and insertion). In the immunoglobulin-binding domain of (B) above, there may be one, more, or several amino acids that are modified, for example, 1 to 13, preferably 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2, or 1.

[0040] In the immunoglobulin-binding domain of (C) above, "corresponding unmodified amino acid sequence" refers to the base amino acid sequence. For example, if the modification is of the amino acid sequence shown in Sequence ID No. 1, it refers to the amino acid sequence shown in Sequence ID No. 1. Furthermore, in the immunoglobulin-binding domain of (C) above, "sequence identity of the unmodified site" refers to the sequence identity calculated by comparing the amino acid sequences obtained by extracting the amino acids other than those substituted by the modification that satisfies the conditions of (i) to (iv) above. For example, if the modification satisfies the conditions of (i) to (iv) above, and only the 41st position is substituted, while the 9th, 15th, and 24th positions are not substituted, the sequence identity calculated by comparing the amino acid sequences obtained by extracting the amino acids other than the 41st position (the amino acid sequence excluding the 41st position) corresponds to "sequence identity of the unmodified site". Furthermore, for example, if a modification satisfying the conditions (i) to (iv) above results in amino acid substitutions at positions 41 and 9, but not at positions 15 and 24, the sequence identity calculated by comparing the amino acid sequence obtained by extracting the amino acids other than those at positions 41 and 9 (the amino acid sequence excluding positions 41 and 9) corresponds to "the sequence identity of the parts where the modification has not been made." Furthermore, for example, if a modification satisfying the conditions (i) to (iv) above results in amino acid substitutions at positions 41, 15 and 24, but not at position 9, the sequence identity calculated by comparing the amino acid sequence obtained by extracting the amino acids other than those at positions 41, 15 and 24 (the amino acid sequence excluding positions 41, 15 and 24) corresponds to "the sequence identity of the parts where the modification has not been made." Furthermore, in the immunoglobulin-binding domain of (C) above, "sequence identity" refers to the value of amino acid sequence identity obtained by the bl2seq program (Tatiana A. Tatsusova, Thomas L. Madden, FEMS Microbiol. Lett., Vol. 174, p247-250, 1999) of BLAST PACKAGE [sgi32 bit edition, Version 2.0.12; available from National Center for Biotechnology Information (NCBI)].The parameters should be set to Gap insertion Cost value: 11 and Gap extension Cost value: 1.

[0041] In the immunoglobulin-binding domain of (C) above, sequence identity should be 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more.

[0042] Furthermore, one embodiment of the immunoglobulin-binding domains of (B) and (C) is that at least the asparagine at position 3 in each amino acid sequence shown in SEQ ID NOs. 1 to 12 is substituted with alanine, valine, or aspartic acid.

[0043] Furthermore, one embodiment of the immunoglobulin-binding domains of (B) and (C) is one in which at least the alanine, glutamine, asparagine, or lysine at position 4 in each amino acid sequence shown in SEQ ID NOs: 1 to 15 is substituted with valine, histidine, arginine, or isoleucine.

[0044] Furthermore, one embodiment of the immunoglobulin-binding domains of (B) and (C) is that at least the asparagine at position 6 in each amino acid sequence shown in SEQ ID NOs. 1 to 12 is substituted with alanine, valine, glutamine, or aspartic acid.

[0045] Furthermore, one embodiment of the immunoglobulin-binding domains of (B) and (C) is one in which at least the threonine or lysine at position 7 in each amino acid sequence shown in SEQ ID NOs: 1-12, 14, and 15 is substituted with glutamic acid, histidine, arginine, or valine.

[0046] Furthermore, one embodiment of the immunoglobulin-binding domains of (B) and (C) is one in which at least the asparagine or serine at position 11 in each amino acid sequence shown in SEQ ID NOs: 1 to 15 is substituted with alanine, valine, histidine, glutamine, or arginine.

[0047] Specific examples of the immunoglobulin-binding domains of (B) and (C) include those in which each amino acid sequence shown in Sequence ID No. 1 to 15 has been modified to satisfy the conditions of (i) to (iv) above, and also has at least the following amino acid substitutions. (i) Position 11 is replaced with alanine, glutamine, histidine, or arginine. (ii) In the case of SEQ ID NOs. 1-12, 14 and 15, the 7th position is substituted with glutamic acid (in the case of SEQ ID NO. 13, the 7th position is unsubstituted). (iii) In the case of SEQ ID NOs. 1-12, 14 and 15, the 7th position is substituted with glutamic acid (the 7th position is unsubstituted in the case of SEQ ID NO. 13), and the 11th position is substituted with alanine or valine. (iv) If the 4th position is substituted with isoleucine, and in the case of SEQ ID NOs: 1-12, 14 and 15, the 7th position is substituted with arginine or glutamic acid, or in the case of SEQ ID NO: 13, the 7th position is unsubstituted or substituted with arginine. (v) The 4th position is substituted with isoleucine, the 7th position is substituted with arginine or glutamic acid in the case of SEQ ID NOs: 1-12, 14 and 15, or the 7th position is unsubstituted or substituted with arginine and the 11th position is substituted with alanine in the case of SEQ ID NO: 13. (vi) If the 4th position is substituted with isoleucine or valine, and if the sequence numbers are 1-12, 14 and 15, the 7th position is substituted with glutamic acid (the 7th position is unsubstituted in the case of sequence number 13). (vii) The 3rd position is substituted with aspartic acid, the 6th position is substituted with aspartic acid in the case of SEQ ID NOs. 1-12 (the 6th position is unsubstituted in the case of SEQ ID NOs. 13-15), the 7th position is substituted with valine or glutamic acid in the case of SEQ ID NOs. 1-12, 14 and 15, or the 7th position is unsubstituted or substituted with valine and the 11th position is substituted with alanine in the case of SEQ ID NOs. 13.

[0048] In the amino acid sequence shown in Sequence ID No. 1, the lysines substituted at positions 40, 43, 46, 49, 50, 53, and 58 play a role in facilitating immobilization to a carrier. Therefore, a preferred embodiment of the immunoglobulin-binding domains of (B) and (C) is that four or more lysines, preferably six or more, and more preferably all lysines, among positions 40, 43, 46, 49, 50, 53, and 58 are not mutated (substituted and / or deleted).

[0049] In the immunoglobulin-binding domains of (B) and (C), "alkali stability is equivalent to or better than that of the polypeptide consisting of the corresponding unmodified amino acid sequence, and IgG elution ability in the weakly acidic region is high" means that there is binding activity to IgG, the residual activity after alkali treatment measured under measurement condition 1 below is equivalent to or better than the residual activity after alkali treatment of the polypeptide consisting of the corresponding unmodified amino acid sequence (any of the amino acid sequences of sequence numbers 1 to 15 that form the basis) measured under the same conditions, and the elution rate in the weakly acidic region measured under measurement condition 2 below is higher than the elution rate in the weakly acidic region when using the polypeptide consisting of the corresponding unmodified amino acid sequence measured under the same conditions. Specifically, if the residual activity after alkali treatment measured under measurement condition 1 below is 1.1 times or more than the residual activity after alkali treatment of the corresponding unmodified amino acid sequence polypeptide measured under the same conditions, and the elution rate in weak acidity measured under measurement condition 2 below is 1.2 times or more than the elution rate in weak acidity when using the corresponding unmodified amino acid sequence polypeptide measured under the same conditions, then it can be said that "compared to the corresponding unmodified amino acid sequence polypeptide, it has equivalent or better alkali stability and high IgG elution ability in the weak acid region." For the immunoglobulin-binding domains of (B) and (C), it is preferable that the residual activity after alkali treatment measured under measurement condition 1 below is 1.14 times or more than the residual activity after alkali treatment of the corresponding unmodified amino acid sequence polypeptide measured under the same conditions, more preferably 1.14 to 1.21 times, and even more preferably 1.15 to 1.21 times. Furthermore, for the immunoglobulin-binding domains of (B) and (C), the elution rate in a weakly acidic environment measured under the following measurement condition 2 is preferably 1.3 times or more, more preferably 1.3 to 1.52 times, and even more preferably 1.4 to 1.55 times, compared to the elution rate in a weakly acidic environment measured under the same conditions using a polypeptide consisting of the corresponding unmodified amino acid sequence.

