Functionalized UBX protein substances for promoting antibody purification

Abstract

Provided herein are methods and compositions for purifying antibodies. Purification is achieved by covalently attaching Protein A domains (E, D, A, B, C) or domain Z, or functional variants thereof, to the Drosophila melanogaster transcription factor Ultrabithorax (Ubx) substrate, thereby increasing the binding capacity of Protein A chromatography. The compositions include fusion proteins comprising the Drosophila melanogaster transcription factor Ultrabithorax (Ubx), or a fragment thereof, and an immunoglobulin-binding protein. In some embodiments, the immunoglobulin-binding protein is a Protein A domain, a Protein Z domain, or a fragment thereof.

Description

Technical Field

[0001] Prior Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 828,142, filed Apr. 2, 2019, which is hereby incorporated by reference in its entirety.

Background Art

[0002] Antibody-based therapeutic agents efficiently target specific molecules or cells with few adverse effects. On the other hand, therapeutic antibodies are costly, and the therapeutic antibodies in clinical trials exceed what can be manufactured using existing facilities. Therefore, it is essential to industrially improve antibody purification methods to suppress manufacturing costs and improve efficiency. Many methods for isolating and purifying antibodies have been established, such methods being class-specific affinity chromatography, anion exchange chromatography, or antigen-specific affinity chromatography, etc. The most common affinity ligand is protein A (a 41 kD cell wall protein derived from Staphylococcus aureus) that binds to the Fc region of human immunoglobulin G (IgG). Additional affinity ligands are protein G (a 65 kD IgG-binding protein of group G streptococcus), protein L (a 76 Kd binding protein of Peptostreptococcus magnus), and protein A / G (a recombinant fusion protein combining the IgG-binding domains of both protein A and protein G), which are functional variants of Staphylococcus aureus protein A. Protein A chromatography is widely recognized as the absolute standard for antibody purification because of its high selectivity (purity 90%) and its large binding capacity (60 mg of antibody per 1 ml of resin).

[0003] U.S. Patent No. 6,127,526 describes a conventional method for isolating and purifying proteins via protein A affinity chromatography. In this purification process, (i.) immobilizing a protein A domain (E, D, A, B, C) or a functional variant thereof on a solid phase, (ii.) removing contaminants by washing the solid phase material with a hydrophobic electrolyte solution, and (iii.) isolating the desired protein from the solid phase are performed. The type of solid phase carrier can dramatically affect the antibody purification process. Commonly used solid phase matrices can consist of substances such as silica or agarose. On the other hand, the method of adding Staphylococcus protein A (SpA) to the solid phase carrier is also important. Protein A chromatography is a powerful antibody purification method, but considering that protein A chromatography is quite costly and there are existing limitations related to throughput, scale-up, and ligand leakage, alternative purification methods are needed.

Summary of the Invention

Means for Solving the Problems

[0004] The present disclosure is directed to compositions and methods for increasing the binding capacity of protein A chromatography by covalently attaching a protein A domain (E, D, A, B, C) or synthetic protein Z (e.g., synthetic Z domain) to a Drosophila melanogaster transcription factor Ultrabithorax (Ubx) substance. The inventors have discovered that the SpA protein incorporated into the Ubx substance is approximately 1,000 times more per gram of the substance compared to that of a commercially available chromatography resin cross-linked with the same SpA protein. The compositions provided herein include a fusion protein comprising a Drosophila melanogaster transcription factor Ultrabithorax (Ubx) or a fragment thereof and an immunoglobulin-binding protein. In some embodiments, the immunoglobulin-binding protein is a protein A domain, a protein Z domain, or a fragment thereof.

[0005] Also provided is an affinity chromatography matrix comprising from about 2 to about 18 immunoglobulin binding domains (e.g., a repeat sequence of SpA domains or Z domains). The binding capacity can be increased by covalently attaching multiple immunoglobulin binding domains (e.g., about 2 to about 18 repeats of SpA domains or Z domains) to a Ubx protein or a fragment thereof. A problem often seen in connection with other SpA applications is that when the SpA domain is chemically immobilized on a solid phase matrix, it is prone to degradation, thereby causing protein A to leak out. See, for example, U.S. Patent Application Publication No. 20050038231. The compositions and methods provided herein make it possible to improve immunoglobulin purity by preventing contamination of SpA due to ligand leakage. By covalently attaching SpA domain repeats (s), Z domain (s), or a fragment thereof to a Ubx substance, leakage or dissociation of the ligand from the matrix is prevented. Accordingly, the present invention provides a capacity-increased antibody affinity separation matrix that enables the isolation of immunoglobulins with improved purity. Also provided herein is a method for isolating or purifying an antibody or an immunoglobulin-containing protein using any of the provided affinity separation matrices. BRIEF DESCRIPTION OF THE DRAWINGS

