Z domain variant of protein a with increased immunoglobulin elution rate, and use thereof

Abstract

The present application relates to a protein A variant with increased immunoglobulin elution efficiency and, more specifically, to: a Z domain variant in which an amino acid at a specific position in the Z domain (B domain variant) of protein A is mutated to have immunoglobulin elution efficiency superior to that of the Z domain; and a use thereof.

Description

Z-domain variant of protein A with increased immunoglobulin elution rate and uses thereof

[0001] Cross-citation with related applications

[0002] This application claims the benefit of priority based on Korean Patent Application No. 10-2025-0006388 dated January 15, 2025, and all contents disclosed in the document of said Korean patent application are incorporated herein as part of this specification.

[0003] The present application relates to a protein A variant with increased immunoglobulin elution efficiency, and more specifically, to a Z domain variant in which an amino acid at a specific position of the Z domain (B domain variant) of protein A is modified, thereby enhancing the immunoglobulin elution efficiency relative to the Z domain, and to the use thereof.

[0004]

[0005] Monoclonal antibodies are secreted into the culture medium during the culture of genetically modified animal cells, and exist at very low concentrations due to the mixing of various proteins secreted by the cells themselves and proteins within the medium. Therefore, removing impurities other than the target monoclonal antibody is an important step in antibody production. The process of separating and purifying monoclonal antibodies mainly utilizes affinity chromatography using antibody affinity ligands that can selectively recover only monoclonal antibodies from the medium, and proteins such as protein A, protein G, and protein L are used as the antibody affinity ligands.

[0006] Immunoglobulin-binding bacterial proteins, such as Protein A, Protein G, Protein A / G, and Protein L, are widely used for the purification and detection of immune proteins (antibodies, antibody fragments, etc.). These proteins exhibit different antibody binding patterns regarding the recognition site on the antibody and the type of antibody to which they bind.

[0007] Among these, protein A is a cell surface protein of Staphylococcus aureus and consists of five domains (E, D, A, B, and C domains) that have high homology. The domains have a structure consisting of three antiparallel helices and two loops located in the middle of the helices, and consist of about 58 amino acid residues.

[0008] To increase antibody purification efficiency, it is required to improve the elution efficiency of protein A-based antibody purification resins.

[0009]

[0010] The present application provides a protein A variant with an improved (increased) elution yield of immunoglobulin (e.g., IgG) and uses thereof. The protein A variant may be a variant of protein A or a functional part thereof (e.g., B domain) and is characterized by having an improved (increased) elution yield of the bound immunoglobulin (e.g., elution yield at pH 2 or higher, pH 2.5 or higher, pH 3 or higher, pH 3.5 or higher, or pH 4 or higher; more specifically, elution yield at pH 3.5 or higher, or pH 4 or higher) compared to a wild-type protein or a wild-type functional part, and / or a non-variant protein or a non-variant functional part.

[0011] One aspect of the present application is that in the Z domain of protein A (SEQ ID NO. 145, SEQ ID NO. 2, or SEQ ID NO. 153),

[0012] One or more, two or more, or three or more, selected from the group consisting of the amino acid corresponding to the 6th position, the amino acid corresponding to the 11th position, the amino acid corresponding to the 18th position, the amino acid corresponding to the 32nd position, the amino acid corresponding to the 36th position, and the amino acid corresponding to the 43rd position, e.g., 1, 2, 3, 4, 5, or 6, are substituted with amino acids different from the original.

[0013] Provides polypeptides.

[0014] The above polypeptide is a variant of the Z domain of protein A, characterized by maintaining the function of the Z domain of protein A while improving the elution efficiency of immunoglobulin.

[0015] In one example, the polypeptide is,

[0016] In the Z domain of protein A (SEQ ID NO. 145, SEQ ID NO. 2, or SEQ ID NO. 153),

[0017] Any one amino acid selected from the group consisting of the amino acid corresponding to the 11th position, the amino acid corresponding to the 18th position, the amino acid corresponding to the 32nd position, the amino acid corresponding to the 36th position, and the amino acid corresponding to the 43rd position may be substituted with an amino acid different from the original.

[0018] In another example, the polypeptide above is,

[0019] In the Z domain of protein A (SEQ ID NO. 145, SEQ ID NO. 2, or SEQ ID NO. 153),

[0020] One or more amino acids selected from the group consisting of the amino acid corresponding to the 11th position, the amino acid corresponding to the 18th position, and the amino acid corresponding to the 43rd position, such as 1, 2, or 3 amino acids, may be substituted with amino acids different from the original.

[0021] More specifically, the polypeptide above is,

[0022] In the Z domain of protein A (SEQ ID NO. 145, SEQ ID NO. 2, or SEQ ID NO. 153),

[0023] The amino acid corresponding to the 11th position is substituted with tryptophan (Trp, W), tyrosine (Tyr, Y), or leucine (Leu, L);

[0024] The amino acid corresponding to the 18th position is substituted with tyrosine (Tyr, Y); and

[0025] The amino acid corresponding to the 43rd position is replaced with tyrosine (Tyr, Y).

[0026] It may include one or more (e.g., one, two, or three) substitutions selected from a group consisting of, or may be mutated into one or more of the above substitutions.

[0027]

[0028] In another example, the polypeptide may be one in which any one amino acid selected from the group consisting of the amino acid corresponding to the 11th position, the amino acid corresponding to the 18th position, and the amino acid corresponding to the 43rd position in the Z domain of protein A (SEQ ID NO. 145, SEQ ID NO. 2, or SEQ ID NO. 153) is substituted with an amino acid different from the original.

[0029] More specifically, the polypeptide above is,

[0030] In the Z domain of protein A (SEQ ID NO. 145, SEQ ID NO. 2, or SEQ ID NO. 153),

[0031] The amino acid corresponding to the 11th position is substituted with tryptophan (Trp, W), tyrosine (Tyr, Y), or leucine (Leu, L) (e.g., tryptophan or tyrosine), or

[0032] The amino acid corresponding to the 18th position is substituted with tyrosine (Tyr, Y), or

[0033] The amino acid corresponding to the 43rd position may be substituted with tyrosine (Tyr, Y).

[0034] In another example, the polypeptide above is,

[0035] In the Z domain of protein A (SEQ ID NO. 145, SEQ ID NO. 2, or SEQ ID NO. 153),

[0036] The amino acid corresponding to the 11th position, the amino acid corresponding to the 18th position, and the amino acid corresponding to the 43rd position may be substituted with amino acids different from the original.

[0037] Specifically, the polypeptide above is,

[0038] In the Z domain of protein A (SEQ ID NO. 145, SEQ ID NO. 2, or SEQ ID NO. 153),

[0039] The amino acid corresponding to the 11th position is substituted with tryptophan (Trp, W), tyrosine (Tyr, Y), or leucine (Leu, L) (e.g., tryptophan), and

[0040] The amino acid corresponding to the 18th position is substituted with tyrosine (Tyr, Y), and

[0041] The amino acid corresponding to the 43rd position may be substituted with tyrosine (Tyr, Y).

[0042]

[0043] In another example, the polypeptide is, in addition to the substitution of one or more amino acids selected from the group consisting of amino acids corresponding to the 11th, 18th, and 43rd positions of the Z domain of protein A (SEQ ID NO. 145, SEQ ID NO. 2, or SEQ ID NO. 153) described above, for example, one, two, or three amino acids different from the original,

[0044] It may additionally include one or more (e.g., one, two, or three) substitutions with amino acids different from the original selected from the group consisting of the amino acid corresponding to the 6th position, the amino acid corresponding to the 32nd position, and the amino acid corresponding to the 36th position, or may be further mutated by said one or more substitutions with amino acids different from the original.

[0045] More specifically, the polypeptide above is,

[0046] In the Z domain of protein A (SEQ ID NO. 145, SEQ ID NO. 2, or SEQ ID NO. 153),

[0047] The amino acid corresponding to the 11th position is substituted with tryptophan (Trp, W), tyrosine (Tyr, Y), or leucine (Leu, L) (e.g., tryptophan or leucine),

[0048] The amino acid corresponding to the 18th position is substituted with tyrosine (Tyr, Y), and

[0049] In addition to the substitution of the amino acid corresponding to the 43rd position with tyrosine (Tyr, Y),

[0050] It may additionally include one or more (1, 2, or 3) substitutions selected from the group consisting of the following, or be additionally mutated with said one or more substitutions:

[0051] The amino acid corresponding to the 6th position is substituted with histidine (His, H);

[0052] The amino acid corresponding to the 32nd position is substituted with methionine (Met, M); and

[0053] The amino acid corresponding to the 36th position is substituted with histidine (His, H) or alanine (Ala, A).

[0054] In one example, the polypeptide is,

[0055] In the Z domain of protein A (SEQ ID NO. 145, SEQ ID NO. 2, or SEQ ID NO. 153),

[0056] The amino acid corresponding to the 11th position, the amino acid corresponding to the 18th position, the amino acid corresponding to the 32nd position, the amino acid corresponding to the 36th position, and the amino acid corresponding to the 43rd position may be substituted with amino acids different from the original.

[0057] Specifically, the polypeptide above is,

[0058] In the Z domain of protein A (SEQ ID NO. 145, SEQ ID NO. 2, or SEQ ID NO. 153),

[0059] The amino acid corresponding to the 11th position is substituted with tryptophan (Trp, W) or leucine (Leu, L), and

[0060] The amino acid corresponding to the 18th position is substituted with tyrosine (Tyr, Y), and

[0061] The amino acid corresponding to the 32nd position is substituted with methionine (Met, M), and

[0062] The amino acid corresponding to the 36th position is substituted with histidine (His, H), and

[0063] The amino acid corresponding to the 43rd position may be substituted with tyrosine (Tyr, Y).

[0064] More specifically, the polypeptide above is,

[0065] In the Z domain of protein A (SEQ ID NO. 145, SEQ ID NO. 2, or SEQ ID NO. 153),

[0066] The amino acid corresponding to the 11th position is substituted with tryptophan (Trp, W), and

[0067] The amino acid corresponding to the 18th position is substituted with tyrosine (Tyr, Y), and

[0068] The amino acid corresponding to the 32nd position is substituted with methionine (Met, M), and

[0069] The amino acid corresponding to the 36th position is substituted with histidine (His, H), and

[0070] The amino acid corresponding to the 43rd position may be substituted with tyrosine (Tyr, Y).

[0071] In another embodiment, the polypeptide is,

[0072] In the Z domain of protein A (SEQ ID NO. 145, SEQ ID NO. 2, or SEQ ID NO. 153),

[0073] The amino acid corresponding to the 11th position is substituted with leucine (Leu, L), and

[0074] The amino acid corresponding to the 18th position is substituted with tyrosine (Tyr, Y), and

[0075] The amino acid corresponding to the 32nd position is substituted with methionine (Met, M), and

[0076] The amino acid corresponding to the 36th position is substituted with histidine (His, H), and

[0077] The amino acid corresponding to the 43rd position may be substituted with tyrosine (Tyr, Y).

[0078] In another example, the polypeptide above is,

[0079] In the Z domain of protein A (SEQ ID NO. 145, SEQ ID NO. 2, or SEQ ID NO. 153),

[0080] The amino acid corresponding to the 6th position, the amino acid corresponding to the 11th position, the amino acid corresponding to the 18th position, and the amino acid corresponding to the 43rd position may be substituted with amino acids different from the original.

[0081] More specifically, the polypeptide above is,

[0082] In the Z domain of protein A (SEQ ID NO. 145, SEQ ID NO. 2, or SEQ ID NO. 153),

[0083] The amino acid corresponding to the 6th position is substituted with histidine (His, H), and

[0084] The amino acid corresponding to the 11th position is substituted with tryptophan (Trp, W), and

[0085] The amino acid corresponding to the 18th position is substituted with tyrosine (Tyr, Y), and

[0086] The amino acid corresponding to the 43rd position may be substituted with tyrosine (Tyr, Y).

[0087]

[0088] Another aspect provides a protein A variant comprising the previously described polypeptide or polypeptide multimer as a B domain.

[0089] Another aspect provides nucleic acid molecules encoding the previously described polypeptide, polypeptide multimer, and / or protein A variant.

[0090] Another aspect provides a recombinant vector comprising the nucleic acid molecule, said recombinant vector may be used as an expression vector for expressing said polypeptide, polypeptide multimer, and / or protein A variant in a suitable host cell.

[0091] Another aspect provides a recombinant cell comprising the nucleic acid molecule or recombinant vector. The recombinant cell may be a host cell transformed with the nucleic acid molecule or recombinant vector, and may be a cell capable of expressing the polypeptide, polypeptide multimer, and / or protein A variant.

[0092] Another aspect provides a matrix (resin) in which a plurality of ligands are coupled to a solid support, the ligands comprising the polypeptide, a polypeptide multimer comprising two or more repeating units of the polypeptide, or a protein A variant comprising the polypeptide or polypeptide multimer as a B domain. The matrix may be intended for use in a technique for separating and / or purifying a target protein by adsorption to the target protein (e.g., a protein comprising the Fc region of an immunoglobulin, more specifically, an immunoglobulin, an Fc-containing fusion protein, etc.), such as chromatography (affinity chromatography).

[0093] Another aspect is,

[0094] (1) The above polypeptide,

[0095] (2) A polypeptide multimer comprising two or more repeating units of the above polypeptide,

[0096] (3) A protein A variant comprising the above polypeptide or polypeptide multimer as a B domain,

[0097] (4) A nucleic acid molecule encoding the polypeptide, polypeptide multimer, or protein A variant,

[0098] (5) A recombinant vector containing the above nucleic acid molecule,

[0099] (6) A recombinant cell containing the nucleic acid molecule or recombinant vector, and

[0100] (7) A matrix in which a plurality of ligands comprising the polypeptide, polypeptide multimer, or protein A variant are coupled to a solid support.

[0101] A composition for binding to an immunoglobulin Fc region and / or a protein comprising said Fc region, comprising one or more selected from the group consisting of said Fc region, and / or a composition for separating and / or purifying an immunoglobulin Fc region and / or a protein comprising said Fc region.

[0102] Another aspect is,

[0103] (1) The above polypeptide,

[0104] (2) A polypeptide multimer comprising two or more repeating units of the above polypeptide,

[0105] (3) A protein A variant comprising the above polypeptide or polypeptide multimer as a B domain,

[0106] (4) A nucleic acid molecule encoding the polypeptide, polypeptide multimer, or protein A variant,

[0107] (5) A recombinant vector containing the above nucleic acid molecule,

[0108] (6) A recombinant cell containing the nucleic acid molecule or recombinant vector, and

[0109] (7) A matrix in which a plurality of ligands comprising the polypeptide, polypeptide multimer, or protein A variant are coupled to a solid support.

[0110] The present invention provides a use for binding to an immunoglobulin Fc region and / or a protein comprising said Fc region of a composition comprising one or more selected from the group consisting of said Fc region, and / or a use for isolation and / or purification of an immunoglobulin Fc region and / or a protein comprising said Fc region.

[0111] Another aspect is,

[0112] (a) The above polypeptide,

[0113] (b) a polypeptide multimer comprising two or more repeating units of the above polypeptide,

[0114] (c) a protein A variant comprising the polypeptide or polypeptide multimer as a B domain, and

[0115] (d) A matrix for chromatography in which a plurality of ligands comprising the polypeptide, polypeptide multimer, or protein A variant are coupled to a solid support

[0116] A composition for the isolation and / or purification of an immunoglobulin Fc region and / or a protein comprising said Fc region, comprising one or more selected from the group consisting of

[0117] Another aspect is,

[0118] (a) The above polypeptide,

[0119] (b) a polypeptide multimer comprising two or more repeating units of the above polypeptide,

[0120] (c) a protein A variant comprising the polypeptide or polypeptide multimer as a B domain, and

[0121] (d) A matrix for chromatography in which a plurality of ligands comprising the polypeptide, polypeptide multimer, or protein A variant are coupled to a solid support

[0122] The present invention provides a use for the isolation and / or purification of the Fc region of an immunoglobulin and / or a protein comprising said Fc region, of a composition comprising one or more selected from the group consisting of

[0123] Another aspect is that, in a sample (e.g., a liquid sample) comprising the Fc region of an immunoglobulin and / or a protein containing said Fc region,

[0124] (a) The above polypeptide,

[0125] (b) a polypeptide multimer comprising two or more repeating units of the above polypeptide,

[0126] (c) a protein A variant comprising the polypeptide or polypeptide multimer as a B domain, and

[0127] (d) A matrix for chromatography in which a plurality of ligands comprising the polypeptide, polypeptide multimer, or protein A variant are coupled to a solid support

[0128] A method for separating and / or purifying an immunoglobulin Fc region and / or a protein containing said Fc region from a sample, comprising the step of contacting one or more selected from the group consisting of said immunoglobulin to adsorb said immunoglobulin.

[0129] The protein containing the Fc region of the above immunoglobulin may be selected from the group consisting of immunoglobulin (antibody), antibody fragment containing the Fc region (e.g., scFv-Fc), fusion protein in which a partner protein is fused to the N-terminus, C-terminus, or both-terminus of the Fc region, but is not limited thereto, and any protein containing the Fc region of the immunoglobulin may be used without limitation.

[0130] Another aspect provides a method for separating or purifying one or more target compounds from a liquid, comprising the step of contacting one or more selected from the group consisting of (a) said polypeptide, (b) a polypeptide multimer comprising two or more repeating units made of said polypeptide, (c) a protein A variant comprising said polypeptide or polypeptide multimer as a B domain, and (d) a chromatographic matrix in which a plurality of ligands comprising said polypeptide, polypeptide multimer, or protein A variant are coupled to a solid support, thereby adsorbing said target compounds to a sample (e.g., a liquid sample) containing said target compounds.