[0050] <Measurement Condition 1 (Measurement of residual activity after alkaline treatment)> Polypeptides immobilized on agarose gel support are washed three times with a 0.1 M NaOH aqueous solution, then replaced with the same alkaline solution, and incubated at 25°C for 68 hours (alkaline treatment). Subsequently, after washing with PBS, the amount of human IgG bound (mg / ml gel) is measured. The amount of human IgG bound to polypeptides immobilized on agarose gel support is also measured for the case without alkaline treatment. The amount of human IgG bound to polypeptides without alkaline treatment is set to 100%, and the percentage of human IgG bound to polypeptides after alkaline treatment is calculated as "residual activity after alkaline treatment (%)".

[0051] <Measurement Condition 2 (Measurement of elution rate in weakly acidic conditions)> Human IgG is bound to a polypeptide immobilized on an agarose gel support. Next, the immobilized polypeptide is washed with PBS, and then the human IgG bound to the immobilized polypeptide is eluted using 0.1 M citrate buffer (pH 4.0). Subsequently, any remaining human IgG bound to the immobilized polypeptide is eluted using 0.1 M glycine hydrochloride buffer (pH 2.8). The amount of human IgG eluted at pH 4.0 and the amount eluted at pH 2.8 are measured. The total amount of human IgG eluted at pH 4.0 and pH 2.8 is taken as 100%, and the percentage of human IgG eluted at pH 4.0 is calculated as the elution rate (%) in a weakly acidic environment.

[0052] The polypeptide of the present invention may be a single-domain polypeptide having one immunoglobulin-binding domain, or a multi-domain polypeptide having two or more immunoglobulin-binding domains linked together. When the polypeptide of the present invention is a multi-domain polypeptide, it has the advantage of having a higher binding ability to immunoglobulins or polypeptides containing the Fc region thereof.

[0053] If the polypeptide of the present invention is a single-domain polypeptide, it is sufficient to include one of the immunoglobulin-binding domains (A) to (C).

[0054] Among the immunoglobulin-binding domains of (A) to (C), the immunoglobulin-binding domains that include the amino acid sequences shown in SEQ ID NOs: 2, 5, 8, 11, and 14 as a base (hereinafter sometimes referred to as "lysine-rich domains") have carrier binding ability. Therefore, when using such immunoglobulin-binding domains as single-domain polypeptides, it is not necessary to add amino acid sequences that have carrier binding ability. However, in order to further improve carrier binding ability, amino acid sequences that have carrier binding ability may be added.

[0055] Furthermore, when using an immunoglobulin-binding domain (hereinafter sometimes referred to as a "lysine-free domain") among the immunoglobulin-binding domains of (A) to (C) that is based on the amino acid sequences shown in SEQ ID NOs: 1, 3, 4, 6, 7, 9, 10, 12, 13, and 15 as a single-domain polypeptide, it is preferable that an amino acid sequence having carrier-binding properties is added in order to confer carrier-binding properties.

[0056] When the polypeptide of the present invention is a multi-domain polypeptide, it is sufficient to include at least one of the immunoglobulin-binding domains (A) to (C), and it may consist of two or more domains from among the immunoglobulin-binding domains (A) to (C), or it may include one or more domains from among the immunoglobulin-binding domains (A) to (C) and one or more of the immunoglobulin-binding domains constituting protein A or protein L. When the polypeptide of the present invention is a multi-domain polypeptide, it is preferable that all of the constituent immunoglobulin-binding domains are composed of any of the immunoglobulin-binding domains (A) to (C) in order to provide even better alkaline stability and antibody elution characteristics in the weakly acidic region.

[0057] When the polypeptide of the present invention is made into a multi-domain polypeptide, the total number of linked immunoglobulin-binding domains may be two or more, but preferably 2 to 10, and more preferably 2 to 6.

[0058] When the polypeptide of the present invention is made into a multi-domain polypeptide, each immunoglobulin-binding domain may be directly linked at its C-terminus and N-terminus, or each immunoglobulin-binding domain may be linked via 1 to 40 amino acid residues, preferably 1 to 10 amino acid residues.

[0059] Furthermore, a suitable example of a polydomain polypeptide of the present invention is a structure in which one or more lysine-unriched domains and one or more lysine-rich domains are linked; more preferably, a structure in which one or more lysine-unriched domains and one lysine-rich domain are linked; even more preferably, a structure in which 2 to 10 lysine-unriched domains and one lysine-rich domain are linked; and particularly preferably, a structure in which 3 to 5 lysine-unriched domains are linked and one lysine-rich domain is linked to the C-terminal or N-terminal side (preferably the C-terminal side). By including one lysine-rich domain in the polydomain polypeptide in this way, it is possible to provide carrier binding ability. Therefore, in a polydomain polypeptide containing a lysine-rich domain, it is not necessary to add an amino acid sequence that provides carrier binding ability. However, even in such a polydomain polypeptide, an amino acid sequence that provides carrier binding ability may be added in order to further improve carrier binding ability.

[0060] Furthermore, in the case of a multi-domain polypeptide, if it contains a lysine-non-rich domain but does not contain a lysine-rich domain, it is preferable that an amino acid sequence having carrier-binding properties is added to confer carrier-binding ability to the polypeptide.

[0061] Examples of the amino acid sequence having carrier binding ability include an amino acid sequence in which 1 to 15 lysine molecules, preferably 3 to 10, and more preferably 4 to 8, are linked together. This linked sequence may also contain amino acids other than lysine. Furthermore, when adding an amino acid sequence having carrier binding ability, it may be added to either the N-terminal or C-terminal end, but the C-terminal end is preferred.

[0062] Furthermore, the polypeptide of the present invention may have other functional polypeptides, peptide tags, etc., attached to its N-terminal or C-terminal side to improve polypeptide expression, facilitate purification, etc. For this purpose, the number of amino acids attached to the N-terminal and / or C-terminal side of the polypeptide of the present invention is not particularly limited, but for example, 1 to 400, preferably 1 to 100, and more preferably 1 to 30.

[0063] 2. DNA The DNA encoding the polypeptide of the present invention (hereinafter sometimes referred to as "the DNA of the present invention") can be obtained, for example, by using DNA encoding protein A derived from Staphylococcus aureus as a template, obtaining the DNA encoding the target immunoglobulin-binding domain by PCR or the like, and then introducing mutations into that DNA so that the desired amino acid substitutions are introduced. Furthermore, the DNA of the present invention can also be artificially synthesized by gene synthesis methods.

[0064] Here, the DNA encoding wild-type protein A from Staphylococcus aureus is known, for example, as the nucleotide sequence shown in Sequence ID No. 16, and can be isolated from Staphylococcus aureus by standard PCR methods. Furthermore, the DNA encoding wild-type protein A can also be artificially synthesized using gene synthesis methods.

[0065] Methods for introducing specific mutations at specific sites in a base sequence are publicly known, and for example, site-directed mutagenesis of DNA can be used. Specific methods for converting bases in DNA can also be carried out using commercially available kits.

[0066] DNA into which mutations have been introduced into the base sequence can be sequenced using a DNA sequencer. Once the base sequence is determined, the DNA encoding the polypeptide can then be obtained by chemical synthesis, PCR using a cloned probe as a template, or hybridization using a DNA fragment containing the sequence as a probe.

[0067] Furthermore, mutant versions of the DNA encoding the peptide that have the same function as the original DNA can be synthesized by site-directed mutagenesis or the like. The mutation can be introduced into the DNA encoding the peptide using known methods such as the Kunkel method, gapped duplex method, or megaprimer PCR.