[0006]

Figure 1

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Figure 7A

Figure 7B

Figure 7C

Figure 7D

DETAILED DESCRIPTION OF THE INVENTION

[0007] Definitions This specification provides an antibody affinity protein immobilized on a Ubx substance backbone. This is achieved via a fusion protein composed of an antibody binding domain and a substance-forming Ubx protein. Also provided is the use of a Ubx substance for purifying immunoglobulins. The present invention is based on the discovery that the SpA protein incorporated into the Ubx substance has 1,000 times more per gram of the substance compared to that of a commercially available chromatography resin cross-linked with the same SpA protein, so that the affinity purification binding capacity can be significantly improved. Further, by covalently binding the antibody affinity domain to the Ubx substance, leakage of the ligand from the carrier is prevented, thereby improving the product purity.

[0008] The terms "polypeptide", "peptide", and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. These terms encompass amino acid chains of any length, including full-length proteins, in which the amino acid residues are linked by covalent peptide bonds. This definition also includes fragments and variants of any polypeptide described herein. A protein can be an antibody. A protein can be one produced by a host cell.

[0009] The terms "antibody affinity domain" and "immunoglobulin binding protein" are used interchangeably and can be, for example, a Protein A domain (e.g., E, D, A, B, C, or Z), or a Protein G domain, Protein L domain, Protein A / G domain, or a functional variant thereof. In some embodiments, the antibody affinity domain can be a Staphylococcus aureus Protein A (SpA) domain. In some embodiments, the antibody affinity domain can comprise, consist essentially of, or consist of the Z domain (e.g., synthetic Z domain (such as SEQ ID NO: 1 (HMVDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK)) or a fragment thereof. In some embodiments, the affinity domain comprises, consists essentially of, or consists of the following: SpA domain E (AQHDEAQQNAFYQVLNMPNLNADQRNGFIQSLKDDPSQSANVLGEAQKLNDSQAPK (SEQ ID NO: 2)), SpA domain A (ADNNFNKEQQNAFYEILNMPNLNEEQRNGFIQSLKDDPSQSANLLSEAKKLNESQAPK (SEQ ID NO: 3)), SpA domain B (ADNKFNKEQQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 4)), SpA domain C (ADNKFNKEQQNAFYEILHLPNLTEEQRNGFIQSLKDDPSVSKEILAEAKKLNDAQAPK (SEQ ID NO: 5)), SpA domain D (SEQ ID NO: 14), Protein G domain (e.g., Protein G C1 domain (TYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTE) (SEQ ID NO: 6)), Protein G C2 domain (TYKLVINGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTE) (SEQ ID NO: 7), or Protein GC3 domain (TYKLVINGKTLKGETTTKAVDAETAEKAFKQYANDNGVDGVWTYDDATKTFTVTE (SEQ ID NO: 8)), Protein L domain (e.g., B1: (VTIKANLIFADGSTQNAEFKGTFAKAVSDAYAYADALKKDNGEYTVDVADKGLTLNIKFAG) (SEQ ID NO: 9)), B2: (VTIKVNLIFADGKTQTAEFKGTFEEATAKAYAYADLLAKENGEYTADLEDGGNTINIKFAG) (SEQ ID NO: 10)), B3: (VTIKVNLIFADGKIQTAEFKGTFEEATAKAYAYANLLAKENGEYTADLEDGGNTINIKFAG) (SEQ ID NO: 11)), or B4: (VTIKVNLIFADGKTQTAEFKGTFEEATAEAYRYADLLAKVNGEYTADLEDGGYTINIKFAG) (SEQ ID NO: 12), Protein A / G domain (e.g., (AQHDEAQQNAFYQVLNMPNLNADQRNGFIQSLKDDPSQSANVLGEAQKLNDSQAPK) (SEQ ID NO: 13)), (ADAQQNNFNKDQQSAFYEILNMPNLNEAQRNGFIQSLKDDPSQSTNVLGEAKKLNESQAPK) (SEQ ID NO: 14)), (ADNNFNKEQQNAFYEILNMPNLNEEQRNGFIQSLKDDPSQSANLLSEAKKLNESQAPK) (SEQ ID NO: 15)), (ADNKFNKEQQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPK) (SEQ ID NO: 16)), (ADNKFNKEQQNAFYEILHLPNLTEEQRNGFIQSLKDDPSVSKEILAEAKKLNDAQAPK) (SEQ ID NO: 17)), (TYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTE) (SEQ ID NO: 18)), or (TYKLVINGKTLKGETTTKAVDAETAEKAFKQYANDNGVDGVWTYDDATKTFTVTE) (SEQ ID NO: 19)), or a fragment thereof. Optionally, any of the fragments of the immunoglobulin-binding proteins described herein can be binding fragments.