[0131]

[0132] The present application provides a Z domain variant of protein A with enhanced immunoglobulin elution efficiency and uses thereof.

[0133]

[0134] Definition of Terms

[0135] In this specification, the phrase “a polynucleotide (which may be conflated with “gene or nucleic acid molecule”) or polypeptide (which may be conflated with “protein”) “comprising a specific nucleic acid sequence or amino acid sequence,” “consisting (essentially) of a specific nucleic acid sequence or amino acid sequence,” or “represented by a specific nucleic acid sequence or amino acid sequence” may mean that the polynucleotide or polypeptide essentially comprises the specific nucleic acid sequence or amino acid sequence, and may be interpreted as comprising (or not excluding) a “substantially equivalent sequence” in which a variation (deletion, substitution, modification, and / or addition) has been applied to the specific nucleic acid sequence or amino acid sequence to the extent that the original function (binding to the Fc region of immunoglobulin) and / or intended function (e.g., increased immunoglobulin elution efficiency compared to a non-variant Z domain of protein A (without the previously described variation introduced) and / or protein A comprising said Z domain) is maintained. there is.

[0136] In this specification, the term "sequence identity" refers to the degree of correspondence with a given nucleic acid sequence or amino acid sequence and may be expressed as a percentage (%). Homology to a nucleic acid sequence can be determined, for example, using the BLAST algorithm described in the literature (cf. Karlin and Altschul, Pro. Natl. Acad. Sci. USA, 90, 5873, 1993) or FASTA by Pearson (cf. Methods Enzymol., 183, 63, 1990). Based on this BLAST algorithm, programs called BLASTN or BLASTX have been developed (cf. http: / www.ncbi.nlm.nih.gov).

[0137] In this specification, "amino acid corresponding to a specific position within the amino acid sequence of a polypeptide" may be interpreted to encompass an amino acid residue at a specific position within said amino acid sequence or an amino acid residue at a position corresponding to said specific position in a homologous isotype protein and / or a heterologous protein having the same or similar activity as said protein.

[0138] In the amino acid sequence of a polypeptide provided herein, if the first N-terminal residue is methionine (M), said methionine may be naturally occurring or generated during the recombination process. When the polypeptide provided herein is produced by recombination, a polypeptide comprising an amino acid sequence excluding methionine (M), which is the first N-terminal residue, and / or a signal peptide and / or a tag for labeling (e.g., His-Tag, etc.) located at the N-terminus is also included within the scope of this specification. Additionally, in the amino acid sequence of a protein, if the first N-terminal residue is not methionine, said protein may be produced recombinantly, and said methionine may be added to the N-terminus of said first residue, but is not limited thereto.

[0139]

[0140] As used in this specification, one (three) letters of amino acids refer to the following amino acids in accordance with standard abbreviation rules in the field of biochemistry:

[0141] A(Ala): Alanine; C(Cys): Cysteine; D(Asp): Aspartic acid; E(Glu): Glutamic acid; F(Phe): Phenylalanine; G(Gly): Glycine; H(His): Histidine; I(IIe): Isoleucine; K(Lys): Lysine; L(Leu): Leucine; M(Met): Methionine; N(Asn): Asparagine; O(Ply) Pyrrolysine; P(Pro): Proline; Q(Gln): Glutamine; R(Arg): Arginine; S(Ser): Serine; T(Thr): Threonine; U(Sec): Selenocysteine, V(Val): Valine; W(Trp): Tryptophan; Y(Tyr): Tyrosine.

[0142] "(1 amino acid character)(amino acid position)(1 amino acid character)" as indicated in this specification means that the preceding amino acid at the corresponding amino acid position of the polypeptide before modification (i.e., the Z domain of protein A (SEQ ID NO. 145, SEQ ID NO. 2, or SEQ ID NO. 153)) is substituted with the following amino acid. For example, R11W means that arginine (R) at residue 11 of the polypeptide before modification is substituted with tryptophan (W).

[0143] Unless otherwise stated, the figures described herein may be interpreted to include, but are not limited to, ranges of ±20%, ±15%, ±10%, ±5%, ±3%, ±2%, ±1%, ±0.7%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, or ±0.1%.

[0144]

[0145] The present invention will be described in more detail below.

[0146]

[0147] Protein A variant: Z domain variant of Protein A, and multimers containing the same, and Protein A variant

[0148] The present application provides a protein A variant with enhanced immunoglobulin elution efficiency and uses thereof. The above protein A variant may be a variant of protein A or a functional part thereof (e.g., Z domain; e.g., SEQ ID NO. 145, SEQ ID NO. 2 (containing an HHHHHHC tag at the C-terminus), or SEQ ID NO. 153 (containing a His-tag (6H) at the C-terminus), and is characterized in that, compared to protein A containing the Z domain or the Z domain, the elution efficiency for immunoglobulin (e.g., elution efficiency at pH 2 or higher, pH 2.5 or higher, pH 3 or higher, pH 3.5 or higher, or pH 4 or higher; more specifically, elution efficiency at pH 3.5 or higher, or pH 4 or higher) is improved (increased) or / or the elution volume (e.g., elution volume at pH 2 or higher, pH 2.5 or higher, pH 3 or higher, pH 3.5 or higher, or pH 4 or higher; more specifically, elution volume at pH 3.5 or higher, or pH 4 or higher) is improved (decreased).

[0149] Protein A of the present application may be derived from Staphylococcus aureus, but is not limited thereto.

[0150] Protein A has binding activity to the Fc region of the heavy chain of an immunoglobulin (e.g., IgG), such as the region between the CH2 domain and the CH3 domain, and can be usefully used for the purification of proteins containing said Fc region, such as antibodies (full length or fragment), antibody fragments containing the Fc region (e.g., scFv-Fc), and fusion proteins in which a partner protein is fused to the N-terminus, C-terminus, or both-terminus of the Fc region.

[0151] The Z domain of the above protein A is a variant of the B domain of protein A derived from Staphylococcus aureus, wherein the alanine (A) at the 1st position and the glycine (G) at the 29th position in the amino acid sequence of the above B domain are substituted with valine (V) and may be represented by the amino acid sequence of SEQ ID NO. 145. The Z domain of the above protein A may have a function as a binding ligand for a target protein (e.g., a protein containing an Fc region such as immunoglobulin (IgG)) that is equivalent to or greater than the B domain of protein A.

[0152] The present application provides a Z domain variant in which a mutation by amino acid substitution is introduced at a predetermined position of the Z domain of protein A, a multimer comprising the same, a protein A variant comprising the Z domain variant as a B domain, and uses thereof.

[0153] The variant of the Z domain of protein A provided in the present application may maintain the function of the Z domain as an immunoglobulin binding ligand (binding function to immunoglobulin (Fc region; e.g., the Fc region of IgG)) at a significant level (e.g., equivalent level). Additionally, the Z domain variant, the multimer containing it, and / or the protein A variant may have improved (increased) immunoglobulin elution efficiency and / or improved (decreased) elution volume compared to the Z domain (e.g., SEQ ID NO. 145, SEQ ID NO. 2, or SEQ ID NO. 153) or protein A containing the Z domain.

[0154]

[0155] One example of the present application provides a polypeptide (a Z domain variant of protein A) in which one or more, two or more, or three or more (e.g., three, four, or five) amino acid variations (substitutions) are introduced into the Z domain of protein A. In one example, the polypeptide serving as the template for the polypeptide (a Z domain variant of protein A) (parent polypeptide: non-variant or pre-variant polypeptide) may include the amino acid sequence of SEQ ID NO. 145, or, for convenience such as labeling and / or purification during recombinant production and / or experimental use, may additionally include a suitable tag at the C-terminus or N-terminus (e.g., C-terminus), such as a His tag (e.g., H6 tag, etc.) and / or cysteine ​​(e.g., corresponding to SEQ ID NO. 2 or SEQ ID NO. 153), but is not limited thereto.

[0156] More specifically, one aspect of the present application is, in the Z domain of protein A (SEQ ID NO. 145, SEQ ID NO. 2, or SEQ ID NO. 153),

[0157] One or more, two or more, or three or more, selected from the group consisting of the amino acid corresponding to the 6th position, the amino acid corresponding to the 11th position, the amino acid corresponding to the 18th position, the amino acid corresponding to the 32nd position, the amino acid corresponding to the 36th position, and the amino acid corresponding to the 43rd position, e.g., 1, 2, 3, 4, 5, or 6, are substituted with amino acids different from the original.

[0158] Provides polypeptides.

[0159] The above polypeptide is a variant of the Z domain of protein A, characterized by an improved elution rate while maintaining the function of the Z domain of protein A. As previously explained, since the Z domain of protein A is a variant of the B domain, the polypeptide provided in this application may be included in the category of variants of the B domain of protein A.

[0160] In the Z domain of the above protein A (SEQ ID NO. 145, SEQ ID NO. 2, or SEQ ID NO. 153),

[0161] The amino acid corresponding to the 6th position is asparagine (Asn, N), and

[0162] The amino acid corresponding to the 11th position is arginine (Arg, R), and

[0163] The amino acid corresponding to the 18th position is histidine (His, H), and

[0164] The amino acid corresponding to the 32nd position is glutamine (Gln, Q), and

[0165] The amino acid corresponding to the 36th position is aspartic acid (Asp, D), and

[0166] The amino acid corresponding to the 43rd position may be asparagine (Asn, N).

[0167] The amino acid different from the original mentioned above is selected from the group consisting of alanine (A, Ala), asparagine (N, Asn), threonine (T, Thr), glutamic acid (E, Glu), serine (S, Ser), valine (V, Val), isoleucine (I, Ile), leucine (L, Leu), aspartic acid (D, Asp), cysteine ​​(C, Cys), glutamine (Q, Gln), methionine (M, Met), phenylalanine (F, Phe), proline (P, Pro), tryptophan (W, Trp), tyrosine (Y, Tyr), arginine (R, Arg), histidine (H, His), lysine (K, Lys), and glycine (G, Gly), and may be an amino acid different from the amino acid at the corresponding position of the Z domain of protein A.

[0168] In the above polypeptide, the amino acid corresponding to the 6th position may be substituted with histidine (His, H).

[0169] In the above polypeptide, the amino acid corresponding to the 11th position may be substituted with tryptophan (Trp, W), tyrosine (Tyr, Y), or leucine (Leu, L).

[0170] In the above polypeptide, the amino acid corresponding to the 18th position may be substituted with tyrosine (Tyr, Y).

[0171] In the above polypeptide, the amino acid corresponding to the 32nd position may be substituted with methionine (Met, M).

[0172] In the above polypeptide, the amino acid corresponding to the 36th position may be substituted with histidine (His, H) or alanine (Ala, A).

[0173] In the above polypeptide, the amino acid corresponding to the 43rd position may be substituted with tyrosine (Tyr, Y).

[0174] More specifically, the polypeptide above is,

[0175] The Z domain of protein A (SEQ ID NO. 145, SEQ ID NO. 2, or SEQ ID NO. 153) may comprise one or more, two or more, three or more, or four or more (e.g., one, two, three, four, five, or six) amino acid substitutions selected from the following, or may be mutated with said amino acid substitutions:

[0176] The amino acid corresponding to the 11th position is substituted with tryptophan (Trp, W) or leucine (Leu, L),

[0177] The amino acid corresponding to the 18th position is replaced with tyrosine (Tyr, Y),

[0178] The amino acid corresponding to the 32nd position is replaced with methionine (Met, M).

[0179] The amino acid corresponding to the 36th position is substituted with histidine (His, H) or alanine (Ala, A),

[0180] The amino acid corresponding to the 43rd position is substituted with tyrosine (Tyr, Y), and

[0181] The amino acid corresponding to the 6th position is replaced with histidine (His, H).

[0182] In one example, the polypeptide is,

[0183] In the Z domain of protein A (SEQ ID NO. 145, SEQ ID NO. 2, or SEQ ID NO. 153),

[0184] Any one amino acid selected from the group consisting of the amino acid corresponding to the 6th position, the amino acid corresponding to the 11th position, the amino acid corresponding to the 18th position, the amino acid corresponding to the 32nd position, the amino acid corresponding to the 36th position, and the amino acid corresponding to the 43rd position may be substituted with an amino acid different from the original.

[0185] More specifically, the polypeptide above is,

[0186] In the Z domain of protein A (SEQ ID NO. 145, SEQ ID NO. 2, or SEQ ID NO. 153),

[0187] The amino acid corresponding to the 11th position is substituted with tryptophan (Trp, W), tyrosine (Tyr, Y), or leucine (Leu, L);

[0188] The amino acid corresponding to the 18th position is substituted with tyrosine (Tyr, Y);

[0189] The amino acid corresponding to the 32nd position is substituted with methionine (Met, M);

[0190] The amino acid corresponding to the 36th position is substituted with histidine (His, H) or alanine (Ala, A); and

[0191] The amino acid corresponding to the 43rd position is substituted with tyrosine (Tyr, Y); and

[0192] The amino acid corresponding to the 6th position is replaced by histidine (His, H).

[0193] It may include any one substitution selected from the group consisting of, or be mutated into any one of the said substitutions.

[0194] In another example, the polypeptide may be one in which any one amino acid selected from the group consisting of the amino acid corresponding to the 11th position, the amino acid corresponding to the 18th position, and the amino acid corresponding to the 43rd position in the Z domain of protein A (SEQ ID NO. 145, SEQ ID NO. 2, or SEQ ID NO. 153) is substituted with an amino acid different from the original.

[0195] More specifically, the polypeptide above is,

[0196] In the Z domain of protein A (SEQ ID NO. 145, SEQ ID NO. 2, or SEQ ID NO. 153),

[0197] The amino acid corresponding to the 11th position is substituted with tryptophan (Trp, W), tyrosine (Tyr, Y), or leucine (Leu, L) (e.g., tryptophan or tyrosine), or

[0198] The amino acid corresponding to the 18th position is substituted with tyrosine (Tyr, Y), or

[0199] The amino acid corresponding to the 43rd position may be substituted with tyrosine (Tyr, Y).

[0200] In another example, the polypeptide above is,

[0201] In the Z domain of protein A (SEQ ID NO. 145, SEQ ID NO. 2, or SEQ ID NO. 153),

[0202] One or more amino acids selected from the group consisting of the amino acid corresponding to the 11th position, the amino acid corresponding to the 18th position, and the amino acid corresponding to the 43rd position, such as 1, 2, or 3 amino acids, may be substituted with amino acids different from the original.

[0203] More specifically, the polypeptide above is,

[0204] In the Z domain of protein A (SEQ ID NO. 145, SEQ ID NO. 2, or SEQ ID NO. 153),

[0205] The amino acid corresponding to the 11th position is substituted with tryptophan (Trp, W), tyrosine (Tyr, Y), or leucine (Leu, L);

[0206] The amino acid corresponding to the 18th position is substituted with tyrosine (Tyr, Y); and

[0207] The amino acid corresponding to the 43rd position is replaced with tyrosine (Tyr, Y).

[0208] It may include one or more (e.g., one, two, or three) substitutions selected from a group consisting of, or may be mutated into one or more of the above substitutions.

[0209] In another example, the polypeptide above is,

[0210] In the Z domain of protein A (SEQ ID NO. 145, SEQ ID NO. 2, or SEQ ID NO. 153),

[0211] The amino acid corresponding to the 11th position, the amino acid corresponding to the 18th position, and the amino acid corresponding to the 43rd position may be substituted with amino acids different from the original.

[0212] Specifically, the polypeptide above is,

[0213] In the Z domain of protein A (SEQ ID NO. 145, SEQ ID NO. 2, or SEQ ID NO. 153),

[0214] The amino acid corresponding to the 11th position is substituted with tryptophan (Trp, W), tyrosine (Tyr, Y), or leucine (Leu, L) (e.g., tryptophan), and

[0215] The amino acid corresponding to the 18th position is substituted with tyrosine (Tyr, Y), and

[0216] The amino acid corresponding to the 43rd position may be substituted with tyrosine (Tyr, Y).

[0217] In another example, the polypeptide is, in addition to the substitution of one or more amino acids selected from the group consisting of amino acids corresponding to the 11th, 18th, and 43rd positions of the Z domain of protein A (SEQ ID NO. 145, SEQ ID NO. 2, or SEQ ID NO. 153) described above, for example, one, two, or three amino acids different from the original,

[0218] It may additionally include one or more (e.g., one, two, or three) substitutions with amino acids different from the original selected from the group consisting of the amino acid corresponding to the 6th position, the amino acid corresponding to the 32nd position, and the amino acid corresponding to the 36th position, or may be further mutated by said one or more substitutions with amino acids different from the original.

[0219] More specifically, the polypeptide above is,

[0220] In the Z domain of protein A (SEQ ID NO. 145, SEQ ID NO. 2, or SEQ ID NO. 153),

[0221] The amino acid corresponding to the 11th position is substituted with tryptophan (Trp, W), tyrosine (Tyr, Y), or leucine (Leu, L) (e.g., tryptophan or leucine),

[0222] The amino acid corresponding to the 18th position is substituted with tyrosine (Tyr, Y), and

[0223] In addition to the substitution of the amino acid corresponding to the 43rd position with tyrosine (Tyr, Y),

[0224] It may additionally include one or more (1, 2, or 3) substitutions selected from the group consisting of the following, or be additionally mutated with said one or more substitutions:

[0225] The amino acid corresponding to the 6th position is substituted with histidine (His, H);

[0226] The amino acid corresponding to the 32nd position is substituted with methionine (Met, M); and

[0227] The amino acid corresponding to the 36th position is substituted with histidine (His, H) or alanine (Ala, A).