[0068] The DNA of the present invention is preferably one whose codon usage frequency is optimized for the host. For example, if E. coli is used as the host, DNA with codon usage frequency optimized for E. coli is preferable.

[0069] 3. Recombinant vectors A recombinant vector containing DNA encoding the polypeptide of the present invention (hereinafter sometimes referred to as "the recombinant vector of the present invention") can be obtained by inserting the DNA of the present invention into an expression vector.

[0070] The recombinant vector of the present invention includes regulatory factors such as promoters operably linked to the DNA of the present invention. Typical regulatory factors include promoters, but may also include enhancers, CCAAT boxes, TATA boxes, SPI sites, and other transcription elements as needed. Operable linkage means that the DNA of the present invention is linked to various regulatory factors such as promoters and enhancers that regulate the DNA of the present invention in a manner that allows it to function within a host cell.

[0071] Preferred expression vectors are those constructed for genetic recombination from phages, plasmids, or viruses that can autonomously proliferate within a host. Such expression vectors are well known, and commercially available examples include pQE vectors (Qiagen Co., Ltd.), pDR540, pRIT2T (GE Healthcare Biosciences Co., Ltd.), and pET vectors (Merck KGaA). The expression vector can be used in any combination with an appropriate host cell. For example, when using Escherichia coli as the host cell, a combination of a pET vector and the BL21(DE3) strain of Escherichia coli, or a combination of a pDR540 vector and the JM109 strain of Escherichia coli, are preferred.

[0072] 4. Transformed organism A transformant (hereinafter sometimes referred to as "the transformant of the present invention") can be obtained by transforming a host using the recombinant vector of the present invention.

[0073] The host used to produce the transformant is not particularly limited as long as the recombinant vector is stable, capable of autonomous replication, and able to express the traits of the exogenous gene. Suitable examples include bacteria belonging to the Escherichia genus, such as Escherichia coli, the Bacillus genus, such as Bacillus subtilis, and the Pseudomonas genus, such as Pseudomonas putida; yeast, etc. Other examples include animal cells, insect cells, plants, etc. Among these, Escherichia coli is particularly preferred.

[0074] The transformants of the present invention can be obtained by introducing the recombinant vector of the present invention into a host, and the conditions for introducing the recombinant vector into the host can be appropriately set according to the type of host, etc. If the host is bacteria, for example, methods using competent cells treated with calcium ions and electroporation can be used. If the host is yeast, for example, electroporation, spheroplast method and lithium acetate method can be used. If the host is animal cells, for example, electroporation, calcium phosphate method and lipofection method can be used. If the host is insect cells, for example, calcium phosphate method, lipofection method and electroporation method can be used. If the host is plants, for example, electroporation, Agrobacterium method, particle gun method and PEG method can be used.

[0075] 5. Polypeptide production The polypeptide of the present invention can be produced by culturing the transformant.

[0076] The culture conditions for the transformants should be set appropriately considering the nutritional and physiological properties of the host, but liquid culture is preferred. Furthermore, in the case of industrial production, aerated and agitated culture is preferred.

[0077] The transformant of the present invention is cultured, and the culture supernatant or bacterial cells are recovered from the culture medium by methods such as centrifugation. If the polypeptide of the present invention is accumulated in the bacterial cells, the bacterial cells are treated by mechanical methods such as ultrasound or French press, or by lytic enzymes such as lysozyme, and if necessary, solubilized by using enzymes such as proteases or surfactants such as sodium dodecyl sulfate (SDS) to obtain a water-soluble fraction containing the polypeptide of the present invention.

[0078] Furthermore, by selecting an appropriate expression vector and host, the expressed polypeptide of the present invention can be secreted into the culture medium.

[0079] The culture medium or water-soluble fraction containing the polypeptide of the present invention obtained as described above may be subjected to purification treatment as is, or the polypeptide of the present invention in the culture medium or water-soluble fraction may be concentrated before being subjected to purification treatment.

[0080] Concentration can be carried out, for example, by vacuum concentration, membrane concentration, salting-out treatment, or fractional precipitation using hydrophilic organic solvents (e.g., methanol, ethanol, and acetone).

[0081] The polypeptide of the present invention can be purified by appropriately combining methods such as gel filtration, hydrophobic chromatography, ion exchange chromatography, and affinity chromatography.

[0082] The polypeptide of the present invention, purified in this manner, may be powdered by freeze-drying, vacuum drying, spray drying, or the like, if necessary.

[0083] 6. Immunoglobulin binding carriers The polypeptide of the present invention is immobilized on an insoluble carrier and used as an immunoglobulin-binding carrier to facilitate the recovery and purification of immunoglobulins. The insoluble carrier used to immobilize the polypeptide of the present invention is not particularly limited, but examples include naturally derived polymer materials such as chitosan, dextran, cellulose, and agarose; synthetic organic materials such as vinyl alcohol, polyimide, and methacrylate; and inorganic materials such as glass and silica.

[0084] The shape of the insoluble carrier is not particularly limited and may be in any form, such as hollow fiber membrane, monolith, or bead. Among these shapes, the bead shape is generally preferred because it has a relatively large surface area per unit volume and is suitable for producing affinity carriers with high immunoglobulin binding ability.

[0085] To immobilize the polypeptide of the present invention onto an insoluble carrier, for example, an amino group, carboxyl group, or thiol group in the polypeptide of the present invention may be coupled with the insoluble carrier. Specifically, immobilization methods include activating the insoluble carrier by reacting it with a coupling agent such as cyanogen bromide, epichlorohydrin, N-hydroxysuccinimide, tosylchloride, tresilchloride, carbodiimide, glutaraldehyde, or hydrazine, or introducing reactive functional groups such as carboxyl groups or thiol groups into the carrier before carrying out a coupling reaction with the polypeptide of the present invention. Such coupling reactions are well known in the art (for example, Janson, J.-C., ed. [Protein purification], 3rd edition, pp. 221-258, ISBN 978-0-471-74661-4) and can be carried out according to commonly used methods.

[0086] Furthermore, when immobilizing the polypeptide of the present invention onto an insoluble carrier via an amino group, it is desirable to use a carrier having a reactive functional group (such as a tresil group, epoxy group, carboxyl group, or formyl group) that can react with the amino group to form a covalent bond. Such insoluble carriers are commercially available as Toyopearl AF-tresil-650, Toyopearl AF-epoxy-650, Toyopearl AF-carboxy-650, Toyopearl AF-formyl-650 (all from Tosoh Corporation), NHS-activated Sepharose, cyanide-bromide-activated Sepharose, epoxy-activated Sepharose (all from GE Healthcare Biosciences Corporation), Profinity Epoxy (Bio-Rad Inc.), Glyoxal-Agarose (Agarose Beads Technologies Inc.), Cellfine Formil (JNC Corporation), etc., and these commercially available products can be used.

[0087] Furthermore, the polypeptide of the present invention can be immobilized on the insoluble carrier by adding a condensation or crosslinking reagent such as carbodiimide or glutaraldehyde to a system in which the polypeptide of the present invention and the insoluble carrier coexist.

[0088] 7. Method for separating immunoglobulins or their fragments Since the polypeptide of the present invention binds to the Fc region of immunoglobulins, it can be used for the separation of immunoglobulins such as IgG, IgM, and IgA, and polypeptides containing their Fc regions (such as Fc fusion proteins).

[0089] To separate immunoglobulins or polypeptides containing their Fc region using the polypeptide of the present invention, an insoluble carrier on which the polypeptide of the present invention is immobilized may be used. Specifically, the separation of immunoglobulins using an insoluble carrier on which the polypeptide of the present invention is immobilized can be performed by affinity column chromatography.