[0010] In some embodiments, any of the fusion proteins described herein includes one or more antibody affinity domains. Optionally, the affinity domain includes an amino acid sequence comprising one or more sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 19 or functional fragments thereof, an amino acid sequence consisting essentially of the one or more sequences, or an amino acid sequence consisting of the one or more sequences.

[0011] The terms "solid phase", "carrier", "scaffold", and "matrix" are used interchangeably herein and refer to a solid substance that provides a physical structure for immobilizing an antibody affinity domain.

[0012] As used herein, the term "antibody" refers to an immunoglobulin (Ig) molecule, an antigen-binding fragment thereof, or a binding derivative thereof. An antigen-binding fragment of an antibody contains an antigen-binding site that specifically binds to an antigen. The antibody (Ab) can be a monoclonal antibody, a polyclonal antibody, or a multispecific antibody (e.g., a bispecific antibody). Examples of antibodies include those of the immunoglobulin (Ig) types IgG, IgD, IgE, IgA, and IgM. The antibody can be a natural antibody or a recombinant antibody. The antibody can be produced by a host cell.

[0013] As used herein, the term "fusion protein" refers to a protein that includes two or more polypeptides, which are derived from different proteins but are produced as a single polypeptide under the control of a single promoter from a polynucleotide that includes nucleotide sequences encoding both proteins and, in some embodiments, a linker sequence. The two or more polypeptides in the fusion proteins described herein are complexed or linked, and this complexing or linking is optionally performed via a linker.

[0014] The terms "nucleic acid" or "nucleotide" refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and their polymers (either in single-stranded or double-stranded form). Unless otherwise specified, a particular nucleic acid sequence implicitly encompasses not only the explicitly shown sequence, but also its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences.

[0015] A "promoter" is defined as one or more nucleic acid control sequences that cause transcription of a nucleic acid. A promoter as used herein includes the necessary nucleic acid sequences near the transcription start site (such as the TATA element in the case of a polymerase II type promoter). A promoter optionally also includes distal enhancer elements or distal repressor elements, and such distal elements can be located thousands of base pairs away from the transcription start site.

[0016] Nucleic acids are "operably linked" when they are arranged so as to bring about a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of that sequence. Alternatively, a ribosome binding site is operably linked to a coding sequence if it is arranged so as to facilitate translation.

[0017] The term "Ubx substance(s)" refers to a biological substance formed by the self - assembly of the Drosophila melanogaster transcription factor Ultrabithorax (US2010 / 0143436) or to an improved synthetic version of Ultrabithorax described in US20180222949A1. In some examples, the Ubx substance comprises two or more self - assembled Ubx protein molecules. For any of the Ubx substances described herein, the Ubx protein may comprise an amino acid sequence or a fragment thereof selected from the group consisting of the amino acid sequences shown in GenBank accession number AAN13717, GenBank accession number AAN13718, GenBank accession number AAN13719, GenBank accession number AAF55355, GenBank accession number AAF55356, and GenBank accession number AAS65158. For the purpose of comparison with other chromatographic carriers, 1 mL of Ubx substance means a mass of Ubx substance corresponding to 1 mL of agarose - based resin in the deposited state.