[0228] In one example, the polypeptide is,

[0229] In the Z domain of protein A (SEQ ID NO. 145, SEQ ID NO. 2, or SEQ ID NO. 153),

[0230] The amino acid corresponding to the 11th position, the amino acid corresponding to the 18th position, and the amino acid corresponding to the 43rd position are replaced with amino acids different from the original, and

[0231] Additionally,

[0232] The amino acid corresponding to the 6th position is substituted with histidine (His, H);

[0233] The amino acid corresponding to the 32nd position is substituted with methionine (Met, M); or

[0234] The amino acid corresponding to the 36th position is replaced with histidine (His, H) or alanine (Ala, A).

[0235] It could be that it happened.

[0236] More specifically, the polypeptide above is,

[0237] In the Z domain of protein A (SEQ ID NO. 145, SEQ ID NO. 2, or SEQ ID NO. 153),

[0238] The amino acid corresponding to the 11th position is substituted with tryptophan (Trp, W) or leucine (Leu, L), and

[0239] The amino acid corresponding to the 18th position is substituted with tyrosine (Tyr, Y), and

[0240] The amino acid corresponding to the 43rd position is substituted with tyrosine (Tyr, Y), and

[0241] Additionally,

[0242] The amino acid corresponding to the 6th position is substituted with histidine (His, H);

[0243] The amino acid corresponding to the 32nd position is substituted with methionine (Met, M); or

[0244] The amino acid corresponding to the 36th position is replaced with histidine (His, H) or alanine (Ala, A).

[0245] It could be that it happened.

[0246]

[0247] In another example, the polypeptide above is,

[0248] In the Z domain of protein A (SEQ ID NO. 145, SEQ ID NO. 2, or SEQ ID NO. 153),

[0249] The amino acid corresponding to the 11th position, the amino acid corresponding to the 18th position, the amino acid corresponding to the 32nd position, the amino acid corresponding to the 36th position, and the amino acid corresponding to the 43rd position may be substituted with amino acids different from the original.

[0250] Specifically, the polypeptide above is,

[0251] In the Z domain of protein A (SEQ ID NO. 145, SEQ ID NO. 2, or SEQ ID NO. 153),

[0252] The amino acid corresponding to the 11th position is substituted with tryptophan (Trp, W) or leucine (Leu, L), and

[0253] The amino acid corresponding to the 18th position is substituted with tyrosine (Tyr, Y), and

[0254] The amino acid corresponding to the 32nd position is substituted with methionine (Met, M), and

[0255] The amino acid corresponding to the 36th position is substituted with histidine (His, H), and

[0256] The amino acid corresponding to the 43rd position may be substituted with tyrosine (Tyr, Y).

[0257] More specifically, the polypeptide above is,

[0258] In the Z domain of protein A (SEQ ID NO. 145, SEQ ID NO. 2, or SEQ ID NO. 153),

[0259] The amino acid corresponding to the 11th position is substituted with tryptophan (Trp, W), and

[0260] The amino acid corresponding to the 18th position is substituted with tyrosine (Tyr, Y), and

[0261] The amino acid corresponding to the 32nd position is substituted with methionine (Met, M), and

[0262] The amino acid corresponding to the 36th position is substituted with histidine (His, H), and

[0263] The amino acid corresponding to the 43rd position may be substituted with tyrosine (Tyr, Y).

[0264] In another embodiment, the polypeptide is,

[0265] In the Z domain of protein A (SEQ ID NO. 145, SEQ ID NO. 2, or SEQ ID NO. 153),

[0266] The amino acid corresponding to the 11th position is substituted with leucine (Leu, L), and

[0267] The amino acid corresponding to the 18th position is substituted with tyrosine (Tyr, Y), and

[0268] The amino acid corresponding to the 32nd position is substituted with methionine (Met, M), and

[0269] The amino acid corresponding to the 36th position is substituted with histidine (His, H), and

[0270] The amino acid corresponding to the 43rd position may be substituted with tyrosine (Tyr, Y).

[0271] In another example, the polypeptide above is,

[0272] In the Z domain of protein A (SEQ ID NO. 145, SEQ ID NO. 2, or SEQ ID NO. 153),

[0273] The amino acid corresponding to the 6th position, the amino acid corresponding to the 11th position, the amino acid corresponding to the 18th position, and the amino acid corresponding to the 43rd position may be substituted with amino acids different from the original.

[0274] More specifically, the polypeptide above is,

[0275] In the Z domain of protein A (SEQ ID NO. 145, SEQ ID NO. 2, or SEQ ID NO. 153),

[0276] The amino acid corresponding to the 6th position is substituted with histidine (His, H), and

[0277] The amino acid corresponding to the 11th position is substituted with tryptophan (Trp, W), and

[0278] The amino acid corresponding to the 18th position is substituted with tyrosine (Tyr, Y), and

[0279] The amino acid corresponding to the 32nd position is substituted with methionine (Met, M), and

[0280] The amino acid corresponding to the 36th position is substituted with histidine (His, H), and

[0281] The amino acid corresponding to the 43rd position may be substituted with tyrosine (Tyr, Y).

[0282]

[0283] The Z domain of protein A of SEQ ID NOs 145, 2, and 153 above serves as a template into which the amino acid variation (substitution) described herein is introduced, wherein SEQ ID NOs 2 and 153 each have an HHHHHHC tag (SEQ ID NO. 146) or an HHHHHH tag (SEQ ID NO. 147) introduced at the C-terminus of the amino acid sequence of SEQ ID NO. 145. Accordingly, the polypeptide provided in this specification may be described as having the amino acid variation (substitution) described above introduced based on SEQ ID NO. 145, or having an HHHHHHC tag (SEQ ID NO. 146) or an HHHHHH tag (SEQ ID NO. 147) introduced at the C-terminus in addition thereto.

[0284]

[0285] In one example, the polypeptide comprises the previously described amino acid substitution and may have sequence identity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, or 94% or more with respect to the amino acid sequence of SEQ ID NO. 145, SEQ ID NO. 2, or SEQ ID NO. 153. The polypeptide is not identical to the amino acid sequence of SEQ ID NO. 145, SEQ ID NO. 2, and SEQ ID NO. 153 (i.e., has less than 100% identity with respect to the amino acid sequence of SEQ ID NO. 145, SEQ ID NO. 2, and SEQ ID NO. 153). The above polypeptide may include the previously described mutation (amino acid substitution) while maintaining the function of the non-mutated protein, i.e., the Z domain (or B domain) of protein A (e.g., binding affinity to immunoglobulin (e.g., Fc region)) at a significant level (e.g., equivalent level).

[0286] For example, the polypeptide may include the amino acid substitution described above and may include an amino acid sequence having 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, or 98% or more sequence homology with an amino acid sequence selected from SEQ ID NOs 146, 149, 150, 4, 138, 140, 154, 155, 156, 248 to 259, and 261 to 264. The amino acid sequence of the polypeptide may be different from SEQ ID NO. 145, SEQ ID NO. 2, and / or SEQ ID NO. 153, that is, having less than 100% sequence identity with SEQ ID NOs 145, 2, and / or 153. For example, the polypeptide may comprise an amino acid sequence selected from SEQ ID NOs 146, 149, 150, 4, 138, 140, 154, 155, 156, 248 to 259, and 261 to 264.

[0287]

[0288] In one embodiment, the polypeptide is,

[0289] In the Z domain of protein A (SEQ ID NO. 145, SEQ ID NO. 2, or SEQ ID NO. 153),

[0290] The amino acid corresponding to the 11th position is substituted with tryptophan (Trp, W) or tyrosine (Tyr, Y), or

[0291] The amino acid corresponding to the 18th position is substituted with tyrosine (Tyr, Y), or

[0292] The amino acid corresponding to the 43rd position is substituted with tyrosine (Tyr, Y), and

[0293] It may include an amino acid sequence having 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, or 98% or more sequence homology with an amino acid sequence selected from SEQ ID NOs 145, 2, 153, and 248 to 259, and more specifically, it may include an amino acid sequence selected from SEQ ID NOs 248 to 259.

[0294] The above polypeptide is,

[0295] In the Z domain of protein A (SEQ ID NO. 145, SEQ ID NO. 2, or SEQ ID NO. 153),

[0296] The amino acid corresponding to the 11th position is substituted with tryptophan (Trp, W), and

[0297] The amino acid corresponding to the 18th position is substituted with tyrosine (Tyr, Y), and

[0298] The amino acid corresponding to the 43rd position is substituted with tyrosine (Tyr, Y), and

[0299] It may include an amino acid sequence having 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, or 98% or more sequence homology with the amino acid sequence of SEQ ID NO. 145, 2, 153, 146, 4, or 154, and more specifically, it may include the amino acid sequence of SEQ ID NO. 146, 4, or 154.

[0300] In another embodiment, the polypeptide is,

[0301] In the Z domain of protein A (SEQ ID NO. 145, SEQ ID NO. 2, or SEQ ID NO. 153),

[0302] The amino acid corresponding to the 11th position is substituted with tryptophan (Trp, W), and

[0303] The amino acid corresponding to the 18th position is substituted with tyrosine (Tyr, Y), and

[0304] The amino acid corresponding to the 32nd position is substituted with methionine (Met, M), and

[0305] The amino acid corresponding to the 36th position is substituted with histidine (His, H), and

[0306] The amino acid corresponding to the 43rd position is substituted with tyrosine (Tyr, Y), and

[0307] It may include an amino acid sequence having 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, or 98% or more sequence homology with the amino acid sequence of SEQ ID NO. 145, 2, 153, 149, 138, or 155, and more specifically, it may include the amino acid sequence of SEQ ID NO. 149, 138, or 155.

[0308] In another embodiment, the polypeptide is,

[0309] In the Z domain of protein A (SEQ ID NO. 145, SEQ ID NO. 2, or SEQ ID NO. 153),

[0310] The amino acid corresponding to the 11th position is substituted with leucine (Leu, L), and

[0311] The amino acid corresponding to the 18th position is substituted with tyrosine (Tyr, Y), and

[0312] The amino acid corresponding to the 32nd position is substituted with methionine (Met, M), and

[0313] The amino acid corresponding to the 36th position is substituted with histidine (His, H), and

[0314] The amino acid corresponding to the 43rd position is substituted with tyrosine (Tyr, Y), and

[0315] It may include an amino acid sequence having 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, or 98% or more sequence homology with the amino acid sequence of SEQ ID NO. 145, 2, 153, 150, 140, or 156, and more specifically, it may include the amino acid sequence of SEQ ID NO. 150, 140, or 156.

[0316]

[0317] In another example, the polypeptide above is,

[0318] In the Z domain of protein A (SEQ ID NO. 145, SEQ ID NO. 2, or SEQ ID NO. 153),

[0319] The amino acid corresponding to the 11th position is substituted with tryptophan (Trp, W) or leucine (Leu, L), e.g., tryptophan (Trp, W), and

[0320] The amino acid corresponding to the 18th position is substituted with tyrosine (Tyr, Y), and

[0321] The amino acid corresponding to the 43rd position is substituted with tyrosine (Tyr, Y), and

[0322] Additionally,

[0323] The amino acid corresponding to the 6th position is substituted with histidine (His, H);

[0324] The amino acid corresponding to the 32nd position is substituted with methionine (Met, M); or

[0325] The amino acid corresponding to the 36th position is substituted with histidine (His, H) or alanine (Ala, A), and

[0326] It may include an amino acid sequence having 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, or 98% or more sequence homology with an amino acid sequence selected from SEQ ID NOs 145, 2, 153, and 261 to 264, and more specifically, it may include an amino acid sequence selected from SEQ ID NOs 261 to 264.

[0327]

[0328] The amino acid sequence (tag-free) of the polypeptide (Z domain variant of protein A) provided in one embodiment of the present application is described in Table 1 below:

[0329] 서열번호설명서열145amino acid sequence of Z domain variant VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK146amino acid sequence of Z domain variant (N11W / H18Y / N43Y; LiSa2-1)VDNKFNKEQQWAFYEILYLPNLNEEQRNAFIQSLKDDPSQSAYLLAEAKKLNDAQAPK149amino acid sequence of Z domain variant (N11W / H18Y / N43Y / Q32M / D36H; LiSa7-3)VDNKFNKEQQWAFYEILYLPNLNEEQRNAFIMSLKHDPSQSAYLLAEAKKLNDAQAPK150amino acid sequence of Z domain variant (N11L / H18Y / N43Y / Q32M / D36H; LiSa7-s3)VDNKFNKEQQLAFYEILYLPNLNEEQRNAFIMSLKHDPSQSAYLLAEAKKLNDAQAPK248amino acid sequence of Z domain variant (N11W; LiSa1-4)VDNKFNKEQQWAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK249amino acid sequence of Z domain variant (N11Y; LiSa1-6)VDNKFNKEQQYAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK250amino acid sequence of Z domain variant (H18Y; LiSa1-13)VDNKFNKEQQNAFYEILYLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK251amino acid sequence of Z domain variant (N43Y;LiSa1-42)VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSAYLLAEAKKLNDAQAPK261amino acid sequence of Z domain variant (N11W / H18Y / N43Y / N6H; LiSa6-52)VDNKFHKEQQWAFYEILYLPNLNEEQRNAFIQSLKDDPSQSAYLLAEAKKLNDAQAPK262amino acid sequence of Z domain variant (N11W / H18Y / N43Y / Q32M; LiSa6-25)VDNKFNKEQQWAFYEILYLPNLNEEQRNAFIMSLKDDPSQSAYLLAEAKKLNDAQAPK263amino acid sequence of Z domain variant (N11W / H18Y / N43Y / D36A; LiSa6-9)VDNKFNKEQQWAFYEILYLPNLNEEQRNAFIQSLKADPSQSAYLLAEAKKLNDAQAPK264amino acid sequence of Z domain variant (N11W / H18Y / N43Y / D36H; LiSa6-17)VDNKFNKEQQWAFYEILYLPNLNEEQRNAFIQSLKHDPSQSAYLLAEAKKLNDAQAPK;

[0330] (In Table 1, mutated amino acids are indicated in bold and underlined)

[0331]

[0332] Another example provides a polypeptide multimer comprising two or more repeating units (monomers) made of the above polypeptide.

[0333] In one embodiment, the polypeptide multimer may comprise two or more repeating units comprising (or composed of) the polypeptide described above, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10. For convenience such as labeling and / or purification during recombinant production and / or experimental use, the multimer may additionally comprise, but is not limited to, a suitable tag, e.g., a His tag (e.g., H6 tag, etc.), and / or cysteine ​​at the C-terminus or N-terminus (e.g., C-terminus) of the multimer.

[0334] The above polypeptide or polypeptide multimer may have a use as the B domain of protein A.

[0335] The above polypeptide is as described above. In one example, the polypeptide multimer may be a tetramer comprising four repeating units (monomers) made of the polypeptide.

[0336] In one example, the polypeptide multimer (tetramer) may have each monomer containing the variant (amino acid substitution) described above and having sequence identity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, or 94% or more with respect to the amino acid sequence of SEQ ID NOs 147, 126, and / or 157. The polypeptide is not identical to the amino acid sequence of SEQ ID NOs 147, 126, and / or 157 (i.e., has less than 100% identity with the amino acid sequence of SEQ ID NOs 147, 126, and 157). The above-mentioned polymer (tetramer) may include the mutation (amino acid substitution) described above, while maintaining the function (e.g., binding affinity to immunoglobulin (e.g., Fc region)) of the non-mutated protein, i.e., the polymer (tetramer) of the Z domain (or B domain) of protein A, at a significant level (e.g., equivalent level). For example, the polypeptide multimer (tetramer) may comprise an amino acid sequence of SEQ ID NOs 148, 151, 152, 128, 142, 144, 158, 159, or 160, wherein each monomer comprises a variation (amino acid substitution) described above, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence homology with said amino acid sequence (provided, it differs from the amino acid sequences of SEQ ID NOs 147, 126, and 157 (having less than 100% sequence homology)). For example, the polypeptide multimer may include the amino acid sequence of SEQ ID NOs 148, 151, 152, 128, 142, 144, 158, 159, or 160.

[0337]

[0338] The polypeptide or polypeptide multimer may further comprise one or more selected from the group consisting of the E, D, A, B, and C domains of protein A. The protein A may be derived from Staphylococcus aureus.

[0339]

[0340] Another example provides a protein A variant comprising the polypeptide or polypeptide multimer described above as the B domain.

[0341] The polypeptide, polypeptide multimer, and / or protein A variant described above may have an elution yield of the target protein (e.g., a protein containing an Fc region such as immunoglobulin) in an affinity resin that is superior (improved, increased) or / or has an improved (decreased) elution volume compared to protein A containing a Z domain (e.g., SEQ ID NO. 145) or a multimer containing the same or a B domain containing a Z domain. The above elution efficiency may be measured under conditions of pH 2 or higher, pH 2.5 or higher, pH 3 or higher, pH 3.5 or higher, pH 3.7 or higher, or pH 4 or higher, e.g., pH 3.7, pH 4, and / or pH 4.3, more specifically, under conditions of pH 3.5 or higher, pH 3.7 or higher, or pH 4 or higher, e.g., pH 3.7, pH 4, and / or pH 4.3, but is not limited thereto.