[0090] To separate immunoglobulins by affinity column chromatography, a solution containing immunoglobulin or a polypeptide containing its Fc region is passed through a column packed with an insoluble carrier immobilized with the polypeptide of the present invention to bind the immunoglobulin or the polypeptide containing its Fc region to the polypeptide of the present invention. After washing the inside of the column as necessary, the immunoglobulin or the polypeptide containing its Fc region is eluted by passing an eluate adjusted to an appropriate pH through the column. The pH of the solution containing immunoglobulin or the polypeptide containing its Fc region should be 6.5 or higher, preferably 6.5 to 8.0. The pH of the eluate should be weakly acidic or lower, specifically pH 5 or lower. However, to prevent the separated immunoglobulin or its fragments from being exposed to strongly acidic conditions, the pH of the eluate is preferably 3 to 5, more preferably 3.4 to 4.5, and even more preferably 3.6 to 4.5. [Examples]

[0091] The present invention will be described in detail below with reference to examples, but the present invention is not limited to the following examples.

[0092] Test Example 1: Production and Evaluation of Monodomain Polypeptides (1) 1. Manufacturing of single-domain polypeptides A single-domain polypeptide with a modified C domain of protein A was produced. The specific production method is as follows.

[0093] <Production of PN-32 expression plasmid> A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 was named PN-32. The expression plasmid of PN-32 was produced by the following method.

[0094] A double-stranded DNA with a recognition sequence of restriction enzyme NdeI at the translation start codon site, a sequence encoding the amino acid sequence of SEQ ID NO: 2, a translation stop codon, and a recognition sequence of restriction enzyme BamHI arranged in this order was prepared using synthetic oligonucleotides. Fragments obtained by cutting both ends of the synthetic oligonucleotide with restriction enzymes NdeI and BamHI, respectively, were ligated into a pET9a plasmid cut with these two restriction enzymes in the same way, and then incorporated into Escherichia coli DH-5α competent cells for transformation. The Escherichia coli was cultured in the presence of kanamycin to purify the expression plasmid of PN-32 (pET9a·PN-32).

[0095] The nucleic acid sequence of pET9a·PN-32 thus obtained was analyzed using a CEQ8000 type DNA sequencer (Beckman Coulter, Inc.) to confirm that it was the designed sequence.

[0096] <Production of PN-32 variant expression plasmid> In addition, an expression plasmid of a PN-32 variant in which the amino acid substitution shown in Table 1 was introduced in the amino acid sequence shown in SEQ ID NO: 2 was produced by the following method.

[0097] Using the pET9a·PN-32 as a template, PCR was performed using oligonucleotide DNA designed and synthesized to have one amino acid substitution of interest as a primer to prepare double-stranded DNA encoding the variant of interest. It was cleaved with restriction enzymes NdeI and BamHI and ligated into the pET9a plasmid by ligation reaction in the same manner as in the preparation of pET9a·PN-32, and an expression plasmid of the PN-32 variant having one amino acid substitution was obtained. Also, using the expression plasmid of the variant having one amino acid substitution as a template, the same operation as above was performed to prepare an expression plasmid of the PN-32 variant having two amino acid substitutions.

[0098] The nucleic acid sequence of the expression plasmid of the PN-32 variant thus obtained was analyzed using a CEQ8000 DNA sequencer (Beckman Coulter, Inc.) to confirm that it was the designed sequence.

[0099] <Production of PN-32 and its variants> Using each of the obtained expression plasmids of PN-32 and its variants, Escherichia coli BL21(DE3) competent cells (Merck KGaA) were transformed to obtain each expression strain of PN-32 and its variants.

[0100] Each expression Escherichia coli strain of PN-32 and its variants was precultured in an LB medium containing 25 mg / L kanamycin and 2.0% glucose for 12 hours. The obtained preculture was inoculated into a 2×TY medium containing 25 mg / L kanamycin and 0.8% glucose and cultured at 37 °C for 16 hours to express the target PN-32 and its variants. Then, Escherichia coli was recovered by centrifugation. Next, the recovered Escherichia coli was suspended in 50 mM sodium phosphate buffer (pH 6.5), disrupted by sonication, and further centrifuged to recover PN-32 and its variants in the supernatant. When each obtained supernatant was subjected to sodium dodecyl sulfate-15% polyacrylamide gel electrophoresis (SDS-PAGE) as a cell extract, it was confirmed that the target PN-32 and its variants were produced.

[0101] Cellular extracts from E. coli strains expressing PN-32 and its variants were adjusted to pH 6.0 and applied to a cation exchange SP-Sepharose Fast Flow (GE Healthcare Corporation) column. After washing with 20 mM phosphate buffer (pH 6.0), proteins were eluted from the column using a linear concentration gradient of 0.5 M NaCl. SDS-PAGE analysis of the eluates showed that PN-32 and its variants eluted between 0.1 and 0.2 M NaCl. Next, the pH of each eluate containing PN-32 and its variants was adjusted to 9 and added to a Gigacap Q (Tosoh Corporation) anion exchange column. After washing with 20 mM phosphate buffer (pH 7.8), PN-32 and its variants were separated using a linear concentration gradient of 0.3 M NaCl. Purity checks of each separation using SDS-PAGE confirmed that PN-32 and its variants were purified as a single band at the theoretical molecular weight position.

[0102] 2. Immobilization of single-domain polypeptides onto gel carriers Purified PN-32 and its variants were immobilized on a formyl-activated agarose gel carrier at a gel concentration of 10 mg / mL according to a conventional method. The reaction solution was recovered after immobilization, and the immobilization rate was measured. All PN-32 and its variants showed an immobilization efficiency of 90% or higher.

[0103] 3. Measurement of immunoglobulin binding activity Each gel carrier immobilized with PN-32 and its variants was washed with PBS. Then, PBS (pH 7.5) containing 40 mg / mL of human polyclonal IgG (obtained from the Japan Blood Products Organization) was added and shaken for 1 hour. After that, the gel carrier was washed with PBS (pH 7.5). Next, the human IgG bound to the gel carrier was eluted from the gel carrier with 0.1 M glycine hydrochloride buffer (pH 2.8). The absorption of the eluate at 280 nm was measured using a spectrophotometer, and 13.8 (1g) was obtained. -1 cm -1 The amount of bound IgG (IgG binding amount) was determined based on the specific absorption coefficient of ).

[0104] 4. Measurement of residual activity after alkaline treatment Each gel support immobilized with PN-32 and its variants was washed with PBS, then washed three times with a 0.1 M NaOH aqueous solution, replaced with the same alkaline solution, and stored at 25°C for 68 hours (alkaline treatment). Subsequently, after washing three times with PBS, the immunoglobulin binding activity was measured under the same conditions as above. The "residual activity after alkaline treatment (%)" was calculated as the percentage of the remaining IgG binding amount after alkaline treatment, with the amount of IgG binding before alkaline treatment set to 100%.

[0105] 5. Measurement of elution rate in weakly acidic conditions Each gel carrier immobilized with PN-32 and its variants was washed with PBS. Then, PBS (pH 7.5) containing 40 mg / mL of human polyclonal IgG (obtained from the Japan Blood Products Organization) was added and shaken for 1 hour. After that, the gel carrier was washed with PBS (pH 7.5). Next, human IgG bound to the gel carrier was eluted using 0.1 M citrate buffer (pH 4.0). Subsequently, remaining human IgG bound to the gel carrier was eluted using 0.1 M glycine hydrochloride buffer (pH 2.8). The absorption at 280 nm of the eluates obtained at pH 4.0 and pH 2.8 was measured using a spectrophotometer, and the value was 13.8 (1g). -1 cm -1 The amount of IgG contained in each eluate was determined based on the specific absorption coefficient of ). The total amount of human IgG in the eluate eluted at pH 4.0 and the human IgG in the eluate eluted at pH 2.8 was set to 100%, and the percentage of human IgG in the eluate eluted at pH 4.0 was calculated as the "elution rate at weak acidity (%)".