[0018] In some embodiments, the full - length Ubx protein is used to create the fusion protein. See, for example, Uniprot number P83949. In other embodiments, the Ubx protein used to generate the Ubx fusion protein is a Ubx protein fragment containing the Ubx homeodomain (e.g., a Ubx protein fragment consisting of or containing SEQ ID NO: 20 (LRRRGRQTYTRYQTLELEKEFHTNHYLTRRRRIEMAHALCLTERQIKIWFQNRRMKLKKEI)). In other embodiments, the Ubx fusion protein comprises a Ubx protein fragment containing SEQ ID NO: 20 and SEQ ID NO: 21 (MNSYFEQA). In other embodiments, the Ubx fusion protein comprises a Ubx protein fragment containing SEQ ID NO: 22 (VRPSACTPDSRVGGYLDTS) and / or SEQ ID NO: 23 (FYPWMAIA). It will be understood that the term "Ubx protein" means the full - length Ubx protein or a fragment thereof.

[0019] The term "functional substance" refers to a Ubx biological substance formed from a fusion protein containing an immunoglobulin-binding protein and a Ubx protein or a fragment thereof. In some embodiments, the Ubx biological substance comprises two or more self-assembling fusion proteins comprising an immunoglobulin-binding protein and a Ubx protein.

[0020] Detailed Description Compositions The present invention relates to a composition comprising a biological substance for purifying an antibody. In some embodiments, the biological substance comprises a Ubx protein or a fragment thereof. The biological substance provided herein has functional properties conferred by fusing an antibody-binding protein or an immunoglobulin-binding protein to a Ubx protein or a fragment thereof. The immunoglobulin-binding protein can be any protein having a natural immunoglobulin-binding capacity, and such proteins include staphylococcal protein A (SpA), streptococcal protein G (SpG), protein L of the genus Peptostreptococcus, protein A / G, or a recombinant protein comprising an IgG-binding domain of such a protein, etc. For a review of such proteins, see, for example, Kronvall and Jonsson (Receptins: a novel term for an expanding spectrum of natural and engineered microbial proteins with binding properties for mammalian proteins, J. Mol. Recognit. 1999 January-February; 12(1):38-44). The immunoglobulin-binding protein can comprise one or more of the E domain, D domain, A domain, B domain, and C domain of SpA. In some embodiments, the immunoglobulin-binding protein comprises domain B of protein A or an engineered synthetic protein Z domain. In some embodiments, a multimeric ligand (Figure 1) comprising two or more (such as 2 to 18) copies of the same monomeric domain derived from domain E, domain D, domain A, domain B, or domain C, or domain Z of protein A. In other embodiments, a multimeric ligand comprising two or more (such as 2 to 18) different monomeric domains selected from domain E, domain D, domain A, domain B, and domain C, or domain Z of protein A can be used. In any of the embodiments described herein, a functional variant of domain E, domain D, domain A, domain B, or domain C, or domain Z of protein A can be used.

[0021] In some embodiments, the functional properties of antibody binding are conferred via a fusion protein comprising an immunoglobulin, an antibody-binding protein, and a Ubx protein or a fragment thereof. The nucleic acid or gene encoding the functional fusion protein is cloned into a plasmid for expressing the protein and is generally ligated 3' of a DNA sequence encoding a peptide tag (e.g., a histidine tag) to facilitate protein purification. Other tags known in the art (e.g., chemical moieties such as fluorescent tags) may also be used. The fusion protein may be expressed in cells (e.g., eukaryotic or prokaryotic cells). Cells comprising a nucleic acid sequence encoding any of the fusion proteins described herein are also provided. Optionally, the nucleic acid sequence is included in a vector. Optionally, the nucleic acid sequence is stably integrated into the genome of the cell. Expression of the fusion protein may be placed under the control of a suitable promoter (e.g., any constitutive or controllable promoter known to those of skill in the art).

[0022] In some embodiments, a linker is placed at the 3' end of the nucleic acid sequence encoding the immunoglobulin-binding protein. The length and sequence of this linker can vary. For example, the length of the linker can be from about 2 to about 20 amino acids. In some embodiments, the linker comprises an alternating repeat sequence of glycine and serine (e.g., (SGSG) n (SEQ ID NO: 24) or (GSGS) n (SEQ ID NO: 25) (wherein n is an integer in the sequence)). When a linker is present, the nucleic acid encoding the Ubx protein or a fragment thereof is located 3' of the linker, and downstream of the nucleic acid, there is one or more stop codons (the only one(s) in the gene fusion). When the fusion gene is expressed in a host cell, a fusion protein is produced, and this fusion protein can be used in the preparation of a functional substance. For methods of preparing fusion proteins and functional substances, see, for example, U.S. Patent Publication No. 20180222949.