[0342] The above elution yield (%) refers to the ratio of eluted protein to injected protein and can be calculated by the following Formula 1, but is not limited thereto:

[0343] (Formula 1)

[0344] Elution yield (%) = [(IgG maxAU* IgG sample injection volume - CIP Area) / (IgG maxAU * IgG sample injection volume)] * 100

[0345] (IgG maxAU : Max AU (Absorbance Unit; UV maximum absorbance of sample solution) of IgG sample solution (Unit: mAU);

[0346] IgG sample injection volume: Amount of IgG sample injected into the equipment (Unit: mL);

[0347] CIP Area: IgG peak area obtained during the Cleaning-In-Place step (Unit: mL*mAU)

[0348]

[0349] In this specification, immunoglobulin may be derived from humans, primates such as monkeys, rodents such as mice and rats, etc., and may be selected from various subtypes such as IgG (IgG1, IgG2, IgG3, or IgG4), IgA, IgD, IgE, IgM, etc., and more specifically may be IgG (IgG1, IgG2, IgG3, or IgG4). The polypeptide (a Z-domain variant of protein A) and / or polypeptide multimers containing the same, protein A, and / or protein A variants provided in this application may bind to a heavy chain region (e.g., Fc region) of immunoglobulin (e.g., IgG). Accordingly, a protein containing the Fc region of immunoglobulin to which the polypeptide (a Z-domain variant of protein A), polypeptide multimers, protein A, and / or protein A variants provided in this application can bind is an immunoglobulin in its complete form; It may be a fragment form containing an Fc region, such as scFv-Fc, (scFv-Fc)2, (scFv)2-Fc, Fab-Fc, Fab'-Fc, etc.; an Fc-containing fusion protein comprising an Fc region and a partner protein bound to the N-terminus, C-terminus, or both-terminus of said Fc region, but is not limited thereto.

[0350]

[0351] In one example, the polypeptide, polypeptide polymer, protein A, and / or protein A variant provided in this application may be non-naturally occurring and may be produced, for example, by recombinant methods or chemical synthesis, but are not limited thereto.

[0352]

[0353] Nucleic acid molecule, recombinant expression vector, recombinant cell

[0354] Other examples provide the previously described polypeptide (a variant of the Z domain of protein A), polypeptide multimer, protein A, or nucleic acid molecules encoding a variant of protein A.

[0355] Another example provides a recombinant vector comprising the above nucleic acid molecule. The recombinant vector may be an expression vector capable of expressing the above nucleic acid molecule as a protein in a suitable host cell. The recombinant vector may be used as an expression vector for expressing the above polypeptide, polypeptide multimer, and / or protein A variant in a suitable host cell.

[0356] Another example provides a recombinant cell comprising the nucleic acid molecule or recombinant vector. The recombinant cell may be a suitable host cell into which the nucleic acid molecule or recombinant vector has been introduced (transformed).

[0357] In addition, the polypeptide, polypeptide polymer, protein A, or protein A variant may be used in an immobilized state rather than in a free state. The immobilization can be prepared by conventional methods known in the art, and as a carrier for immobilization, natural polymers such as cellulose, starch, dextran, and agarose; synthetic polymers such as polyacrylamide, polyacrylate, polymethacrylate, and Eupergit C; or minerals such as silica, bentonite, and metals may be used. Furthermore, it is possible to bind the polypeptide, polypeptide polymer, protein A, or protein A variant to these carriers by covalent bonding, ionic bonding, hydrophobic bonding, physical adsorption, microencapsulation, etc. In addition, it is possible to immobilize the polypeptide, polypeptide polymer, protein A, or protein A variant by forming a covalent bond through the action of glutaraldehyde, cyanogen bromide, etc., in these carrier-enzyme conjugates. Furthermore, it is possible to immobilize and use microbial cells containing the polypeptide, polypeptide polymer, protein A, or protein A variant directly without the need to separately purify the polypeptide, polypeptide polymer, protein A, or protein A variant. In such whole cell immobilization, techniques such as creating holes in the cells or surface expression may be applied to increase the reactivity of the polypeptide, polypeptide polymer, protein A, or protein A variant contained in the microorganism.

[0358]

[0359] Due to the degeneracy of codons, the nucleic acid sequences described herein may undergo various modifications to the coding region within a range that does not alter the amino acid sequence and / or function of the protein expressed from the coding region, taking into account the codons preferred by the microorganism intended to express the polypeptide, polypeptide multimer, protein A, or protein A variant.

[0360] The introduction of the above nucleic acid molecule or vector may be performed by a person skilled in the art by appropriately selecting a known transformation method. In this specification, the term “transformation” means introducing a vector containing a nucleic acid molecule encoding a target protein (foreign protein) into a host cell so that the protein encoded by said nucleic acid molecule can be expressed within the host cell. The transformed nucleic acid molecule may be inserted into the chromosomes of the host cell and / or located outside the chromosomes, provided that it can be expressed within the host cell. Furthermore, said nucleic acid molecule comprises DNA and / or RNA encoding the target protein (said polypeptide, polypeptide multimer, protein A, or protein A variant). There is no limitation on the form in which said nucleic acid molecule is introduced, as long as it can be introduced into the host cell and expressed. For example, said nucleic acid molecule may be introduced into the host cell in the form of an expression cassette, which is a genetic structure containing all the elements necessary for self-expression. The above expression cassette may include expression regulatory elements, such as a promoter, a transcription termination signal, a ribosome binding site, and / or a translation termination signal, which are typically operably linked to the nucleic acid molecule. The above expression cassette may be in the form of a self-replicating expression vector. Additionally, the nucleic acid molecule may be introduced into the host cell genome in its own form and operably linked to the sequence required for expression in the host cell. In the above, the term "operably linked" may mean that the expression regulatory element (e.g., a promoter) and the nucleic acid molecule are functionally linked so that the expression regulatory element can perform transcriptional regulation (e.g., transcription initiation) of the nucleic acid molecule encoding the target protein (foreign protein). Operable linkage may be performed using gene recombination techniques known in the art, for example, by conventional site-specific DNA cleavage and linkage, but is not limited thereto.

[0361] The method of transforming the above nucleic acid molecule into a host cell can be performed by any method of introducing the nucleic acid into the cell (microorganism), and depending on the host cell, transformation techniques known in the art can be appropriately selected and performed. Examples of the above-known transformation methods include electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG)-mediated uptake, DEAE-dextran method, cationic liposome method, lipofection, lithium acetate-DMSO method, heat shock method, particle gun bombardment, silicon carbide whiskers, sonication, etc., but are not limited thereto.

[0362] The introduction (insertion) of the above nucleic acid molecule into the host cell genome (chromosome) may be performed by a person skilled in the art by appropriately selecting a known method, for example, using one or more selected from the group consisting of an RNA-guided endonuclease system (RNA-guided endonuclease system or CRISPR system; for example, (a) an RNA-guided endonuclease (e.g., Cas9 protein, etc.), a gene encoding the same, or a vector containing said gene; and (b) a mixture containing a guide RNA (e.g., single guide RNA (sgRNA), etc.), DNA encoding the same, or a vector containing said DNA (e.g., a mixture of an RNA-guided endonuclease protein and a guide RNA, etc.), a complex (e.g., a ribonucleic acid fusion protein (RNP)), a recombinant vector (e.g., a vector containing both an RNA-guided endonuclease gene and guide RNA encoding DNA, etc.), but is not limited thereto.

[0363] In this specification, the term “vector” means a DNA product containing a sequence of nucleotides of a nucleic acid molecule encoding said target protein, which is operably linked to a suitable regulatory sequence to enable the expression of said target protein within a suitable host. The regulatory sequence may include a promoter capable of initiating transcription, any operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and / or a sequence regulating the termination of transcription and / or translation. After being transformed into a suitable host cell, the vector may be expressed independently of the host cell’s genome or incorporated into the host cell’s genome.

[0364] Vectors available in this specification are not particularly limited as long as they are capable of replicating within a host cell and may be selected from all commonly used vectors. Examples of commonly used vectors include plasmids, cosmids, viruses, bacteriophages, etc., in their natural or recombinant state. For example, as the above vectors, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A, etc. may be used as phage vectors or cosmid vectors, and as plasmid vectors, pBC series (e.g., pBC-KS(+)), pBR series (e.g., pBR322, pBR325), pUC series (e.g., pUC118 and pUC119), pBluescriptII series, pGEM series, pTZ series, pCL series, pET series (e.g., pET-22b(+)), plasmids derived from Bacillus subtilis (e.g., pUB110, pTP5), plasmids derived from Bacillus subtilis (e.g., pUB110, pTP5), plasmids derived from animal viruses such as retroviruses, adenoviruses, or vaccinia viruses, and plasmids derived from insect viruses such as baculoviruses. The back may be used, but is not limited thereto.

[0365] The above host cell may be selected from a group consisting of all types of commonly used single-celled organisms, such as prokaryotic microorganisms like various bacteria (e.g., Escherichia genus, Clostridia genus, etc.) and eukaryotic microorganisms like yeast, and may be selected from a group consisting of microorganisms of the genus Clostridia (e.g., Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium saccharoperbutylacetonicum, or Clostridium saccharobutylicum), microorganisms of the genus Escherichia (e.g., Escherichia coli, etc.), but is not limited thereto.

[0366] The vectors available in this specification may be known expression vectors and / or vectors for inserting nucleic acid molecules into host cell chromosomes. The insertion of said nucleic acid molecules into the host cell chromosomes may be achieved by any method known in the art, for example, homologous recombination or a CRISPR system, but is not limited thereto. The vector may further include a selection marker to confirm whether the insertion into the chromosome has occurred. The selection marker is intended to select cells transformed with the vector, that is, to confirm whether the polynucleotide has been inserted, and may be selected from genes conferring selectable phenotypes such as drug resistance, nutritional requirements, resistance to cytotoxic agents, or expression of surface proteins. Since only cells expressing the selection marker survive or exhibit other phenotypes in an environment treated with a selective agent, the transformed cells can be selected.

[0367]

[0368] Another example provides a method for producing the previously described polypeptide (Z domain variant), polypeptide multimer, protein A, or protein A variant, comprising the step of expressing the nucleic acid molecule in a suitable host cell. The method for producing may include the step of culturing the previously described recombinant cell and may optionally further include the step of isolating and / or purifying the polypeptide, polypeptide multimer, protein A, or protein A variant from the culture.

[0369]

[0370] Matrix (resin) containing Z domain variants

[0371] Another example provides a matrix (resin) in which the previously described polypeptide (a Z-domain variant of protein A), polypeptide polymer, protein A, or protein A variant is coupled to a solid support. The matrix may be a chromatographic matrix for use in techniques for separating and / or purifying proteins by adsorption, e.g., (affinity) chromatography, but is not limited thereto.

[0372] In the above matrix, the polypeptide, polypeptide polymer, protein A, or protein A variant is used as a ligand that is coupled to a solid support, and may be used as two or more multiple ligands. In one embodiment, the chromatography matrix may have multiple polypeptide polymers coupled to a solid support, but is not limited thereto.

[0373] The solid support may be appropriately selected from any suitable known type capable of coupling polypeptides, for example, from solid supports used in conventional affinity separation matrices. For example, the solid support may be an organic or inorganic material.

[0374] In one example, the solid support may be an organic material and may be based on a polymer that exposes a hydrophilic surface to an aqueous medium and / or exposes hydroxyl (-OH), carboxyl (-COOH), carboxyamide (-CONH2, possibly in an N-substituted form), amino (-NH2, possibly in a substituted form), oligo-, or polyethyleneoxy groups on an internal surface. The polymer may be based on polysaccharides such as dextran, starch, cellulose, pullulan, and agarose, such as polysaccharides crosslinked with bis-epoxides, epihalohydrins, or lower hydrocarbons substituted with 1,2,3-trihals, to provide suitable porosity and strength. In one embodiment, the solid support may be porous agarose beads, but is not limited thereto. The solid supports used herein are prepared according to standard methods such as inverse suspension gelatinization (S. Hjerten: Biochem Biophys Acta 79 (2), 393-398 (1964)), or are commercially available products, such as cephalosporin. TM FF (Amersham Biosciences, Uppsala, Sweden) and others may be used, but are not limited thereto.

[0375] In other examples, the solid support may be based on synthetic polymers such as polyvinyl alcohol, polyhydroxyalkyl acrylate, polyhydroxyalkyl methacrylate, polyacrylamide, and polymethacrylamide. In the case of hydrophobic polymers such as matrices based on divinyl and monovinyl-substituted benzene, the matrix surface is often hydrophilized to expose the hydrophilic groups as described above to the surrounding aqueous liquid. The polymer may be prepared according to standard methods ("Styrene based polymer supports developed by suspension polymerization", R. Arshady: Chimica eL'Industria 70 (9), 70-75 (1988)), or commercially available products, such as Source (trademark) (Amersham Biosciences, Uppsala, Sweden), but is not limited thereto.

[0376] In another example, the solid support may include an inorganic support such as silica or zirconium oxide, or the solid support may be in other forms such as a surface, chip, capillary, or filter.

[0377] In one example, the matrix may be in the form of a porous monolith, or in the form of porous or non-porous beads or particles. The bead or particle matrix may be used as a packed bed or in a suspended form. Suspended forms include those known as expanded beds and pure suspensions, in which particles or beads can move freely. In the case of monoliths, packed beds, and expanded beds, the separation process is typically performed after conventional chromatography through a concentration gradient.

[0378] The above ligand (the polypeptide, polypeptide polymer, protein A, or protein A variant) can be attached to a support via conventional coupling techniques using, for example, amino groups and / or carboxyl groups present on the ligand. Bis-epoxides, epichlorohydrin, CNBr, N-hydroxysuccinimide (NHS), etc. are well-known coupling agents. A molecule known as a spacer can be introduced between the support and the ligand, which will improve the utilization of the ligand and facilitate the chemical coupling of the ligand to the support. Alternatively, the ligand can be attached to the support by non-covalent bonding, such as physical adsorption or biospecific adsorption.

[0379]

[0380] Uses for the binding, separation, and purification of the Fc region of immunoglobulins or proteins containing it

[0381] One or more target compounds (e.g., the Fc region of an immunoglobulin (e.g., IgG) or a protein containing said Fc region, etc.) are adsorbed onto a polypeptide (a Z domain variant of protein A), a polypeptide polymer, protein A, a protein A variant, and / or a matrix for chromatography provided herein, thereby allowing the target compound to be separated and / or purified from a liquid.

[0382] Accordingly, other examples provide for the binding of target proteins to the previously described polypeptide (a Z-domain variant of protein A), polypeptide multimer, protein A, protein A variant, and / or a matrix for chromatography, and / or for the separation and / or purification of target proteins.

[0383] More specifically, one example provides a composition for binding a target protein comprising one or more selected from the group consisting of the previously described polypeptide (a Z domain variant of protein A), polypeptide multimer, protein A, a protein A variant, nucleic acid molecules encoding them, a recombinant vector and a recombinant cell, and a matrix for chromatography.

[0384] One example provides a use for binding a target protein of one or more selected from the group consisting of the previously described polypeptide (a Z domain variant of protein A), polypeptide multimer, protein A, protein A variant, nucleic acid molecules encoding them, recombinant vectors and recombinant cells, and a matrix for chromatography, or a composition comprising the same.

[0385] Another example provides a composition for the separation and / or purification of a target protein comprising one or more selected from the group consisting of the previously described polypeptide (a Z domain variant of protein A), polypeptide multimer, protein A, a protein A variant, and a matrix for chromatography.

[0386] Another example provides for the separation and / or purification of a target protein by one or more selected from the group consisting of the previously described polypeptide (a Z domain variant of protein A), polypeptide multimer, protein A, a protein A variant, and a matrix for chromatography, or a composition containing the same.

[0387] Another example provides a method for separating and / or purifying a target protein, comprising the step of contacting one or more selected from the group consisting of the previously described polypeptide (a Z domain variant of protein A), polypeptide polymer, protein A, a protein A variant, and a matrix for chromatography with a sample containing the target protein to adsorb the target protein.

[0388] Another example provides a method for separating and / or purifying one or more target compounds from a liquid, comprising the step of contacting one or more selected from the group consisting of the previously described polypeptide (a Z domain variant of protein A), polypeptide polymer, protein A, a protein A variant, and a matrix for chromatography with a liquid sample containing a target compound to adsorb said target compound.

[0389] The above chromatography may be affinity chromatography.

[0390] In the compositions, uses, and methods for binding, isolating, and / or purifying a target protein provided herein, the target protein may be selected from the Fc region of an immunoglobulin (e.g., IgG) and a protein comprising said Fc region. The Fc region of said immunoglobulin may be capable of binding to a polypeptide (a Z domain variant of protein A), a polypeptide multimer, protein A, and / or a protein A variant provided herein. The protein comprising said immunoglobulin's Fc region may be selected from immunoglobulin (antibody; full length or fragment), antibody fragment comprising said immunoglobulin's Fc region (e.g., scFv-Fc, (scFv-Fc)2, (scFv)2-Fc, Fab-Fc, Fab'-Fc, etc.), an Fc-containing fusion protein comprising an immunoglobulin's Fc region and a partner protein bound to said Fc region's N-terminus, C-terminus, or both-terminus.

[0391] The above immunoglobulin may be derived from humans, primates such as monkeys, rodents such as mice and rats, etc., and may be selected from various subtypes such as IgG (IgG1, IgG2, IgG3, or IgG4), IgA, IgD, IgE, and IgM, and more specifically may be IgG (IgG1, IgG2, IgG3, or IgG4). The immunoglobulin subject to purification and / or isolation may be in a complete form or in a fragment form containing an Fc region, e.g., scFv-Fc, (scFv-Fc)2, (scFv)2-Fc, Fab-Fc, Fab'-Fc, etc., but is not limited thereto.

[0392] The method for separating and / or purifying the above target protein or target compound may be chromatography, e.g., affinity chromatography.