[0106] 6.Results The results are shown in Table 1. The results showed that in PN-32 (SEQ ID NO: 2), the modified form in which the serine at position 41 was replaced with an amino acid having a hydrophobic side chain (valine, phenylalanine), histidine, or tyrosine exhibited residual activity more than 1.1 times that of PN-32 after alkaline treatment, and improved elution rate in weakly acidic conditions to more than 1.2 times that of PN-32. Position 41 was the only single mutation site exhibiting both of these effects.

[0107] In the variant where the 9 - glutamine is substituted with an amino acid having a hydrophobic aliphatic side chain (alanine, valine, leucine, or isoleucine) and the 41 - serine is substituted with an amino acid having a hydrophobic side chain (alanine, valine, leucine, phenylalanine), tyrosine, or histidine, it was confirmed that the residual activity after alkali treatment was higher than that of the single - mutation variant at position 41, and the elution rate in weak acidity was dramatically improved compared to PN - 32. In the variant where the 41 - serine was substituted with alanine or leucine, further substitution of the 9 - glutamine with an amino acid having a hydrophobic aliphatic side chain or histidine resulted in high residual activity after alkali treatment, and the elution rate in weak acidity was dramatically improved compared to the single - mutation substitution variant. Note that in the variant where the 9 - glutamine was substituted with phenylalanine and the 41 - serine was substituted with an amino acid having a hydrophobic side chain, tyrosine, or histidine, the residual activity after alkali treatment was not higher than that of the variant with substitution only at position 41.

[0108]

Table 1

[0109] Test Example 2: Production and Evaluation of Monodomain Polypeptides (2) 1. Manufacturing of single-domain polypeptides In PN - 32 (SEQ ID NO: 2), the expression plasmids of PN - 32 variants into which the amino acid substitutions shown in Table 2 were introduced were produced by the same method as in Test Example 1 above.

[0110] The nucleic acid sequences of the expression plasmids of the PN - 32 variants thus obtained were analyzed using a CEQ8000 type DNA sequencer (Beckman Coulter, Inc.), and it was confirmed that they had the designed sequences.

[0111] <Production of PN - 32 Variants>[ Using the expression plasmids of the modified PN-32 obtained above, compliant cells of *Escherichia coli* BL21(DE3) (Merck KGaA) were transformed to obtain expression strains of PN-32 and its modified counterparts.

[0112] Each strain of E. coli expressing a variant of PN-32 was seed cultured for 12 hours in LB medium containing 25 mg / L kanamycin and 2.0% glucose. The resulting seed culture was inoculated into 2×TY medium containing 25 mg / L kanamycin and 0.8% glucose and cultured at 37°C for 16 hours to express the target PN-32 and its variants. The E. coli were then collected by centrifugation. Next, the collected E. coli were suspended in 50 mM sodium phosphate buffer (pH 6.5), sonicated to disrupt the E. coli, and the variant of PN-32 was collected in the supernatant by further centrifugation. Each of the resulting supernatants was subjected to sodium dodecyl sulfate-15% polyacrylamide gel electrophoresis (SDS-PAGE) as a cell extract, confirming that the desired variant of PN-32 was produced.

[0113] Cellular extracts from each E. coli strain expressing a modified PN-32 were adjusted to pH 6.0 and then applied to a cation exchange SP-Sepharose Fast Flow column (GE Healthcare Corporation). After washing with 20 mM phosphate buffer (pH 6.0), the protein was eluted from the column using a linear concentration gradient of 0.5 M NaCl. Analysis of the eluate by SDS-PAGE showed that the modified PN-32 eluted between 0.1 and 0.2 M NaCl. Next, the pH of each eluate containing the PN-32 variant was adjusted to 9, and then it was added to an anion exchange Gigacap Q (Tosoh Corporation) column. After washing with 20 mM phosphate buffer (pH 7.8), the PN-32 variant was separated using a 0.3 M NaCl linear concentration gradient. Purity of each separation was confirmed by SDS-PAGE, and it was confirmed that the PN-32 variant was purified as a single band at the position of the theoretical molecular weight.

[0114] 2. Immobilization of single-domain polypeptides onto gel carriers Modified forms of purified PN-32 were immobilized on a formyl-activated agarose gel carrier at a gel concentration of 10 mg / mL according to a conventional method. The reaction solution was collected after immobilization, and the immobilization rate was measured. All modified forms of PN-32 showed an immobilization efficiency of 90% or higher.

[0115] 3. Measurement of immunoglobulin binding activity Using each gel carrier on which PN-32 and its modified counterparts were immobilized, the amount of IgG bound was determined under the same conditions as in Test Example 1.

[0116] 4. Measurement of residual activity after alkaline treatment Each gel support immobilized with the modified PN-32 was washed with PBS, then washed three times with a 0.5 M NaOH aqueous solution, replaced with the same alkaline solution, and stored at 25°C for 17 hours (alkaline treatment). Subsequently, after washing three times with PBS, the immunoglobulin binding activity was measured under the same conditions as above. The "residual activity after alkaline treatment (%)" was calculated as the percentage of the remaining IgG binding amount after alkaline treatment, with the amount of IgG binding before alkaline treatment set to 100%.

[0117] 5. Measurement of elution rate in weakly acidic conditions The elution rate in a weakly acidic environment was determined using each gel carrier on which PN-32 and its modified counterparts were immobilized, under the same conditions as in Test Example 1.

[0118] 6.Results The results are shown in Table 2. From these results, it was also confirmed that in the variant of PN-32 (SEQ ID NO: 2) where the 9th position is substituted with an amino acid having a hydrophobic aliphatic side chain, the 41st position is substituted with an amino acid having a hydrophobic side chain, and the 11th position is substituted with alanine, the residual activity after alkali treatment is 1.49 times or more that of PN-32, and the elution rate in weakly acidic conditions is dramatically improved to 1.27 times that of PN-32. Furthermore, in the variant of PN-32 (SEQ ID NO: 2) where the 9th position is substituted with an amino acid having a hydrophobic aliphatic side chain, the 41st position is substituted with an amino acid having a hydrophobic side chain, the 15th position is substituted with alanine, and the 11th position is substituted with arginine, the elution rate in weakly acidic conditions could be further improved along with the improvement of alkali stability. Also, in the variant where the 9th position is unsubstituted, the 15th position is substituted with alanine or histidine, the 24th position is substituted with glutamine, and the 41st position is substituted with an amino acid having a hydrophobic side chain, the residual activity after alkali treatment is 1.40 times or more that of PN-32, and the elution rate in weakly acidic conditions is dramatically improved to 1.40 times that of PN-32.

[0119]

Table 2

[0120] Test Example 3: Production and Evaluation of Monodomain Polypeptides (3) 1. Manufacturing of single-domain polypeptides A single-domain polypeptide obtained by modifying the C domain of Protein A was produced. The specific production method is as follows.

[0121] <Production of PN-128 expression plasmid> A polypeptide consisting of an amino acid sequence in which threonine at the 7th position in the amino acid sequence shown in SEQ ID NO: 2 is substituted with glutamic acid was named PN-128. The expression plasmid of PN-128 was produced by the same method as in Test Example 1 above.

[0122] The nucleic acid sequence of the PN-128 expression plasmid thus obtained was analyzed using a CEQ8000 DNA sequencer (Beckman Coulter, Inc.) to confirm that it had the designed sequence.

[0123] <Production of PN-128 variant expression plasmid> Also, an expression plasmid of a PN-128 variant in which the amino acid substitutions shown in Table 3 were introduced in the amino acid sequence shown in SEQ ID NO: 2 was produced by the same method as in Test Example 1 above.

[0124] The nucleic acid sequence of the PN-128 variant expression plasmid thus obtained was analyzed using a CEQ8000 DNA sequencer (Beckman Coulter, Inc.) to confirm that it had the designed sequence.