[0023] By using the compositions and methods described herein, the proteins incorporated into the Ubx substance can bind antibodies to functional biomolecules at a higher capacity compared to those of commercially available chromatography resins cross-linked with the same protein, being more than 1,000-fold greater per gram of the substance, and thus can bind antibodies to functional biomolecules at a higher capacity compared to the case of using antibody binding techniques as reported previously (Figures 2 and 3).

[0024] Provided herein is a fusion protein comprising at least one Ubx protein and an immunoglobulin-binding protein. In some embodiments, the immunoglobulin-binding protein comprises a single domain of staphylococcal protein A (SpA), domain Z, or a functional variant thereof. In some embodiments, the domain of staphylococcal protein A (SpA) is the E domain, D domain, A domain, B domain, or C domain. In some embodiments, the immunoglobulin-binding protein comprises two or more different monomeric domains of staphylococcal protein A (SpA), domain Z, or a functional variant thereof. In some embodiments, the two or more different monomeric domains of staphylococcal protein A (SpA) are the E domain, D domain, A domain, B domain, or C domain. In some embodiments, the immunoglobulin-binding protein comprises two or more monomeric domains of staphylococcal protein A (SpA), domain Z, or a functional variant thereof, and these monomeric domains are the same. In some embodiments, the two or more different monomeric domains of staphylococcal protein A (SpA) are the E domain, D domain, A domain, B domain, or C domain.

[0025] Also provided herein is a solid-phase matrix comprising any of the fusion proteins described herein. In some embodiments, leakage of the immunoglobulin-binding protein from the solid-phase matrix is prevented or reduced. For example, the reduction rate of leakage can be at least 55, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%. In some embodiments, the binding capacity of the substance is more than 1 g / mL per 1 mL.

[0026] In some embodiments, the fusion protein comprises a Ubx protein fragment, and this Ubx protein fragment is composed of or contains SEQ ID NO: 20 (LRRRGRQTYTRYQTLELEKEFHTNHYLTRRRRIEMAHALCLTERQIKIWFQNRRMKLKKEI). In other embodiments, the Ubx fusion protein is composed of a Ubx protein fragment that includes SEQ ID NO: 20 and SEQ ID NO: 21 (MNSYFEQA). In other embodiments, the Ubx fusion protein includes a Ubx protein fragment that includes SEQ ID NO: 22 (VRPSACTPDSRVGGYLDTS) and / or SEQ ID NO: 23 (FYPWMAIA). Also provided are fusion proteins comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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% identity to any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 23. The term "identity" as used in the context of a polynucleotide sequence or polypeptide sequence refers to the sequence identity percentage between the sequence and a reference sequence being at least 60%. Alternatively, the percentage of identity can be any integer from 60% to 100%. As examples of embodiments, when compared to a reference sequence using the programs described herein, those having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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% identity are included, and this comparison is preferably performed by BLAST using standard parameters as described below.One of ordinary skill in the art will recognize that such values can be appropriately adjusted so that the percent identity of the proteins encoded by two nucleotide sequences is determined by considering codon degeneracy, amino acid similarity, reading frame positioning, and the like.

[0027] For sequence comparisons, typically one sequence serves as a reference sequence to which test sequences are compared. When sequence comparison algorithms are used, the test and reference sequences are input into a computer, subsequence coordinates are designated (if necessary), and sequence algorithm program parameters are designated. Default program parameters can be used or alternative parameters can be designated. Next, the percent sequence identity is calculated for the test and reference sequences by a sequence comparison algorithm based on the program parameters.

[0028] As used herein, "comparison window" includes reference to any one segment of a number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence is compared to a reference sequence of the same number of contiguous positions in this segment, and the comparison can be conducted after the two sequences are optionally aligned. Methods of sequence alignment for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted by the local homology algorithm of Smith and Waterman Add.APL.Math.2:482 (1981), the homology alignment algorithm of Needleman and Wunsch J.Mol.Biol.48:443 (1970), the search for similarity method of Pearson and Lipman Proc.Natl.Acad.Sci.(U.S.A.) 85:2444 (1988), by computerized implementations of these algorithms (e.g., BLAST), or by manual alignment and visual inspection.