[0393] To briefly explain the affinity chromatography described above, in the first step, a target compound, e.g., a target protein as described above (e.g., a solution containing an immunoglobulin or a fragment thereof (e.g., a cell culture medium expressing the same immunoglobulin or its fragment, etc.)) is passed through a separation matrix under conditions where said target compound (target protein) can be adsorbed to a ligand present on the separation matrix. These conditions are controlled, for example, by pH and / or salt concentration, i.e., by the ionic strength in the solution. Care must be taken not to exceed the capacity of the matrix, that is, the flow must be slowed sufficiently to allow for satisfactory adsorption. At this step, other components of the solution will, in principle, pass through without clogging. Although not essential, the matrix is ​​then washed, such as by using an aqueous solution, to remove retained material and / or loosely bound material. The matrix provided in this application has the advantage of maintaining its binding ability to the target compound (e.g., a target protein such as an immunoglobulin or its fragment) even after performing a washing step using an alkaline agent as described above. In the next step, a second solution, referred to as an eluent, is passed over the matrix under conditions that enable the desorption, or release, of the target compound. These conditions are typically provided by changes in pH, salt concentration, i.e., ionic strength, hydrophobicity, etc. Various elution methods are known, such as gradient elution and stepwise elution. Elution may also be possible by a second solution containing a competing substance to replace the desired antibody on the matrix.

[0394]

[0395] By providing a protein A or its functional domain with enhanced elution efficiency of a target protein (e.g., immunoglobulin), it can be advantageously applied as an immunoglobulin-binding ligand in immunoglobulin purification technology using chromatography.

[0396]

[0397] Figure 1 schematically shows the process of fabricating a Z domain variant (LiSa2-1) according to one embodiment.

[0398] Figure 2 is a graph showing the elution efficiency of immunoglobulin (IgG1 Fc region) in elution buffer (0.1 M Glycine) and pH 4.0 for four Z domain variants (N11W (LiSa1-4), N11Y (LiSa1-6), H18Y (LiSa1-13), N43Y (LiSa1-42)) tested in Example 3.2.

[0399] Figure 3 is a graph showing the elution efficiency of immunoglobulin (IgG1 Fc region) in elution buffer (0.1 M Glycine) and pH 4.1 conditions for two Z domain variants (H18Y(LiSa1-13), N11W / H18Y / N43Y (LiSa2-1)) tested in Example 3.3.

[0400] Figures 4a to 4j are graphs showing the elution patterns of the tetramer of the Z domain variant (LiSa2-1) (4a, 4b: Elution Profile, 50 mM Citrate, pH 3.7; 4c, 4d: Elution Profile, 50 mM Citrate, pH 4.0; 4e, 4f: Elution Profile, 50 mM Citrate, pH 4.3; 4g, 4h: Elution Profile, 100 mM Glycine, pH 3.7; 4i, 4j: Elution Profile, 100 mM Glycine, pH 4.0; solid line: UV, dotted line: pH).

[0401] Figure 5 schematically shows the process of fabricating Z domain variants (LiSa7-3 and LiSa7-s3) according to the embodiment.

[0402] Figure 6 is a graph showing the elution efficiency of immunoglobulin (IgG1 Fc region) in elution buffer (0.1 M Glycine) and pH 4.3 for five Z domain variants (LiSa2-1, LiSa6-52 (N6H addition variant in LiSa2-1), LiSa6-25 (Q32M addition variant in LiSa2-1), LiSa6-9 (D36A addition variant in LiSa2-1), LiSa6-17 (D36H addition variant in LiSa2-1)) tested in Example 5.2.

[0403] Figure 7 is a graph showing the elution efficiency of immunoglobulin (IgG1 Fc region) in elution buffer (0.1 M Glycine) and pH 4.5 for four Z domain variants (LiSa2-1, LiSa6-25 (Q32M addition variant in LiSa2-1), LiSa7-3 (Q32M, D36H addition variant in LiSa2-1), LiSa7-s3 (N11L introduction into LiSa7-3)) tested in Examples 5.3 and 5.4.

[0404] Figures 8a to 8f are graphs showing the elution patterns of tetramers of Z domain variants (LiSa2-1, LiSa7-3, LiSa7-s3) (8a, 8b: Elution profile, 50 mM Citrate, pH 3.7; 8c, 8d: Elution profile, 50 mM Citrate, pH 4.0; 8e, 8f: Elution profile, 50 mM Citrate, pH 4.3; solid line: UV, dotted line: pH).

[0405]

[0406] The present invention will be described more specifically below with reference to examples, but these are merely illustrative and are not intended to limit the scope of the invention. It is obvious to those skilled in the art that the examples described below can be modified without departing from the essential essence of the invention.

[0407]

[0408] <Example 1> Preparation of the Z domain of protein A derived from Staphylococcus aureus

[0409] 1.1. Synthesis of the Z domain gene of Protein A derived from Staphylococcus aureus

[0410] The Z domain (Sequence No. 2) refers to a mutation in which the 1st amino acid residue A (Ala) is substituted with V (Val) and the 29th amino acid residue G (Gly) is substituted with A (Ala), using the B domain of wild-type protein A from Staphylococcus aureus as a template. In the embodiments of the present application, for ease of experimentation, a protein (Sequence No. 2) containing a His-tag (6H) and C (Cys) at the end of the Z domain was constructed and used. To this end, the gene (Sequence No. 1) encoding the protein was synthesized by commissioning Bionia (Daejeon, Korea).

[0411]

[0412] 1.2. Preparation of a recombinant vector (pBC-Z) expressing the Z domain

[0413] The Z domain gene (Sequence No. 1) obtained in Example 1.1 above was inserted into the XbaⅠ and NotⅠ restriction enzyme recognition sites of the pBC-KS(+) vector (Stratagene, USA) to construct a pBC-Z plasmid for expressing the Z domain. The specific method of preparation is as follows.

[0414] The above Z domain gene DNA product (Sequence No. 1; size approximately 0.2 kb) was cleaved with restriction enzymes XbaI and NotI, purified using a purification kit (QIAquick Gel Extraction Kit; QIAGEN, Germany), and used as the insert DNA. Additionally, the DNA fragment obtained by cleaving the pBC KS(+) vector (Stratagene, USA) DNA with restriction enzymes XbaI and NotI and dephosphorylating it with CIP (Calf intestinal alkaline phosphatase; Quick CIP, New England BioLabs) was used as the vector DNA. The above insert DNA and vector DNA were ligated using T4 DNA ligase (New England Biolabs, Sweden) at 16°C for 18 hours, and then transformed into the E. coli TG1 strain by electrophoresis using the ligation solution. Transformers were selected by plating the above strain onto LB agar medium containing 20 μg / ml of chloramphenicol antibiotic and incubating overnight at 37°C. Plasmids were isolated from these transformers, and the base sequence of the inserted DNA was confirmed to finally obtain the pBC-Z plasmid containing a Z domain. The pBC-Z plasmid expressed the Z domain protein (Sequence No. 2).

[0415] The nucleic acid sequence and amino acid sequence of the Z domain obtained above are listed in Table 2 below:

[0416] Nucleic acid sequence (5'→3'), amino acid sequence (N→C) Sequence number Z Domain nucleic acid sequence (with HHHHHHC tag) GTAGATAACAAATTCAACAAAGAACAACAAAACGCTTTCTATGAAATCTTACATTTACCTAACTTAAATGAAGAACAACGCAATGCCTTCATCCAAAGCTTAAAGGATGACCCAAGCCAAAGCGCTAACCTTTTAGCAGAAGCTAAATGATGCACAAGCACCAAAACACCACCACCACCACCACTGT1Z Domain amino acid sequence (with HHHHHHC tag) VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKHHHHHHC2Z Domain amino acid sequence (with HHHHHH tag)VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKHHHHHH153Z Domain amino acid sequence (tag removed)VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK145

[0417]

[0418] <Example 2> Preparation of Fc protein of recombinant immunoglobulin G for improvement

[0419] 2.1. Synthesis of the Fc Domain Gene of Immunoglobulin G

[0420] The sequence encoding the Fc region of human IgG1 was found via Blast on the NCBI site (GenBank accession no. Y14735) and synthesized by Cosmogenetech (Daejeon, Korea).

[0421]

[0422] 2.2. Preparation of pET-Fc Plasmid

[0423] The Fc gene (SEQ No. 129) synthesized in Example 2.1 above was inserted into the NdeI and XhoI restriction enzyme recognition sites of the pET29a(+) vector (Stratagene, USA) to prepare a pET-Fc plasmid. Details are as follows.

[0424] The Fc gene DNA product obtained through synthesis in Example 2.1 above was cleaved with restriction enzymes NdeI and XhoI, purified using a purification kit (QIAEX Gel Extraction Kit; QIAGEN, Germany), and used as the insert DNA. Additionally, the DNA fragment obtained by cleaving pET29a(+) vector DNA with restriction enzymes NdeI and XhoI and dephosphorylating it with CIP (New England Biolabs) was used as the vector DNA. The insert DNA and vector DNA were ligated using T4 DNA ligase (Roche, Germany) at 16°C for 18 hours, and then the ligation solution was used to perform transformation on the E. coli BL21(DE3) strain by electrophoresis. Transformers were selected by plating the strain onto LB agar medium containing 50 μg / ml of kanamycin antibiotic and incubating overnight at 37°C. By isolating a plasmid from this transformant and confirming the base sequence of the inserted DNA, a pET-Fc plasmid containing an Fc gene having the nucleic acid sequence of SEQ ID NO. 129 was prepared. The pET-Fc plasmid expresses the wild-type Fc protein represented by SEQ ID NO. 130.

[0425] The nucleic acid sequence and amino acid sequence of the Fc region obtained above are listed in Table 3 below:

[0426] 핵산 서열(5'→3'), 아미노산 서열(N→C)서열번호Fc 핵산서열ATGGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCACGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAACTCGAGCACCACCACCACCACCACTGA129Fc 아미노산 서열MDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKLEHHHHHH130。

[0427]

[0428] 2.3. Protein Purification Using Nickel-Affinity Resin

[0429] To culture Escherichia coli BL21(DE3) transformants, 5 mL of LB liquid medium containing kanamycin antibiotic was dispensed into a 50 mL conical tube, and the mixture was incubated with shaking at 37°C and 200 rpm for 16 hours. 1% (v / v) of the culture solution was then inoculated into a 500 mL Erlenmeyer flask dispensed with 200 mL of LB liquid medium, and the mixture was incubated with shaking at 37°C and 200 rpm. IPTG (isopropyl-β-D-thio-galactopyranoside) was added to the mixture to reach a final concentration of 1 mM at approximately OD600=0.6, and the mixture was further incubated with shaking at 37°C and 200 rpm for 18 hours. After recovering the bacterial cells by centrifuging the culture medium in this flask (4°C, 10,000 rpm, 30 min), the cells were suspended in 10 mL of PBS buffer (pH 7.4) (Intron Biotechnology, Korea), crushed using an ultrasonic grinder at 4°C for 15 minutes, and centrifuged at 4°C, 10,000 rpm for 30 minutes to collect only the supernatant. 5 mL of binding buffer (20 mM NaH2PO4, 30 mM NaCl, 10 mM Imidazole pH 7.4) was flowed through a column packed with 1 mL of the nickel-affinity resin NiNTA Chelating Agarose CL-6B (Incospharm, Korea), and 2.5 mL of the supernatant was mixed with 5 mL of binding buffer and flowed through the column. After running 5 mL of washing buffer (20 mM NaH2PO4, 30 mM NaCl, 20 mM Imidazole, pH 7.4), 2.5 mL of elution buffer (20 mM NaH2PO4, 30 mM NaCl, 300 mM Imidazole, pH 7.4) was collected in a 15 mL conical tube. The purified protein was desalted using a PD10 column.

[0430]

[0431] <Example 3> Preparation and Selection of Variants with High Elution Efficiency at Mild pH

[0432] 3.1. Construction of a variant library by site-saturation mutagenesis

[0433] To prepare mutant protein A for increased elution efficiency, a library of mutant protein A was prepared by inducing a local saturation mutation in the entire nucleic acid sequence of the pBC-Z gene prepared in Example 1. Specifically, to prepare a library with a mutation in the first amino acid, the Z domain gene inserted into the pBC-KS(+) vector was used as a template, and the reaction was performed with A1-F primer (SEQ No. 5), A1-R primer (SEQ No. 6), Pfu-X reaction buffer (Solgent), 10 mM dNTP, and Pfu-X polymerase (Solgent), with the final volume adjusted to 50 μL. The reaction conditions involved pre-denaturing the reaction mixture at 95°C for 2 minutes, repeating the reaction 18 times with denaturation at 95°C for 30 seconds, annealing at 52°C for 40 seconds, and polymerization at 72°C for 4 minutes, followed by post-polymerization at 72°C for 7 minutes. The PCR product obtained under the above conditions was treated with restriction enzyme DpnⅠ for 18 hours, purified using a purification kit (QIAquick PCR purification Kit; QIAGEN, Germany), and immediately transformed into the E. coli BL21 (DE3) strain by electrophoresis. The strain was plated onto LB agar medium containing 50 μg / ml of kanamycin antibiotic and incubated overnight at 37°C to prepare a position-saturated mutant library.

[0434] After that, a regiosaturation mutant library was constructed using pBC-Z as a template and primers from the 2nd amino acid to the 58th amino acid in the same manner as described above.

[0435] The sequences of the primers used in this example are listed in Table 4 below:

[0436]

[0437]

[0438] 3.2. Selection of variants with increased elution efficiency at mild pH

[0439] The E. coli BL21(DE3) transformants containing the mutant Z domain gene induced in Example 3-1 were inoculated into 96-deep well plates (Bionia, Korea) dispensed with 600 μL of LB liquid medium containing chloramphenicol antibiotics, and then cultured with shaking at 37°C and 280 rpm for 18 hours. Protein purification was performed using the Promega HisLink™ 96 Protein Purification System (Promega, USA). More specifically, 60 μL of FastBreak™ Cell Lysis Reagent, 10X / DNase I solution was added to 600 μL of culture medium, 45 μL of HisLink™ Resin was loaded into each well, and then mixed at 100 rpm for 30 minutes. The reaction solution and resin were transferred to a filtration plate and filtered using a Vac-Man®96 Vacuum Manifold (Promega, USA) by applying a vacuum for 10 seconds. Next, 250 μL of wash buffer was added to the 96-well plate and a vacuum was applied for 10 seconds. This process was repeated three times. 200 μL of elution buffer (100 mM HEPES, 50 mM Imidazole, pH 7.5) was added to the plate and incubated for 10 minutes, after which a vacuum was applied for 1 minute to collect the purified protein in a new 96-well plate.

[0440] The purified protein variants were coupled to NHS-activated sepharose beads [NHS(N-hydroxysuccinimide)-Activated sepharose 4 Fast flow; GE Healthcare, Sweden] in a 96-well plate. 150 μL (59.5 μg / mL) of the Fc purified in Examples 2-3 was transferred to a filtration plate containing the variants coupled to the NHS-activated sepharose beads and incubated at 100 rpm for 1 hour at room temperature. Unbound Fc protein was removed by vacuum, 150 μL of PBS buffer was dispensed, and the mixture was washed by vacuuming. This washing process was repeated three times. 150 μL of elution buffer (0.1 M Glycine, pH 3.0) was added and incubated at room temperature for 30 seconds, after which the protein was collected in a new 96-well plate by vacuuming for 1 minute. The filtration plates treated with the elution buffer were washed by dispensing 150 μL of PBS buffer and applying a vacuum. This washing process was repeated three times. The amount of Fc protein at OD280 was measured on a new 96-well plate to which the supernatant had been transferred using a Synergy HTX multi-mode reader (BioTek, USA). To confirm the elution efficiency of the variants at mild pH, the variants contained in the filtration plates were bound to the resin with Fc protein in the same manner as above, and the pH of the elution buffer (0.1 M Glycine) was raised to 4.0 to measure the amount of eluted protein, thereby confirming the protein elution efficiency at pH 4.0 compared to pH 3.0.

[0441] Four variants with increased elution efficiency compared to the Z domain (Sequence No. 1) were selected using the method described above (see Fig. 2). Through gene sequencing, it was confirmed that the four variants were mutated into N11W (LiSa1-4; Sequence No. 252), N11Y (LiSa1-6; Sequence No. 253), H18Y (LiSa1-13; Sequence No. 254), and N43Y (LiSa1-42; Sequence No. 255), respectively.

[0442]

[0443] 3.3. Preparation of a Variant Library by DNA Shuffling and Selection of LISa2-1 Improved Strains

[0444] Additionally, to improve variant protein A with increased elution efficiency, a positional mutant library for three amino acid residues [N11W(LiSa1-4; SEQ NO 252), N11Y(LiSa1-6; SEQ NO 253), H18Y(LiSa1-13; SEQ NO 254), N43Y(LiSa1-42; SEQ NO 255)] was prepared based on the activity of the improved strain obtained from the positional saturated mutant library.

[0445] Specifically, to prepare a library with N11, H18 amino acid residues mutated, PCR was performed using the Z domain and LiSa1-4, LiSa1-6 as templates and the T3 primer (Sequence No. 121) and H18Y-R primer (Sequence No. 124) to recover a PCR product of approximately 210 bp, and to prepare a library with H18, N43 amino acid residues mutated, PCR was performed using the Z domain and LiSa1-42 as templates and the H18Y-F primer (Sequence No. 123) and T7 primer (Sequence No. 122) to recover a PCR product of approximately 230 bp.

[0446] The PCR reaction mixture was composed of the respective template DNA, primer, pfu-x buffer, dNTPs mix, and pfu-x polymerase, with a final volume of 100 μL. The PCR reaction conditions involved pre-denaturing the reaction mixture at 96°C for 3 minutes, followed by 18 cycles of denaturation at 96°C for 30 seconds, annealing at 52°C for 30 seconds, and polymerization at 68°C for 1 minute, after which post-polymerization was performed at 68°C for 5 minutes.

[0447] A PCR product of approximately 210 bp and a PCR product of 230 bp obtained under the above conditions were mixed, and PCR was performed using T3 primer (SEQ No. 121) and T7 primer (SEQ No. 122) to amplify a multiple variant DNA fragment of approximately 400 bp. The PCR product of approximately 400 bp obtained in this way was inserted into the pBC-KS(+) vector DNA in the same manner as in Example 1-2, and transformation was performed on the E. coli TG1 strain to construct a localized mutant library.