[0125] <Production of PN-128 and its variants> Using each of the above-obtained expression plasmids of PN-128 and its variants, Escherichia coli BL21(DE3) competent cells (Merck KGaA) were transformed to obtain each expression strain of PN-32 and its variants.

[0126] Each expression Escherichia coli strain of PN-128 and its variants was precultured for 12 hours in an LB medium containing 25 mg / L kanamycin and 2.0% glucose. The obtained preculture was inoculated into a 2×TY medium containing 25 mg / L kanamycin and 0.8% glucose, cultured at 37°C for 16 hours to express the target PN-128 and its variants, and then the Escherichia coli was recovered by centrifugation. Next, the recovered Escherichia coli was suspended in 50 mM sodium phosphate buffer (pH 6.5), disrupted by ultrasonic treatment, and further centrifuged to recover PN-32 and its variants in the supernatant. When each obtained supernatant was subjected to sodium dodecyl sulfate - 15% polyacrylamide gel electrophoresis (SDS-PAGE) as a cell extract, it was confirmed that the target PN-128 and its variants were produced.

[0127] Cellular extracts from E. coli strains expressing PN-128 and its variants were adjusted to pH 6.0 and applied to a cation exchange SP-Sepharose Fast Flow (GE Healthcare Corporation) column. After washing with 20 mM phosphate buffer (pH 6.0), proteins were eluted from the column using a linear concentration gradient of 0.5 M NaCl. SDS-PAGE analysis of the eluates showed that PN-128 and its variants eluted between 0.1 and 0.2 M NaCl. Next, the pH of each eluate containing PN-128 and its variants was adjusted to 9 and added to a Gigacap Q (Tosoh Corporation) anion exchange column. After washing with 20 mM phosphate buffer (pH 7.8), PN-128 and its variants were separated using a linear concentration gradient of 0.3 M NaCl. Purity checks of each separation using SDS-PAGE confirmed that PN-128 and its variants were purified as a single band at the theoretical molecular weight position.

[0128] 2. Immobilization of single-domain polypeptides onto gel carriers Purified PN-128 and its variants were immobilized on a formyl-activated agarose gel carrier at a gel concentration of 10 mg / mL according to a conventional method. The reaction solution was recovered after immobilization, and the immobilization rate was measured. All of PN-128 and its variants showed an immobilization efficiency of 90% or higher.

[0129] 3. Measurement of immunoglobulin binding activity Using each gel carrier on which PN-128 and its variants were immobilized, the amount of IgG bound was determined under the same conditions as in Test Example 1.

[0130] 4. Measurement of residual activity after alkaline treatment The residual activity after alkali treatment was determined using each gel carrier on which PN-128 and its modified counterparts were immobilized, under the same conditions as in Test Example 2.

[0131] 5. Measurement of elution rate in weakly acidic conditions The elution rate in a weakly acidic environment was determined using each gel carrier on which PN-128 and its modified counterparts were immobilized, under the same conditions as in Test Example 1.

[0132] 6.Results The results are shown in Table 3. As a result, in PN-128 (an amino acid sequence in which the 7th position is substituted with glutamic acid in the amino acid sequence shown in SEQ ID NO: 2), in addition to the 9th position being substituted with an amino acid having a hydrophobic aliphatic side chain and the 41st position being substituted with an amino acid having a hydrophobic side chain or histidine, (i) substitution of the 11th position with an amino acid having a hydrophobic side chain, (ii) substitution of the 15th position with an amino acid, tyrosine, or histidine having a hydrophobic side chain, (iii) substitution of the 24th position with alanine, glutamine, or histidine. In at least one variant having any of these substitutions, it was confirmed that the residual activity after alkali treatment was equal to or higher than that of PN-128, and the elution rate in weakly acidic conditions was significantly improved compared to PN-128.

[0133]

Table 3

[0134] Test Example 4: Production and Evaluation of Multi-Domain Polypeptides 1. Production of multi-domain polypeptides A multi-domain polypeptide having six modified sequences of the C domain of protein A was produced. The specific production method is as follows.

[0135] <Production of PN-621 expression plasmid> A polypeptide consisting of an amino acid sequence in which five amino acid sequences shown in SEQ ID NO: 3 (sequence α) and one amino acid sequence shown in SEQ ID NO: 2 (sequence β) are directly linked in this order from the N-terminal side was named PN-621. The expression plasmid of PN-621 was produced by the method described in Patent Document 3.

[0136] [[ID=***]] The nucleic acid sequence of the PN-621 expression plasmid thus obtained was analyzed using a CEQ8000 type DNA sequencer (Beckman Coulter, Inc.), and it was confirmed that the sequence was as designed.

[0137] <Production of PN-621 modified variant expression plasmid> An expression plasmid of the PN-621 variant into which a substitution having the amino acid sequence shown in Table 4 was introduced was produced using the methods described in Test Example 1 and Patent Document 3 above.

[0138] The nucleic acid sequence of the expression plasmid of the PN-621 variant thus obtained was analyzed using a CEQ8000 DNA sequencer (Beckman Coulter, Inc.), and it was confirmed that the sequence was as designed.

[0139] <Production of PN-621 and its variants> Using each of the expression plasmids of PN-621 and its variants obtained above, Escherichia coli BL21(DE3) competent cells (Merck KGaA) were transformed to obtain each expression strain of PN-621 and its variants.

[0140] Each expression Escherichia coli strain of PN-621 and its variants was precultured for 12 hours in an LB medium containing 25 mg / L kanamycin and 2.0% glucose. The obtained preculture solution was inoculated into a 2×TY medium containing 25 mg / L kanamycin and 0.8% glucose, cultured at 37°C for 16 hours to express the target PN-621 and its variants, and then Escherichia coli was recovered by centrifugation. Next, the recovered Escherichia coli was suspended in 50 mM sodium phosphate buffer (pH 6.5), disrupted by ultrasonic treatment, and PN-621 and its variants were recovered in the supernatant by further centrifugation. When each of the obtained supernatants was subjected to sodium dodecyl sulfate-15% polyacrylamide gel electrophoresis (SDS-PAGE) as a cell extract, it was confirmed that the target PN-621 and its variants were being produced.

[0141] Cellular extracts from E. coli strains expressing PN-621 and its variants were adjusted to pH 6.0 and applied to a cation exchange SP-Sepharose Fast Flow (GE Healthcare Corporation) column. After washing with 20 mM phosphate buffer (pH 6.0), the proteins were eluted from the column using a linear concentration gradient of 0.5 M NaCl. After adjusting the pH of each eluate containing PN-621 and its variants to 8, they were added to a Gigacap Q (Tosoh Corporation) anion exchange column. After washing with 20 mM phosphate buffer (pH 7.8), PN-621 and its variants were separated using a linear concentration gradient of 0.3 M NaCl. Purity of each separation was confirmed by SDS-PAGE, and it was confirmed that PN-621 and its variants were purified as a single band at the theoretical molecular weight position.

[0142] 2. Immobilization of multi-domain polypeptides onto gel carriers Purified PN-621 and its variants were immobilized on a formyl-activated agarose gel carrier at a gel concentration of 10 mg / mL according to a conventional method. The reaction solution was recovered after immobilization, and the immobilization rate was measured. All of PN-621 and its variants showed an immobilization efficiency of 90% or higher.

[0143] 3. Measurement of immunoglobulin binding activity 0.5 mL of each gel carrier immobilized with PN-621 and its variants was packed into a Tricorn 5 / 20 column (GE Healthcare Corporation). The column was set in an AKTA pure25 liquid chromatography system (GE Healthcare Corporation), and after equilibrating with PBS, a 3 mg / mL human polyclonal IgG solution (obtained from the Japan Blood Products Organization) was flowed through it at a flow rate with a residence time of 5 minutes. The absorbance of the eluent at 280 nm was monitored, and the amount of IgG added when the IgG concentration in the eluent reached 10% of the IgG solution used was determined. From this value, the amount of IgG dynamically bound per 1 mL of gel (mg / mL gel) was calculated according to the following formula.

number

[0144] 4. Measurement of residual activity after alkaline treatment The residual activity after alkali treatment was determined using each gel carrier on which PN-621 and its modified forms were immobilized, under the same conditions as in Test Example 2.