[0448] The sequences used in this example are listed in Table 5 below:

[0449] Nucleic acid sequence (5'→3'), amino acid sequence (N→C) Sequence number LiSa1-4 Nucleic acid sequence (with HHHHHHC tag) GTAGATAACAAATTCAACAAAGAACAACAATGGGCTTTCTATGAAATCTTACATTTACCTAACTTAAATGAAGAACAACGCAATGCCTTCATCCAAAGCTTAAAGGATGACCCAAGCCAAAGCGCTAACCTTTTAGCAGAAGCTAAAAAGCTAAATGATGCACAAGCACCAAAACACCACCACCACCACCACCACTGT169LiSa1-6 Nucleic acid sequence (with HHHHHHC tag)GTAGATAACAAATTCAACAAAGAACAACAATACGCTTTTCTATGAAATCTTACATTTACCTAACTTAAATGAAGAACAACGCAATGCCTTCATCCAAAGCTTAAAGGATGACCCAAGCCAAAGCGCTAACCTTTTAGCAGAAGCTAAAAAGCTAAATGATGCACAAGCACCAAAACACCACCACCACCACCACTGT170LiSa1-42 Nucleic acid sequence (with HHHHHHC tag)GTAGATAACAAATTCAACAAAGAACAACAAAACGCTTTTCTATGAAATCTTACATTTACCTAACTTAAAATGAAGAACAACGCAATGCCTTCATCCAAAGCTTAAAGGATGACCCAAGCCAAAGCGCTTACCTTTTAGCAGAAGCTAAAAAGCTAAATGATGCACAAGCACCAAAACACCACCACCACCACCACTGT260 Primer SEQ ID NO:T3AATTAACCCTCACTAAAGGG121T7GTAATACGACTCACTATAGGGC122H18Y-FAACGCTTTCTATGAAATCTTAYATTTACCTAACTTAAATGAAGAAC123H18Y-RGTTCTTCATTTAAGTTAGGTAAATRTAAGATTTCATAGAAAGCGTT124

[0450] Variants with increased elution efficiency from the above-mentioned positional mutation library were searched by adjusting the pH of 0.1 M Glycine buffer from 4.0 to 4.1 in reference to the method of Example 3-2. As a result, a triple variant (N11W, H18Y, N43Y) with increased elution efficiency compared to the Z domain and the previously selected LiSa 1-13 was selected and named LiSa2-1 (see Fig. 3). The amino acid sequence of LiSa2-1 (Sequence No. 4) and the nucleic acid sequence encoding it (Sequence No. 3) are shown in Table 6 below:

[0451] Nucleic acid sequence (5'→3'), amino acid sequence (N→C) Sequence number LiSa2-1 Nucleic acid sequence GTAGATAACAAATTCAACAAAGAACAACAATGGGCTTTCTATGAAATCTTATATTTACCTAACTTAAATGAAGAACAACGCAATGCCTTCATCCAAAGCTTAAAGGATGACCCAAGCCAAAGCGCTTACCTTTTAGCAGAAGCTAAAAAGCTAAATGATGCACAAGCACCAAAACACCACCACCACCACCACCACTGT3LiSa2-1 Amino acid sequence (with HHHHHHC tag)VDNKFNKEQQWAFYEILYLPNLNEEQRNAFIQSLKDDPSQSAYLLAEAKKLNDAQAPKHHHHHHC4LiSa2-1 Amino acid sequence (with HHHHHH tag)VDNKFNKEQQWAFYEILYLPNLNEEQRNAFIQSLKDDPSQSAYLLAEAKKLNDAQAPKHHHHHH154LiSa2-1 amino acid sequence (tag removed)VDNKFNKEQQWAFYEILYLPNLNEEQRNAFIQSLKDDPSQSAYLLAEAKKLNDAQAPK146

[0452]

[0453] <Example 4> Investigation of Elution Efficiency of Variant Tetramers by pH

[0454] 4.1. Synthesis of Tetrameric Genes

[0455] To verify the precise elution efficiency of the previously selected LiSa2-1, we commissioned Bionia (Daejeon, Korea) to synthesize a Z domain containing a His tag and a tetramer of LiSa2-1 for purification.

[0456]

[0457] 4.2. Preparation of Tetrameric Plasmids

[0458] The coding genes for the tetrameric Z domain and LiSa2-1 obtained in Example 4.1 above were prepared by inserting them into the NdeI and XhoI restriction enzyme recognition sites of the pET29a(+) vector (Stratagene, USA). Details are as follows.

[0459] The Z domain and LiSa2-1 gene DNA products were cleaved with restriction enzymes NdeI and XhoI, purified using a purification kit (QIAEX Gel Extraction Kit; QIAGEN, Germany), and used as insertion DNA. Additionally, the DNA fragment obtained by cleaving pET29a(+) vector DNA with restriction enzymes NdeI and XhoI and dephosphorylating it with CIP (New England Biolabs) was used as vector DNA. The above insertion DNA and vector DNA were ligated using T4 DNA ligase (Roche, Germany) at 16°C for 18 hours, and then the ligation solution was used to perform transformation into the E. coli BL21(DE3) strain by electrophoresis. Transformers were selected by plating the strain onto LB agar medium containing 50 μg / ml of kanamycin antibiotic and incubating overnight at 37°C. By isolating plasmids from this transformant and confirming the base sequence of the inserted DNA, pET-Z (containing SEQ ID NO. 125 as the coding DNA sequence) and pET-LiSa2-1 (containing SEQ ID NO. 127 as the coding DNA sequence) plasmids were prepared. The above plasmids each express Z tetramer (SEQ ID NO. 126) and LiSa2-1 tetramer (SEQ ID NO. 128) proteins.

[0460] Nucleic acid sequence (5'→3'), amino acid sequence (N→C) Sequence number Z Tetrameric nucleic acid sequence (with HHHHHHC tag) GTCGACAACAAATTTAACAAAGAGCAGCAGAACGCATTCTACGAAATTCTGCATCTGCCAAATCTGAACGAGGAGCAGCGTAATGCATTCATCCAGTCTCTGAAGGATGACCCGTCTCAGTCTGCGAACCTGCTGGCTGAAGCTAAGAAACTGAACGACGCCCAGGCCCCTAAGGTGGATAACAAGTTTAACAAGGAACAGCAGAATGCGTTCTATGAAATTCTGCACCTGCCAAACCTGAATGAAGAACAGCGTAACGCATTTATTCAGTCTCTGAAAGACGACCCGTCCCAGAGCGGAAGCGGAAGCTGAACGATGCGCAGGCACCAAAAGTTGACAACA AGTTCAACAAGGAGGCAGCAGAACGCCTTCTACGAGATCCTGCACCTGCCTAACCTGAACGAAGAGCAGCGCAATGCCTTTATCCAAAGCCTGAAAGATGATCCGTCTCAGAGCGCTAACCTGCTGGCCGAAGCGAAGAAACTGAATGATGCGCAGGCGCCGAAAGTTGATAACAAATTCAA CAAAGAACAGCAAAACGCTTTTTACGAAATCCTGCATCTGCCGAACCTGAACGAGGAACAGCGCAACGCTTTCATCCAATCTCTGAAGGACGATCCTAGCCAGTCTGCAAATCTGCTGGCAGAAGCGAAAAAGCTGAACGACGCTCAGGCTCCGAAACACCACCACCACCACCACTGT125Z Tetrameric amino acid sequence (with HHHHHHCtag)VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKHHHHHHHC126LiSa2-1 사량체 핵산 서열 (with HHHHHHCtag)GTCGACAACAAATTTAACAAAGAGCAGCAGTGGGCATTCTACGAAATTCTGTATCTGCCAAATCTGAACGAGGAGCAGCGTAATGCATTCATCCAGTCTCTGAAGGATGACCCGTCTCAGTCTGCGTATCTGCTGGCTGAAGCTAAGAAACTGAACGACGCCCAGGCCCCTAAGGTGGATAACAAGTTTAACAAGGAACAGCAGTGGGCGTTCTATGAAATTCTGTACCTGCCAAACCTGAATGAAGAACAGCGTAACGCATTTATTCAGTCTCTGAAAGACGACCCGTCCCAGAGCGCGTACCTGCTGGCGGAAGCGAAGAAGCTGAACGATGCGCAGGCACCAAAAGTTGACAACAAGTTCAACAAGGAGCAGCAGTGGGCCTTCTACGAGATCCTGTACCTGCCTAACCTGAACGAAGAGCAGCGCAATGCCTTTATCCAAAGCCTGAAAGATGATCCGTCTCAGAGCGCTTATCTGCTGGCCGAAGCGAAGAAACTGAATGATGCGCAGGCGCCGAAAGTTGATAACAAATTCAACAAAGAACAGCAATGGGCTTTTTACGAAATCCTGTATCTGCCGAACCTGAACGAGGAACAGCGCAACGCTTTCATCCAATCTCTGAAGGACGATCCTAGCCAGTCTGCATACCTGCTGGCAGAAGCGAAAAAGCTGAACGACGCTCAGGCTCCGAAACACCACCACCACCACCACTGT127LiSa2-1 사량체 아미노산 서열(with HHHHHHCtag)VDNKFNKEQQWAFYEILYLPNLNEEQRNAFIQSLKDDPSQSAYLLAEAKKLNDAQAPKVDNKFNKEQQWAFYEILYLPNLNEEQRNAFIQSLKDDPSQSAYLLAEAKKLNDAQAPKVDNKFNKEQQWAFYEILYLPNLNEEQRNAFIQSLKDDPSQSAYLLAEAKKLNDAQAPKVDNKFNKEQQWAFYEILYLPNLNEEQRNAFIQSLKDDPSQSAYLLAEAKKLNDAQAPKHHHHHHC128Z Tetrameric Amino Acid Sequence(with HHHHHH tag)VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFN KEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKHHHHHHC157LiSa2-1 Tetrameric amino acid sequence (with HHHHHH tag)VDNKFNKEQQWAFYEILYLPNLNEEQRNAFIQSLKDDPSQSAYLLAEAKKLNDAQAPKVDNKFNKEQQWAFYEILYLPNLNEEQRNAFIQSLKDDPSQSAYLLAEAKKLNDAQAPKVDNKFNKEQQWAFYEILYLPNLNEEQRNAFIQSLKDDPSQSAYLLAEAKKLNDAQAPKVDNKFNKEQQWAFYEILYLPNLNEEQRNAFIQSLKDDPSQSAYLLAEAKKLNDAQAPKHHHHHH158Z tetrameric amino acid sequence (tagREMOVE)VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDDN KFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK147LiSa2-1 Tetramer Amino Acid Sequence (Tick Remove)VDNKFNKEQQWAFYEILYLPNLNEEQRNAFIQSLKDDPSQSAYLLAEAKKLNDAQAPKVDNKFNKEQQWAFYEILYLPNLNEEQRNAFIQSLKDDPSQSAYLLAEAKKLNDAQAPK VDNKFNKEQQWAFYEILYLPNLNEEQRNAFIQSLKDDPSQSAYLLAEAKKLNDAQAPKVDNKFNKEQQWAFYEILYLPNLNEEQRNAFIQSLKDDPSQSAYLLAEAKKLNDAQAPK148

[0461]

[0462] 4.3. Purification and Immobilization of Tetramers Using Nickel-Affinity Resin

[0463] To culture Escherichia coli BL21(DE3) transformants, 5 mL of TB liquid medium containing kanamycin antibiotic was dispensed into a 50 mL test tube and then incubated with shaking at 37°C and 200 rpm for 16 hours. 1% (v / v) of the culture solution was then inoculated into a 2000 mL Erlenmeyer flask dispensed with 500 mL of TB liquid medium and incubated with shaking at 37°C and 200 rpm. Then, IPTG (isopropyl-β-D-thio-galactopyranoside) was added to reach a final concentration of 1 mM at approximately OD600=0.6, and the mixture was incubated with shaking at 37°C and 200 rpm for 18 hours. After recovering the bacterial cells by centrifuging the culture medium in this flask (4°C, 10,000 rpm, 30 min), the cells were suspended in 20 mL of PBS buffer (pH 7.4) (Intron Biotechnology, Korea), crushed using an ultrasonic grinder at 4°C for 30 minutes, and centrifuged at 4°C, 10,000 rpm for 30 minutes to collect only the supernatant. 25 mL of binding buffer (20 mM NaH2PO4, 30 mM NaCl, 10 mM Imidazole pH 7.4) was flowed through a column packed with 5 mL of Ni NTA Chelating Agarose CL-6B (Incospharm, Korea), and 20 mL of the above supernatant was mixed with 40 mL of binding buffer and flowed through the column. After running 25 mL of wash buffer (20 mM NaH2PO4, 30 mM NaCl, 20 mM Imidazole pH 7.4), 40 mL of elution buffer (20 mM NaH2PO4, 30 mM NaCl, 300 mM Imidazole pH 7.4) was collected in a 50 mL conical tube. The purified protein was desalted using a PD10 desalting column.

[0464]

[0465] Purified tetramers Z4 and LiSa2-1 were coupled to an epoxy-activated resin (Puriose AP HP-30, Furiogen). The specific procedure is as follows. First, a wet cake was prepared by repeating the washing process three times by flowing 90 mL of ultrapure water through vacuum filtration into 60 mL of resin, followed by maintaining reduced pressure for 10 minutes. 7.2 g of the prepared wet cake, ligand protein, sodium phosphate buffer, and sodium sulfate were added to a reactor, and the reaction volume was set to 15 mL. After 16 hours of reaction, the mixture was prepared in the form of a wet cake by vacuum filtration and washing three times with three times the volume of ultrapure water, followed by vacuuming. The washed wet cake was placed in a reactor, and an equal volume of epoxy inactivator was added to initiate the reaction. After vacuum filtration and washing three times with three times the volume of ultrapure water, the mixture was washed with two times the volume of washing buffer for 30 minutes. After removing the washing buffer by vacuum filtration again, the product was washed three additional times with three times the amount of ultrapure water, and then 20% ethanol was added to prepare a slurry, which was then refrigerated.

[0466]

[0467] 4.4. Comparison of Elution Efficiency of Mutant Protein LiSa2-1 Tetramer by pH

[0468] Using the resin prepared in Example 4.3, the elution efficiency and elution volume at different pH levels were compared with commercial resins Mabselect Sure and MabSelect PrismA (GE Healthcare Life Sciences, USA).

[0469] 2 mL of the prepared resin was packed into a Tricorn, and the operating linear velocity was 100 cm / h with a volumetric flow rate of 0.33 mL / min. The column packed with resin was connected to an FPLC (AKTA Pure 25, Cytiva), and 20 mM PBS (pH 7.4) buffer (using PBS tablets, Millipore) was flowed in five volumetric indices (based on volume; unless otherwise noted, the indices of buffer or solution are based on volume). After binding with 2.5 mg / mL IgG (Livgamma SN, SK Plasma), the column was washed with 10 volumetric indices of 20 mM PBS (pH 7.4) buffer. The antibody bound to the resin was eluted by flowing 7 times the amount of elution buffer, and the process was performed with different compositions depending on the conditions (50 mM Citrate pH 3.7, pH 4.0, pH 4.3; or 100 mM Glycine pH 3.7, pH 4.0). After elution was complete, 5 times the amount of 0.1 M NaOH solution was flowed first, followed by 10 times the amount of 20 mM PBS (pH 7.4) buffer to wash and regenerate.

[0470] The elution profiles obtained above are shown in FIGS. 4a to 4e (4a: Elution Profile, 50 mM Citrate, pH 3.7; 4b: Elution Profile, 50 mM Citrate, pH 4.0; 4c: Elution Profile, 50 mM Citrate, pH 4.3; 4d: Elution Profile, 100 mM Glycine, pH 3.7; 4e: Elution Profile, 100 mM Glycine, pH 4.0).

[0471] In addition, the following formula (Equation 1) was used to calculate the dissolution efficiency.

[0472] (Formula 1)

[0473] (IgG maxAU: Maximum AU of IgG (Absorbance Unit; referring to the UV absorbance of a sample solution, meaning the maximum UV absorbance at 280 nm detected by a UV detector when an IgG sample is injected into an elution efficiency measuring device (AKTA)) (Unit: mAU);

[0474] IgG sample injection volume: Amount of IgG sample injected into the equipment (Unit: mL);

[0475] CIP Area: IgG peak area obtained during the Cleaning-In-Place step (Unit: mL*mAU)

[0476]

[0477] The above-mentioned dissolution rates are shown in Table 8 below:

[0478]

[0479] As shown in Table 8 and Figures 4a to 4j, it was confirmed that the tetrameric LiSa2-1 resin of the present disclosure has superior elution efficiency and reduced elution volume compared to MabSelect Sure and MabSelect PrismA, which are widely used in industry. When eluted using 50 mM Citrate, pH 4.0 buffer, the elution efficiency of MabSelect PrismA was 88% and the elution volume was 15 mL or more, whereas in the case of LiSa2-1, the elution efficiency increased to 96.8% and the elution volume decreased to 13.2 mL.

[0480]

[0481] <Example 5> Preparation and Selection of Variants with High Elution Efficiency at Mild pH

[0482] 5.1. Construction of a variant library by site-saturation mutagenesis

[0483] To prepare variant protein A for increased elution efficiency, a library was prepared by inducing a site-saturation mutation in the entire nucleic acid sequence of the pBC-LiSa2-1 (SEQ No. 3) gene prepared in Example 3.3. Specifically, to prepare a library with a mutation in the first amino acid, Z (SEQ No. 1) inserted into the pBC-KS(+) vector was used as a template, and the reaction was carried out with A1-F primer (SEQ No. 5), A1-R primer (SEQ No. 6), Pfu-X reaction buffer, 10 mM dNTPs, and Pfu-X polymerase, with a final volume adjusted to 50 μL. The reaction conditions involved pre-denaturing the reaction mixture at 95°C for 2 minutes, repeating the reaction 18 times with denaturation at 95°C for 30 seconds, annealing at 52°C for 40 seconds, and polymerization at 72°C for 4 minutes, followed by post-polymerization at 72°C for 7 minutes. The PCR product obtained under the above conditions was treated with restriction enzyme DpnⅠ for 18 hours and purified using a purification kit (QIAquick PCR purification Kit; QIAGEN, Germany), and then immediately transformed into the E. coli TG1 strain by electrophoresis. The strain was plated onto LB agar medium containing 50 μg / ml of kanamycin antibiotic and incubated overnight at 37°C to prepare a position-saturated mutant library.

[0484] After that, a regiosaturation mutant library was constructed using pBC-LiSa2-1 as a template and primers from the 2nd amino acid to the 58th amino acid in the same manner as described above.

[0485] The coding nucleic acid sequence and amino acid sequence of LiSa2-1 used in this example, and the sequences of the primers are listed in Table 9 below:

[0486] Nucleic acid sequence (5'→3'), amino acid sequence (N→C) Sequence number LiSa2-1 Nucleic acid sequence (with HHHHHHC tag)GTAGATAACAAATTCAACAAAGAACAACAATGGGCTTTCTATGAAATCTTATATTTACCTAACTTAAATGAAGAACAACGCAATGCCTTCATCCAAAGCTTAAAGGATGACCCAAGCCAAAGCGCTTACCTTTTAGCAGAAGCTAAAAAGCTAAATGATGCACAAGCACCAAAACACCACCACCACCACCACCACTGT3LiSa2-1 Amino acid sequence (with HHHHHHC tag)VDNKFNKEQQWAFYEILYLPNLNEEQRNAFIQSLKDDPSQSAYLLAEAKKLNDAQAPKHHHHHHC4

[0487]

[0488]

[0489] 5.2. Selection of variants with increased elution efficiency at mild pH

[0490] Escherichia coli TG1 transformants containing the coding gene of the mutation-induced variant LiSa2-1 were inoculated into 96-deep-well plates (Bionia, Korea) dispensed with 600 μL of LB liquid medium containing chloramphenicol antibiotics, and then cultured with shaking at 37°C and 280 rpm for 18 hours. Protein purification was performed using the Promega HisLink™ 96 Protein Purification System (Promega, USA). More specifically, 60 μL of FastBreak™ Cell Lysis Reagent, 10X / DNase I solution was added to 600 μL of culture medium, 45 μL of HisLink™ Resin was mounted in each well, and the mixture was stirred at 100 rpm for 30 minutes. The reaction mixture and resin were transferred to a filtration plate and filtered using a Vac-Man®96 Vacuum Manifold (Promega, USA) by applying a vacuum for 10 seconds. Next, 250 μL of wash buffer was added to the 96-well plate, and a vacuum was applied for 10 seconds. Washing was performed three times in the same manner. 200 μL of elution buffer (100 mM HEPES, 50 mM Imidazole, pH 7.5) was added to the plate and reacted for 10 minutes, after which a vacuum was applied for 1 minute to collect the purified protein in a new 96-well plate.

[0491] The purified variants were coupled to NHS(N-hydroxysuccinimide)-Activated sepharose 4 Fast Flow (GE Healthcare, Sweden) in a 96-well plate. 150 μL (59.5 μg / mL) of the Fc purified in Examples 2-3 was transferred to a filtration plate containing the variants coupled to the NHS-activated sepharose beads and incubated at 100 rpm for 1 hour at room temperature. Unbound Fc proteins were removed by vacuum, 150 μL of PBS buffer was dispensed, and the mixture was washed under vacuum. This washing process was repeated three times. 150 μL of elution buffer (0.1 M Glycine, pH 3.0) was added and incubated at room temperature for 30 seconds, after which the protein was collected in a new 96-well plate under vacuum for 1 minute. The filtration plates treated with the elution buffer were washed by dispensing 150 μL of PBS buffer and applying a vacuum. This washing process was repeated three times. The amount of Fc protein at OD280 was measured on a new 96-well plate to which the supernatant had been transferred using a Synergy HTX multi-mode reader (BioTek, USA). To confirm the elution efficiency of the variants at mild pH, Fc protein was bound to the variant resin in the filtration plates in the same manner as above, and the amount of eluted protein was measured by raising the pH of the elution buffer (0.1 M Glycine) to 4.3 to confirm the protein elution efficiency at pH 4.3 compared to pH 3.0.

[0492] Four variants with increased elution efficiency compared to LiSa2-1 were selected using the method described above (Fig. 6). Through gene sequencing, it was confirmed that the four variants were mutated into N6H (LiSa6-52), Q32M (LiSa6-25), D36A (LiSa6-9), and D36H (LiSa6-17).

[0493]

[0494] 5.3. Preparation of a Variant Library by DNA Shuffling and Selection of LiSa7-3 Improved Strains

[0495] Additionally, to improve variant protein A with increased elution efficiency, a positional mutant library was prepared based on the activity of the improved strain obtained from the positional saturated mutant library, using LiSa6-25 as a template, fixing the 32nd amino acid to methionine, and positioning the mutant library for two amino acid residues (N6H, D36A, or D36H).

[0496] Specifically, to prepare a library with N6 amino acid mutations, PCR was performed using LiSa6-25 as a template with T3 primers (SEQN 123) and N6H-R primers (SEQN 126) to recover a PCR product of approximately 170 bp; to prepare a library with N6 and D36 amino acid mutations, PCR was performed using LiSa6-25 as a template with N6H-F primers (SEQN 125), D36H-R primers (SEQN 128), and D36A-R primers (SEQN 130) to recover a PCR product of 130 bp; and to prepare a library with D36 amino acid mutations, PCR was performed using LiSa6-25 as a template with D36H-F primers (SEQN 127), D36A-F primers (SEQN 129), and T7 primers (SEQN 124) to recover a product of approximately 170 bp PCR products of that size were recovered.

[0497] The PCR reaction mixture was composed of the respective template DNA, primer, pfu-x buffer, dNTPs mix, and pfu-x polymerase, with a final volume of 100 μL. The PCR reaction conditions involved pre-denaturing the reaction mixture at 96°C for 3 minutes, followed by 18 cycles of denaturation at 96°C for 30 seconds, annealing at 52°C for 30 seconds, and polymerization at 68°C for 1 minute, after which post-polymerization was performed at 68°C for 5 minutes.

[0498] Two PCR products of approximately 170 bp and a PCR product of 130 bp obtained under the above conditions were mixed, and PCR was performed using T3 primer (SEQ No. 121) and T7 primer (SEQ No. 122) to amplify a multiple variant DNA fragment of approximately 400 bp. The PCR product of approximately 400 bp obtained in this way was inserted into the pBC-KS(+) vector DNA in the same manner as in Example 2-1, and transformation was performed on the E. coli TG1 strain to prepare a localized mutant library.

[0499] Variants with increased elution efficiency from the above-mentioned positional mutation library were searched based on the method of Example 5.2 and by adjusting the pH of 0.1 M Glycine buffer from pH 4.3 to pH 4.5 (see Fig. 7). As a result, a strain expressing a protein in which Q32M and D36H were additionally mutated in LiSa2-1 was selected and named the mutant protein LiSa7-3. The coding color sequence of the mutant protein LiSa7-3 is represented by SEQ ID NO. 137 and the amino acid sequence by SEQ ID NO. 138.

[0500] 핵산 서열(5'→3'), 아미노산 서열(N→C)서열번호LiSa6-25 핵산서열GTAGATAACAAATTCAACAAAGAACAACAATGGGCTTTCTATGAAATCTTATATTTACCTAACTTAAATGAAGAACAACGCAATGCCTTCATCATGAGCTTAAAGGATGACCCAAGCCAAAGCGCTTACCTTTTAGCAGAAGCTAAAAAGCTAAATGATGCACAAGCACCAAAACACCACCACCACCACCACTGT171프라이머 서열서열번호T3AATTAACCCTCACTAAAGGG121T7GTAATACGACTCACTATAGGGC122N6H-FCATATGGTAGATAACAAATTCMACAAAGAACAACAATGGGCTT131N6H-RAAGCCCATTGTTGTTCTTTGTKGAATTTGTTATCTACCATATG132D36H-FCTTCATCATGAGCTTAAAGSATGACCCAAGCCAAAGC133D36H-RGCTTTGGCTTGGGTCATSCTTTAAGCTCATGATGAAG134D36A-FCTTCATCATGAGCTTAAAGGCTGACCCAAGCCAAAGC135D36A-RGCTTTGGCTTGGGTCAGCCTTTAAGCTCATGATGAAG136

[0501] Nucleic acid sequence (5'→3'), amino acid sequence (N→C) Sequence number LiSa7-3 Nucleic acid sequence GTAGATAACAAATTCAACAAAGAACAACAATGGGCTTTCTATGAAATCTTATATTTACCTAACTTAAATGAAGAACAACGCAATGCCTTCATCATGAGCTTAAAGCATGACCCAAGCCAAAGCGCTTACCTTTTAGCAGAAGCTAAAAAGCTAAATGATGCACAAGCACCAAAACACCACCACCACCACCACCACTGT137LiSa7-3 Amino acid sequence (with HHHHHHC tag)VDNKFNKEQQWAFYEILYLPNLNEEQRNAFIMSLKHDPSQSAYLLAEAKKLNDAQAPKHHHHHHC138LiSa7-3 Amino acid sequence (with HHHHHH tag)VDNKFNKEQQWAFYEILYLPNLNEEQRNAFIMSLKHDPSQSAYLLAEAKKLNDAQAPKHHHHHH155LiSa7-3 amino acid sequence (tag removed)VDNKFNKEQQWAFYEILYLPNLNEEQRNAFIMSLKHDPSQSAYLLAEAKKLNDAQAPK149

[0502]

[0503] 5.4. Construction of a variant library by site-saturation mutagenesis

[0504] To obtain a variant with further enhanced elution efficiency, a regiosaturation mutant library was prepared for amino acid residues 11, 18, 32, 36, and 43 of the variant amino acid residues of LiSa7-3 using LiSa7-3 as a template. In this example, the library preparation method was performed in the same manner as described in Example 5.1 above.

[0505] AA site primer sequence Sequence number 11A11-F2CAAATTCAACAAAGAACAACAANNKGCTTTCTATGAAATCTTACATT188A11-R2AATGTAAGATTTCATAGAAAGCMNNTTGTTGTTCTTTGTTGAATTTG18918A18-F2AACGCTTTCTATGAAATCTTANNKTTACCTAACTTAAATGAAGAAC202A18-R2GTTCTTCATTTAAGTTAGGTAAMNNTAAGATTTCATAGAAAGCGTT20332A32-F2AACGCAATGCCTTCATC NNKAGCTTAAAGCATGACCC242A32-R2GGGTCATGCTTTAAGCTMNNGATGAAGGCATTGCGTT24336A36-F2CTTCATCATGAGCTTAAAGNNKGACCCAAGCCAAAGC244A36-R2GCTTT GGCTTGGGTCMNNCTTTTAAGCTCATGATGAAG24543A43-FCCAAGCCAAAGCGCTNNKCTTTTAGCAGAAGCTAAAAAG89A43-RCTTTTTAGCTTCTGCTAAAAGMNNAGCGCTTTGGCTTGG90

[0506] As a result of searching the above-mentioned regiosaturation mutant library using the same method as in Example 5.3, a nucleic acid molecule encoding the variant LiSa7-s3 protein, in which the 11th amino acid of the LiSa7-3 protein is mutated to leucine (Leu, L), was selected (see FIG. 7). The coding nucleic acid sequence of the variant LiSa7-s3 is represented by SEQ ID NO. 139, and the amino acid sequence by SEQ ID NO. 140, respectively:

[0507] Nucleic acid sequence (5'→3'), amino acid sequence (N→C) Sequence number LiSa7-s3 Nucleic acid sequence GTAGATAACAAATTCAACAAAGAACAACAACTGGCTTTCTATGAAATCTTATATTTACCTAACTTAAATGAAGAACAACGCAATGCCTTCATCATGAGCTTAAAGCATGACCCAAGCCAAAGCGCTTACCTTTTAGCAGAAGCTAAAAAGCTAAATGATGCACAAGCACCAAAACACCACCACCACCACCACCACTGT139LiSa7-s3 amino acid sequence (with HHHHHHC tag)VDNKFNKEQQLAFYEILYLPNLNEEQRNAFIMSLKHDPSQSAYLLAEAKKLNDAQAPKHHHHHHC140LiSa7-s3 amino acid sequence (with HHHHHH tag)VDNKFNKEQQLAFYEILYLPNLNEEQRNAFIMSLKHDPSQSAYLLAEAKKLNDAQAPKHHHHHH156LiSa7-s3 Amino acid sequence (tags removed)VDNKFNKEQQLAFYEILYLPNLNEEQRNAFIMSLKHDPSQSAYLLAEAKKLNDAQAPK150

[0508]

[0509] <Example 6> Investigation of Mutant Tetramer Elution Efficiency

[0510] 6.1. Synthesis of Tetrameric Genes

[0511] In order to confirm the exact elution efficiency of LiSa2-1, LiSa7-3, and LiSa7-s3 selected in Examples 3.3 and 5.3 and 5.4, a tetramer containing a His tag was synthesized for purification by commissioning Bionia (Daejeon, Korea).

[0512]

[0513] 6.2. Preparation of Tetrameric Plasmids

[0514] The tetrameric genes LiSa2-1, LiSa7-3, and LiSa7-s3 obtained in Example 6.1 above were prepared by inserting them into the NdeI and XhoI restriction enzyme recognition sites of the pET29a(+) vector (Stratagene, USA). Details are as follows.

[0515] The DNA products of the LiSa2-1, LiSa7-3, and LiSa7-s3 genes were cleaved with restriction enzymes NdeI and XhoI, purified using a purification kit (QIAEX Gel Extraction Kit; QIAGEN, Germany), and used as insertion DNA. Additionally, the DNA fragment obtained by cleaving pET29a(+) vector DNA with restriction enzymes NdeI and XhoI and dephosphorylating it with CIP (New England Biolabs) was used as vector DNA. The above insertion DNA and vector DNA were ligated using T4 DNA ligase (Roche, Germany) at 16°C for 18 hours, and then the ligation solution was used to perform transformation into the E. coli BL21(DE3) strain by electrophoresis. Transformers were selected by plating the strain onto LB agar medium containing 50 μg / ml of kanamycin antibiotic and incubating overnight at 37°C. By isolating plasmids from this transformant and confirming the base sequence of the inserted DNA, pET-LiSa2-1 (containing SEQ ID NO. 127 as the protein-coding nucleic acid sequence), pET-LiSa7-3 (containing SEQ ID NO. 141 as the protein-coding nucleic acid sequence), and pET-LiSa7-s3 (containing SEQ ID NO. 143 as the protein-coding nucleic acid sequence) plasmids were prepared. Each of the above plasmids expresses the proteins LiSa2-1 tetramer (SEQ ID NO. 128), LiSa7-3 tetramer (SEQ ID NO. 142), and LiSa7-s3 tetramer (SEQ ID NO. 144).