[0145] 5. Measurement of elution rate in weakly acidic conditions The elution rate in a weakly acidic environment was determined using each gel carrier on which PN-621 and its modified counterparts were immobilized, under the same conditions as in Test Example 1.

[0146] 6.Results The results are shown in Table 4. These results indicate that polydomain polypeptides having multiple amino acid sequences in SEQ ID NO: 2 or 3 where position 41 is substituted with an amino acid or histidine having a hydrophobic side chain; polydomain polypeptides having multiple amino acid sequences where position 9 is substituted with an amino acid or histidine having a hydrophobic aliphatic side chain, and position 41 is substituted with an amino acid or histidine having a hydrophobic side chain; and polydomain polypeptides having multiple amino acid sequences where position 9 is unsubstituted, position 15 is substituted with histidine, position 24 is substituted with glutamine, and position 41 is substituted with histidine also exhibit high residual activity after alkaline treatment and high IgG elution ability in weakly acidic conditions.

[0147] [Table 4]

[0148] #1 The sequences α, β, α1-12, and β1-10 shown in Table 4 are as follows: Array α : Amino acid sequence shown in Sequence ID No. 3 Sequence β : Amino acid sequence shown in Sequence ID No. 2 Array α1 : Amino acid sequences in which the amino acid substitution A4I / T7R / N11A / S41V is introduced in the amino acid sequence shown in Sequence ID No. 3 Sequence β1: Amino acid sequences in which the amino acid substitution A4I / T7R / N11A / S41V is introduced in the amino acid sequence shown in Sequence ID No. 2 Array α2 : Amino acid sequences in which the amino acid substitution A4I / T7R / Q9V / N11A / S41V is introduced in the amino acid sequence shown in Sequence ID No. 3 Sequence β2 : Amino acid sequences in which the amino acid substitution A4I / T7R / Q9V / N11A / S41V is introduced in the amino acid sequence shown in Sequence ID No. 2 Array α3 : The amino acid sequence shown in Sequence ID No. 3, in which the amino acid substitution A4V / T7E / Q9L / E15H / S41V is introduced. Sequence β3 : The amino acid sequence shown in Sequence ID No. 2, in which the amino acid substitution A4V / T7E / Q9L / E15H / S41V is introduced. Array α4 : The amino acid sequence shown in Sequence ID No. 3, in which the amino acid substitution A4I / T7E / N11H / E15L / S41V is introduced. Sequence β4 : The amino acid sequence shown in Sequence ID No. 2, in which the amino acid substitution A4I / T7E / N11H / E15L / S41V has been introduced. Array α5 : Amino acid sequences in which the amino acid substitution A4I / T7E / Q9A / N11A / S41V is introduced in the amino acid sequence shown in Sequence ID No. 3 Sequence β5 : Amino acid sequences in which the amino acid substitution A4I / T7E / Q9A / N11A / S41V is introduced in the amino acid sequence shown in Sequence ID No. 2 Array α6 : Amino acid sequences in which the amino acid substitution A4I / T7E / Q9V / N11A / S41V is introduced in the amino acid sequence shown in Sequence ID No. 3 Sequence β6 : Amino acid sequences in which the amino acid substitution A4I / T7E / Q9V / N11A / S41V is introduced in the amino acid sequence shown in Sequence ID No. 2 Array α7: The amino acid sequence shown in Sequence ID No. 3, in which the amino acid substitution A4I / T7E / Q9H / N11A / S41V has been introduced. Sequence β7 : The amino acid sequence shown in Sequence ID No. 2, in which the amino acid substitution A4I / T7E / Q9H / N11A / S41V has been introduced. Array α8 : The amino acid sequence shown in Sequence ID No. 3, in which the amino acid substitution A4I / T7R / Q9V / N11A / E15H / S41V has been introduced. Sequence β8 : The amino acid sequence shown in Sequence ID No. 2, in which the amino acid substitution A4I / T7R / Q9V / N11A / E15H / S41V has been introduced. Array α9 : The amino acid sequence shown in Sequence ID No. 3, in which the amino acid substitution A4I / T7E / Q9V / N11A / E15A / S41V has been introduced. Sequence β9 : The amino acid sequence shown in Sequence ID No. 2, in which the amino acid substitution A4I / T7E / Q9V / N11A / E15A / S41V has been introduced. Sequence α10 : The amino acid sequence shown in Sequence ID No. 3, in which the amino acid substitution A4I / T7E / Q9L / N11H / E15L / S41V has been introduced. Sequence β10 : The amino acid sequence shown in Sequence ID No. 2, in which the amino acid substitution A4I / T7E / Q9L / N11H / E15L / S41V has been introduced. Sequence α11 : Amino acid sequences in which the amino acid substitution N3D / A4A / N6D / T7V / Q9A / N11A / S41V is introduced in the amino acid sequence shown in Sequence ID No. 3. Sequence α12 : Amino acid sequences in which the amino acid substitution N3D / A4A / N6D / T7E / Q9V / N11A / S41V is introduced in the amino acid sequence shown in Sequence ID No. 3. Sequence α13 : Amino acid sequences in which the amino acid substitution N3V / A4A / N6Q / T7V / Q9I / N11Q / S41V has been introduced, as shown in SEQ ID NO: 3 Sequence α14 : Amino acid sequences in which the amino acid substitution N3D / A4V / N6Q / T7V / N11Q / E15H / E24Q / S41H has been introduced in the amino acid sequence shown in Sequence ID No. 3.

[0149] Test Example 5: IgG Elution Profile Based on pH Gradient Human polyclonal IgG (obtained from the Japan Blood Products Organization) was injected and bound to a column packed with a gel support immobilized with each of the multi-domain polypeptides of Example 56 (PN-621 modified) and Comparative Example 50 (PN-621). Then, human IgG was eluted by applying a linear pH gradient using 0.1 M citrate buffer at pH 7.2 and 0.1 M citrate buffer at pH 2.3, and the absorbance of the eluate at 280 nm was monitored.

[0150] The results are shown in Figure 1. In Figure 1, (A) shows the results using Example 56 (PN-621 modified), and (B) shows the results using Comparative Example 50 (PN-621). Human IgG was eluted at pH 4.02 in the gel carrier immobilized with the multi-domain polypeptide of Example 56. On the other hand, human IgG was eluted at pH 3.19 in the gel carrier immobilized with the multi-domain polypeptide of Comparative Example 50 (PN-621). These results clearly demonstrate that the protein A modified of the present invention can purify IgG at a higher pH value under weakly acidic conditions.

[0151] Test Example 6: Production and Evaluation of Wild-Type Single-Domain Polypeptides and Their Modified Forms 1. Production of wild-type, monomutant, and dimutant monodomain polypeptides Expression plasmids of wild-type single-domain polypeptides for each of the five domains (A, B, C, D, E) of protein A, and their variants, were prepared. Specifically, expression plasmids of wild-type domain A (SEQ ID NO: 4), wild-type domain B (SEQ ID NO: 7), wild-type domain C (SEQ ID NO: 1), wild-type domain D (SEQ ID NO: 10), wild-type domain E (SEQ ID NO: 13), single-mutant variants in which position 41 of each wild-type domain was substituted with histidine, and bi-mutant variants in which position 9 of each wild-type domain was substituted with leucine and position 41 with histidine were prepared using the same method as in Test Example 1. These wild-type domains and their variants were given sequences with five lysine units ligated to the C-terminus for use in immobilization on a gel support.

[0152] The nucleic acid sequences of wild-type single-domain polypeptides and their modified expression plasmids were analyzed using a CEQ8000 DNA sequencer (Beckman Coulter, Inc.) to confirm that the sequences were as designed.