[0516] Nucleic acid sequence (5'→3'), amino acid sequence (N→C) Sequence number LiSa2-1 tetramer nucleic acid sequence (with HHHHHHC tag)-1 tetramer amino acid sequence (with HHHHHHCtag)VDNKFNKEQQWAFYEILYLPNLNEEQRNAFIQSLKDDPSQSAYLLAEAKKLNDAQAPKVDNKFNKEQQWAFYEILYLPNLNEEQRNAFIQSLKDDPSQSAYLLAEAKKLNDAQAPKVDNKFNKEQQWAFYEILYLPNLNEEQRNAFIQSLKDDPSQSAYLLAEAKKLNDAQAPKVDNKFNKEQQWAFYEILYLPNLNEEQRNAFIQSLKDDPSQSAYLLAEAKKLNDAQAPKHHHHHHHC128LiSa7-3 사량체 핵산 서열(with HHHHHHHCtag)GTCGATAACAAATTTAACAAAGAGCAGCAGTGGGCATTCTACGAAATTCTGTATCTGCCAAATCTGAACGAGGAGCAGCGTAATGCATTCATCATGTCTCTGAAGCACGACCCGTCTCAGTCTGCGTATCTGCTGGCTGAAGCTAAGAAACTGAACGACGCCCAGGCCCCTAAGGTGGACAACAAGTTTAACAAGGAACAGCAGTGGGCGTTCTATGAAATTCTGTACCTGCCAAACCTGAATGAAGAACAGCGTAACGCATTTATTATGTCTCTGAAACATGACCCGTCCCAGAGCGCGTACCTGCTGGCGGAAGCGAAGAAGCTGAACGATGCGCAGGCACCAAAAGTTGATAACAAGTTCAACAAGGAGCAGCAGTGGGCCTTCTACGAGATCCTGTACCTGCCTAACCTGAACGAAGAGCAGCGCAATGCCTTTATCATGAGCCTGAAACACGATCCGTCTCAGAGCGCTTATCTGCTGGCCGAAGCGAAGAAACTGAATGATGCGCAGGCGCCGAAAGTTGACAACAAATTCAACAAAGAACAGCAATGGGCTTTTTACGAAATCCTGTATCTGCCGAACCTGAACGAGGAACAGCGCAACGCTTTCATCATGTCTCTGAAGCATAATCCTAGCCAGTCTGCATACCTGCTGGCAGAAGCGAAAAAGCTGAACGACGCTCAGGCTCCGAAACACCACCACCACCACCACTGT141LiSa7-3 사량체 아미노산 서열(with HHHHHHCtag)VDNKFNKEQQWAFYEILYLPNLNEEQRNAFIMSLKHDPSQSAYLLAEAKKLNDAQAPKVDNKFNKEQQWAFYEILYLPNLNEEQRNAFIMSLKHDPSQSAYLLAEAKKLNDAQAPKVDNKFNKEQQWAFYEILYLPNLNEEQRNAFIMSLKHDPSQSAYLLAEAKKLNDAQAPKVDNKFNKEQQWAFYEILYLPNLNEEQRNAFIMSLKHNPSQSAYLLAEAKKLNDAQAPKHHHHHHC142LiSa7-3 tetramer amino acid sequence(with HHHHHH tag)VDNKFNKEQQWAFYEILYLPNLNEEQRNAFIMSLKHDPSQSAYLLAEAKKLNDAQAPKVDNKFNKEQQWAFYEILYLPNLNEEQRNAFIMSLKHDPSQSAYLLAEAKKLNDAQAPKVDNKFNKEQQWAFYEILYLPNLNEEQRNAFIMSLKHDPSQSAYLLAEAKKLNDAQAPKVDNKFNKEQQWAFYEILYLPNLNEEQRNAFIMSLKHNPSQSAYLLAEAKKLNDAQAPKHHHHHH159LiSa7-3 tetrameric amino acid sequence (tag Removal)VDNKFNKEQQWAFYEILYLPNLNEEQRNAFIMSLKHDPSQSAYLLAEAKKLNDAQAPKVDNKFNKEQQWAFYEILYLPNLNEEQRNAFIMSLKHDPSQSAYLLAEAKKLNDAQAPKVDNKFNKEQQWAFYEILYLPNLNEEQRNAFIMSLKHDPSQSAYLLAEAKKLNDAQAPKVDNKFNKEQQWAFYEILYLPNLNEEQRNAFIMSLKHNPSQSAYLLAEAKKLNDAQAPK151LiSa7-s3 tetrameric nucleic acid sequence (with HHHHHHCtag)GTCGATAACAAATTTAACAAAGAGCAGCAGCTGGCATTCTACGAAATTCTGTATCTGCCAAATCTGAACGAGGAGCAGCGTAATGCATTCATCATGTCTCTGAAGCACGACCCGTCTCAGTCTGCGTATCTGCTGGCTGAAGCTAAGAAACTGAACGACGCCCAGGCCCCTAAGGTGGACAACAAGTTTAACAAGGAACAGCAGCTGGCGTTCTATGAAATTCTGTACCTGCCAAACCTGAATGAAGAACAGCGTAACGCATTTATTATGTCTCTGAAACATGACCCGTCCCAGAGCGCGTACCTGCTGGCGGAAGCGAAGAAGCTGAACGATGCGCAGGCACCAAAAGTTGATAACAAGTTCAACAAGGAGCAGCAGCTGGCCTTCTACGAGATCCTGTACCTGCCTAACCTGAACGAAGAGCAGCGCAATGCCTTTATCATGAGCCTGAAACACGATCCGTCTCAGAGCGCTTATCTGCTGGCCGAAGCGAAGAAACTGAATGATGCGCAGGCGCCGAAAGTTGACAACAAATTCAACAAAGAACAGCAACTGGCTTTTTACGAAATCCTGTATCTGCCGAACCTGAACGAGGAACAGCGCAACGCTTTCATCATGTCTCTGAAGCATAATCCTAGCCAGTCTGCATACCTGCTGGCAGAAGCGAAAAAGCTGAACGACGCTCAGGCTCCGAAACACCACCACCACCACCACTGT143LiSa7-s3 사량체 아미노산 서열(with HHHHHHCtag)VDNKFNKEQQLAFYEILYLPNLNEEQRNAFIMSLKHDPSQSAYLLAEAKKLNDAQAPKVDNKFNKEQQLAFYEILYLPNLNEEQRNAFIMSLKHDPSQSAYLLAEAKKLNDAQAPKVDNKFNK EQQLAFYEILYLPNLNEEQRNAFIMSLKHDPSQSAYLLAEAKKLNDAQAPKVDNKFNKEQQLAFYEILYLPNLNEEQRNAFIMSLKHNPSQSAYLLAEAKKLNDAQAPKHHHHHHC144LiSa7-s3 Tetrameric amino acid sequence (with HHHHHH tag)VDNKFNKEQQLAFYEILYLPNLNEEQRNAFIMSLKHDPSQSAYLLAEAKKLNDAQAPKVDNKFNKEQQLAFYEILYLPNLNEEQRNAFIMSLKHDPSQSAYLLAEAKKLNDAQAPKVDNKFNKEQQLAFYEILYLPNLNEEQRNAFIMSLKHDPSQSAYLLAEAKKLNDAQAPKVDNKFNKEQQLAFYEILYLPNLNEEQRNAFIMSLKHNPSQSAYLLAEAKKLNDAQAPKHHHHHH160LiSa7-s3 tetramer amino acid sequence (tag Remove)VDNKFNKEQQLAFYEILYLPNLNEEQRNAFIMSLKHDPSQSAYLLAEAKKLNDAQAPKVDNKFNKEQQLAFYEILYLPNLNEEQRNAFIMSLKHDPSQSAYLLAEAKKLNDAQAPK VDNKFNKEQQLAFYEILYLPNLNEEQRNAFIMSLKHDPSQSAYLLAEAKKLNDAQAPKVDNKFNKEQQLAFYEILYLPNLNEEQRNAFIMSLKHNPSQSAYLLAEAKKLNDAQAPK152

[0517]

[0518] 6.3. Purification and Immobilization of Tetramers Using Nickel-Affinity Resin

[0519] To culture Escherichia coli BL21(DE3) transformants, 5 mL of TB liquid medium containing kanamycin antibiotic was dispensed into a 50 mL test tube and incubated with shaking at 37°C and 200 rpm for 16 hours. 1% (v / v) of the culture solution was then inoculated into a 2000 mL Erlenmeyer flask dispensed with 500 mL of TB liquid medium and incubated with shaking at 37°C and 200 rpm. IPTG (isopropyl-β-D-thio-galactopyranoside) was added to achieve a final concentration of 1 mM at approximately OD600=0.6 and incubated with shaking at 37°C and 200 rpm for 18 hours. After recovering the bacterial cells by centrifuging the culture medium in this flask (4°C, 10,000 rpm, 30 min), the cells were suspended in 20 mL of PBS buffer (pH 7.4) (Intron Biotechnology, Korea), crushed using an ultrasonic grinder at 4°C for 30 minutes, and centrifuged at 4°C, 10,000 rpm for 30 minutes to collect only the supernatant. 25 mL of binding buffer (20 mM NaH2PO4, 30 mM NaCl, 10 mM Imidazole pH 7.4) was flowed through a column packed with 5 mL of Ni NTA Chelating Agarose CL-6B (Incospharm, Korea), and 20 mL of the above supernatant was mixed with 40 mL of binding buffer and flowed through the column. After running 25 mL of wash buffer (20 mM NaH2PO4, 30 mM NaCl, 20 mM Imidazole pH 7.4), 40 mL of elution buffer (20 mM NaH2PO4, 30 mM NaCl, 300 mM Imidazole pH 7.4) was collected in a 50 mL conical tube. The purified protein was desalted using a PD10 desalting column.

[0520] Purified tetramers LiSa2-1, LiSa7-3, and LiSa7-s3 were coupled to an epoxy-activated resin (Puriose AP HP-30, Furiogen). The specific procedure is as follows. First, a wet cake was prepared by washing 60 mL of resin with 90 mL of ultrapure water via vacuum filtration, repeating this process three times, and then maintaining the vacuum for 10 minutes. 7.2 g of the prepared wet cake, ligand protein, sodium phosphate buffer, and sodium sulfate were added to a reactor, and the reaction volume was set to 15 mL. After 16 hours of reaction, the mixture was washed three times with vacuum filtration and three times the volume of ultrapure water, followed by vacuuming to prepare a wet cake. The washed wet cake was placed in a reactor, and an equal volume of epoxy inactivator was added to initiate the reaction. After vacuum filtration and washing three times with triple-dip ultrapure water, the sample was washed for 30 minutes with double-dip washing buffer. After removing the washing buffer by vacuum filtration again, the sample was washed three more times with triple-dip ultrapure water, and then 20% ethanol was added to prepare a slurry, which was stored under refrigeration.

[0521]

[0522] 6.4. Comparison of Elution Efficiency of Mutant Proteins LiSa7-3 and LiSa7-s3 Tetramers by pH

[0523] Using the resin prepared in Example 4.3, the elution efficiency and elution volume at different pH levels were compared with commercial resins Mabselect Sure and MabSelect PrismA (GE Healthcare Life Sciences, USA).

[0524] 2 mL of the prepared resin was packed into a Tricorn, with a linear velocity of 100 cm / h and a volumetric flow rate of 0.33 mL / min. The column packed with resin was connected to an FPLC (AKTA Pure 25, Cytiva), and 20 mM PBS (pH 7.4) buffer (using PBS tablets, Millipore) was flowed in five times. After binding with 2.5 mg / mL IgG (Livgamma SN, SK Plasma), the column was washed with 20 mM PBS (pH 7.4) buffer in ten times. The antibody bound to the resin was eluted by flowing elution buffer in seven times, with the composition varying for each condition (50 mM Citrate pH 3.7, pH 4.0, and pH 4.3), and the process was performed accordingly. After elution was complete, 5 times the amount of 0.1M NaOH solution was first run, followed by 10 times the amount of 20 mM PBS (pH 7.4) buffer to wash and regenerate.

[0525] The elution profiles obtained above are shown in FIGS. 8a to 8c (8a: Elution profile, 50 mM Citrate, pH 3.7; 8b: Elution profile, 50 mM Citrate, pH 4.0; 8c: Elution profile, 50 mM Citrate, pH 4.3).

[0526] In addition, the following formula (Equation 1) was used to calculate the dissolution efficiency.

[0527] (Formula 1)

[0528] (IgG maxAU : Maximum AU of IgG (Absorbance Unit; referring to the UV absorbance of a sample solution, meaning the maximum UV absorbance at 280 nm detected by a UV detector when an IgG sample is injected into an elution efficiency measuring device (AKTA)) (Unit: mAU);

[0529] IgG sample injection volume: Amount of IgG sample injected into the equipment (Unit: mL);

[0530] CIP Area: IgG peak area obtained during the Cleaning-In-Place step (Unit: mL*mAU)

[0531]

[0532] The above-mentioned dissolution rates are shown in Table 16 below:

[0533]

[0534] As shown in Table 16 and Figures 8a to 8f above, it was confirmed that the tetramer LiSa7-3 and LiSa7-s3 resins of the present invention exhibit superior elution efficiency and reduced elution volume compared to MabSelect Sure, PrismA, which are widely used in industry, and LiSa2-1, an existing improved strain. When eluted using a 50 mM citrate, pH 4.3 buffer, the elution efficiency of PrismA and LiSa2-1 was 90% or less, whereas LiSa7-3 and LiSa7-s3 showed an elution efficiency of 97% or more; furthermore, while the elution volume of PrismA and LiSa2-1 was 15 mL or more, the elution volume of LiSa7-3 and LiSa7-s3 was reduced to 5 mL or less.

Claims

1. In the amino acid sequence of SEQ ID NO. 145, SEQ ID NO. 2, or SEQ ID NO. 153, Polypeptide comprising one or more amino acid substitutions selected from the following: The amino acid corresponding to the 11th position is substituted with tryptophan (Trp, W), tyrosine (Tyr, Y), or leucine (Leu, L), The amino acid corresponding to the 18th position is replaced with tyrosine (Tyr, Y), The amino acid corresponding to the 43rd position is replaced with tyrosine (Tyr, Y), The amino acid corresponding to the 32nd position is replaced with methionine (Met, M). The amino acid corresponding to the 36th position is substituted with histidine (His, H) or alanine (Ala, A), and The amino acid corresponding to the 6th position is replaced with histidine (His, H).

2. The polypeptide of claim 1, comprising any one of the following amino acid substitutions in the amino acid sequence of SEQ ID NO. 145, SEQ ID NO. 2, or SEQ ID NO. 153: The amino acid corresponding to the 11th position is substituted with tryptophan (Trp, W), tyrosine (Tyr, Y), or leucine (Leu, L), The amino acid corresponding to the 18th position is substituted with tyrosine (Tyr, Y), and The amino acid corresponding to the 43rd position is substituted with tyrosine (Tyr, Y).

3. The polypeptide of claim 1, wherein the amino acid sequence of SEQ ID NO. 145, SEQ ID NO. 2, or SEQ ID NO. 153 comprises the following amino acid substitutions: The amino acid corresponding to the 11th position is substituted with tryptophan (Trp, W), leucine (Leu, L), or tyrosine (Tyr, Y), The amino acid corresponding to the 18th position is substituted with tyrosine (Tyr, Y), and The amino acid corresponding to the 43rd position is substituted with tyrosine (Tyr, Y).

4. The polypeptide of claim 3, wherein in the amino acid sequence of SEQ ID NO. 145, SEQ ID NO. 2, or SEQ ID NO. 153, it further comprises one or more amino acid substitutions selected from the following: The amino acid corresponding to the 6th position is replaced with histidine (His, H), The amino acid corresponding to the 32nd position is substituted with methionine (Met, M), and The amino acid corresponding to the 36th position is substituted with histidine (His, H) or alanine (Ala, A).

5. In paragraph 3, a polypeptide further comprising the following amino acid substitutions in the amino acid sequence of SEQ ID NO. 145, SEQ ID NO. 2, or SEQ ID NO. 153: The amino acid corresponding to the 32nd position is substituted with methionine (Met, M), and The amino acid corresponding to the 36th position is replaced with histidine (His, H).

6. The polypeptide of claim 1, represented by an amino acid sequence selected from the group consisting of SEQ ID NOs 146, 149, 150, 4, 138, 140, 154, 155, 156, 248 to 259, and 261 to 264.

7. A polypeptide having an increased immunoglobulin elution efficiency compared to the polypeptide of SEQ ID NO. 145, 2, or 153, in any one of claims 1 to 6.

8. A polypeptide according to any one of claims 1 to 6, further comprising one or more selected from the group consisting of the E, D, A, and C domains of protein A.

9. A polypeptide multimer comprising two or more repeating units composed of the polypeptide of any one of claims 1 to 6.

10. A polypeptide multimer according to claim 9, further comprising one or more selected from the group consisting of the E, D, A, and C domains of protein A.

11. As a B domain, a protein A variant comprising a polypeptide of any one of claims 1 to 6 or a polypeptide multimer comprising two or more repeating units of said polypeptide.

12. A nucleic acid molecule encoding a polypeptide of any one of claims 1 to 6, a polypeptide multimer comprising two or more repeating units of said polypeptide, or a protein A variant comprising said polypeptide or polypeptide multimer as a B domain.

13. A recombinant vector comprising the nucleic acid molecule of claim 12.

14. A recombinant cell comprising the nucleic acid molecule of paragraph 12 or a recombinant vector containing the same.

15. A matrix for chromatography, wherein a polypeptide of any one of claims 1 to 6, a polypeptide multimer comprising two or more repeating units of said polypeptide, or a protein A variant comprising said polypeptide or polypeptide multimer as a B domain is coupled to a solid support. 16.(1) A polypeptide of any one of paragraphs 1 to 6, (2) A polypeptide multimer comprising two or more repeating units of the above polypeptide, (3) A protein A variant comprising the above polypeptide or polypeptide multimer as a B domain, (4) A nucleic acid molecule encoding the polypeptide, polypeptide multimer, or protein A variant, (5) A recombinant vector containing the above nucleic acid molecule, (6) A recombinant cell containing the nucleic acid molecule or recombinant vector, and (7) A matrix for chromatography in which the polypeptide, polypeptide multimer, or protein A variant is coupled to a solid support. A composition for binding a protein comprising an Fc region of an immunoglobulin or said Fc region, comprising one or more selected from the group consisting of 17.(a) A polypeptide of any one of paragraphs 1 to 6, (b) a polypeptide multimer comprising two or more repeating units of the above polypeptide, (c) a protein A variant comprising the above polypeptide or polypeptide multimer as a B domain, or (d) A matrix for chromatography in which a plurality of ligands comprising the polypeptide, polypeptide multimer, or protein A variant are coupled to a solid support A composition for the separation or purification of a protein comprising the Fc region of an immunoglobulin, comprising 18. A method comprising the step of adsorbing the protein comprising the Fc region of the immunoglobulin to a sample comprising the protein comprising the Fc region of the immunoglobulin by contacting a chromatographic matrix, wherein the matrix comprises (a) a polypeptide of any one of claims 1 to 6, (b) a polypeptide multimer comprising two or more repeating units formed of said polypeptide, (c) a protein A variant comprising said polypeptide or polypeptide multimer as a B domain, or (d) a plurality of ligands comprising said polypeptide, polypeptide multimer, or protein A variant, coupled to a solid support. Method for isolating or purifying a protein containing the Fc region of an immunoglobulin.

19. A method comprising the step of adsorbing the target compound by contacting a chromatographic matrix, wherein the matrix comprises (a) a polypeptide of any one of claims 1 to 6, (b) a polypeptide multimer comprising two or more repeating units of said polypeptide, (c) a protein A variant comprising said polypeptide or polypeptide multimer as a B domain, or (d) a plurality of ligands comprising said polypeptide, polypeptide multimer, or protein A variant, coupled to a solid support. A method for separating or purifying one or more target compounds from a liquid.

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