[0153] <Manufacturing of Domain Modifications> Using the expression plasmids obtained above, E. coli BL21(DE3) competent cells (Merck KGaA) were transformed to obtain each wild-type single-domain polypeptide and each expression strain of their variants.

[0154] Each of the expression-producing E. coli strains obtained above was seed cultured for 12 hours in LB medium containing 25 mg / L kanamycin and 2.0% glucose. The resulting seed culture was inoculated into 2×TY medium containing 25 mg / L kanamycin and 0.8% glucose and cultured at 37°C for 16 hours to express the target wild-type single-domain polypeptides and their variants, after which the E. coli were recovered by centrifugation. Next, the recovered E. coli were suspended in 50 mM sodium phosphate buffer (pH 6.5), sonicated to disrupt the E. coli, and then the wild-type single-domain polypeptides and their variants were recovered in the supernatant by further centrifugation. The obtained supernatants were subjected to sodium dodecyl sulfate-15% polyacrylamide gel electrophoresis (SDS-PAGE) as cell extracts, confirming that the target wild-type single-domain polypeptides and their variants were produced.

[0155] The cell extracts of each E. coli strain obtained above were adjusted to pH 6.0 and then applied to a cation exchange SP-Sepharose Fast Flow (GE Healthcare Corporation) column. After washing with 20 mM phosphate buffer (pH 6.0), linear chromatography was performed using 0.5 M NaCl. Proteins were eluted from the column using a linear concentration gradient. Analysis of the eluates by SDS-PAGE revealed that each wild-type single-domain polypeptide and its variants eluted between 0.1 and 0.2 M NaCl concentrations. Next, the pH of each eluate containing each wild-type single-domain polypeptide and its variants was adjusted to 9, and then added to a Gigacap Q anion exchange column (Tosoh Corporation). After washing with 20 mM phosphate buffer (pH 7.8), each wild-type single-domain polypeptide and its variants were separated using a linear concentration gradient of 0.3 M NaCl. Purity of each separation was confirmed by SDS-PAGE, and it was confirmed that each wild-type single-domain polypeptide and its variants were purified as a single band at the theoretical molecular weight position.

[0156] 2. Immobilization of single-domain polypeptides onto gel carriers Each purified wild-type single-domain polypeptide and its variants were immobilized on a formyl-activated agarose gel carrier at a gel concentration of 10 mg / mL according to a conventional method. The reaction solution was collected after immobilization, and the immobilization rate was measured. All wild-type single-domain polypeptides and their variants showed an immobilization efficiency of 90% or higher.

[0157] 3. Measurement of immunoglobulin binding activity The amount of IgG bound to each wild-type single-domain polypeptide and their variants was determined under the same conditions as in Test Example 1 using each gel carrier on which these polypeptides were immobilized.

[0158] 4. Measurement of residual activity after alkaline treatment Each gel support immobilized with wild-type single-domain polypeptides and their variants was washed with PBS, then washed three times with 0.1 M NaOH aqueous solution, and replaced with the same alkaline solution. The gels were then stored at 25°C for 6, 17, or 68 hours (alkaline treatment). Subsequently, after washing three times with PBS, the immunoglobulin binding activity was measured under the same conditions as above. The "residual activity after alkaline treatment (%)" was calculated as the percentage of the remaining IgG binding amount after alkaline treatment, with the amount of IgG binding before alkaline treatment set to 100%.

[0159] 5. Measurement of elution rate in weakly acidic conditions Using each gel carrier on which each wild-type single-domain polypeptide and its variants were immobilized, the elution rate in a weakly acidic environment was determined under the same conditions as in Test Example 1, except that 0.1 M citrate buffer (pH 5.0) was used for IgG elution.

[0160] 6.Results The results are shown in Tables 5 and 6. From these results, it was confirmed that in the five domains of protein A, the single mutant variant in which the 41st position was substituted with histidine, and the bimutant mutant variant in which the 9th position was substituted with leucine and the 41st position with histidine, exhibited residual activity equal to or greater than that of the wild type after alkaline treatment, and the elution rates in weak acidity were also improved in each case.

[0161] Table 5

[0162] Table 6

Claims

1. A polypeptide comprising at least one immunoglobulin-binding domain as shown in (A) to (C) below. (A) An immunoglobulin-binding domain containing an amino acid sequence in which a modification satisfying the following conditions (i) to (iv) has been introduced in any of the amino acid sequences shown in Sequence ID No. 1 to 15: (i) The serine at position 41 is replaced with valine, tyrosine, or histidine. (ii) The glutamine at position 9 is unsubstituted, or substituted with an amino acid or histidine having a hydrophobic aliphatic side chain. (iii) The glutamic acid or glutamine at position 15 is unsubstituted or substituted with alanine, histidine, tyrosine, or leucine. (iv) The glutamic acid or alanine at position 24 is unsubstituted, substituted with glutamine, histidine, or alanine in the case of SEQ ID NOs. 1-12, or substituted with glutamine or histidine in the case of SEQ ID NOs. 13-15. (B) An immunoglobulin-binding domain having an amino acid sequence shown in any of Sequence IDs 1 to 15, in which a modification satisfying the conditions of (i) to (iv) above has been introduced, and one or more amino acids in the unmodified site have been substituted, added, inserted and / or deleted, and which has alkaline stability equal to or better than that of a polypeptide consisting of the corresponding unmodified amino acid sequence, and high IgG elution activity in the weakly acidic region. (C) An immunoglobulin-binding domain in which a modification satisfying the conditions of (i) to (iv) above is introduced in any of the amino acid sequences shown in Sequence ID No. 1 to 15, the sequence identity of the unmodified region with respect to the corresponding unmodified amino acid sequence is 90% or more, and the alkali stability is equal to or better than that of a polypeptide consisting of the corresponding unmodified amino acid sequence, and the IgG elution activity in the weakly acidic region is high.

2. The polypeptide according to claim 1, which is a single-domain peptide containing one immunoglobulin-binding domain selected from among the immunoglobulin-binding domains shown in (A) to (C) above.

3. The polypeptide according to claim 1, which is a polydomain peptide comprising two or more immunoglobulin-binding domains selected from the immunoglobulin-binding domains shown in (A) to (C) above, linked together.

4. The polypeptide according to any one of claims 1 to 3, wherein the glutamine at position 9 in the amino acid sequence is substituted with an amino acid having a hydrophobic aliphatic side chain or histidine.

5. In the aforementioned amino acid sequence, glutamic acid or glutamine at position 15 is substituted with alanine or histidine, and The polypeptide according to any one of claims 1 to 4, wherein in the case of SEQ ID NOs. 1 to 12, the glutamic acid at position 24 is substituted with glutamine, or in the case of SEQ ID NOs. 13 to 15, the alanine at position 24 is substituted with glutamine.

6. The polypeptide according to any one of claims 1 to 5, wherein the glutamine at position 9 in the amino acid sequence is substituted with alanine, valine, leucine, isoleucine, or histidine.

7. DNA encoding a polypeptide according to any one of claims 1 to 6.

8. A recombinant vector comprising the DNA described in claim 7.

9. A transformant obtained by transforming a host using the recombinant vector described in claim 8.

10. A method for producing a polypeptide according to any one of claims 1 to 6, comprising the step of culturing the transformant according to claim 9.

11. An immunoglobulin-binding carrier comprising a polypeptide according to any one of claims 1 to 6 immobilized on an insoluble carrier.

12. A method for separating an immunoglobulin or a fragment thereof, comprising separating an immunoglobulin or a polypeptide containing its Fc region using the immunoglobulin-binding carrier described in claim 11.

13. The separation method according to claim 12, wherein an immunoglobulin or a polypeptide containing its Fc region is first bound to the immunoglobulin-binding carrier, and then the immunoglobulin or the polypeptide containing its Fc region is eluted under pH conditions of 3 to 5.

Images