[0029] Algorithms suitable for determining percent sequence identity and percent sequence similarity are the BLAST algorithm and the BLAST 2.0 algorithm, which are described in Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1977) Nucleic Acids Res. 25:3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) website. In such algorithms, high-scoring sequence pairs (HSPs) are first identified by identifying short strings of length W in the query sequence, which strings of length W match or satisfy some positive-valued threshold score T when aligned with strings of the same length in the database sequences. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits serve as seeds for initiating searches to find longer HSPs that contain them. Next, the word hits are extended in both directions along each sequence as long as the cumulative alignment score can increase. The calculation of the cumulative score is performed using, for nucleotide sequences, parameters M (reward score for a pair of matching residues (always >0)) and N (penalty score for a mismatched residue (always <0)). For amino acid sequences, the cumulative score is calculated using a scoring matrix. Extension of the word hits in each direction is stopped when: the cumulative alignment score decreases by an amount X from its maximum achieved value; the cumulative score becomes 0 or less due to the accumulation of one or more negatively scored residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. In the BLASTN program (for nucleotide sequences), word length (W) 28, expectation value (E) 10, M = 1, N = -2, and a two-stranded comparison are used as defaults.For amino acid sequences, in the BLASTP program, a string length (W) of 3, an expectation value (E) of 10, and the BLOSUM62 scoring matrix are used as defaults (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0030] In the BLAST algorithm, statistical analysis of the similarity between two sequences is also performed (see, for example, Karlin & Altschul, Proc. Nat’l. Acad. Sci. USA 90:5873 - 5787 (1993)). One similarity measure provided by the BLAST algorithm is the minimum total probability (P(N)), and P(N) is an indicator of the probability that a chance match will occur between two nucleotide sequences or amino acid sequences. For example, a nucleic acid is considered to be similar to a reference sequence if the minimum total probability is less than about 0.01, more preferably about 10 -5 less, and most preferably about 10 -20 less in a comparison of the test nucleic acid with the reference nucleic acid.

[0031] The amino acids in the polypeptides described herein can be any of the 20 naturally occurring amino acids, D-stereoisomers of naturally occurring amino acids, non-naturally occurring or unnatural amino acids, and chemically modified amino acids. Non-natural amino acids (i.e., those not naturally found in proteins) are well known in the art and are described, for example, in Zhang et al., “Protein engineering with unnatural amino acids,” Curr. Opin. Struct. Biol. 23(4):581-587 (2013), Xie et la., “Adding amino acids to the genetic repertoire,” 9(6):548-54(2005)), and all references cited therein. Β-amino acids and γ-amino acids are known in the art and are also contemplated herein as non-natural amino acids. Any of the synthetic peptides described herein (e.g., synthetic domain Z) can be derived from or engineered from a domain of SpA (e.g., domain E, domain D, domain A, domain B, or domain C).

[0032] As used herein, a chemically modified amino acid refers to an amino acid whose side chain is chemically modified. For example, the side chain can be modified to include a signaling moiety (such as a fluorophore or radiolabel). The side chain can also be modified to include a new functional group (such as a thiol group, carboxylic acid group, or amino group). Post-translationally modified amino acids are also included in the definition of chemically modified amino acids.

[0033] One or more conservative amino acid substitutions to any one of the polypeptides described herein are also contemplated. By way of example, conservative amino acid substitutions can be made to one or more of the amino acid residues (e.g., one or more lysine residues of any one of the polypeptides provided herein). Those skilled in the art will know that a conservative substitution is one in which one amino acid residue is replaced with another amino acid residue that is biologically and / or chemically similar. Shown below are eight groups, each of which includes amino acids that are conservative substitutions for one another:

[0034] 1) Alanine (A), Glycine (G),

[0035] 2) Aspartic acid (D), Glutamic acid (E),

[0036] 3) Asparagine (N), Glutamine (Q),

[0037] 4) Arginine (R), Lysine (K),

[0038] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V),

[0039] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W),

[0040] 7) Serine (S), Threonine (T), and

[0041] 8) Cysteine (C), Methionine (M)

[0042] By way of example, when a substitution from arginine to serine is mentioned, conservative substitutions to serine (e.g., to threonine) are also contemplated. Non-conservative substitutions (e.g., those that substitute lysine with asparagine) are also contemplated.

[0043] Method This specification also provides a method for separating one or more immunoglobulin-containing proteins from a liquid or solution. The method includes (a) contacting a separation matrix containing a ligand (i.e., an immunoglobulin-binding protein) immobilized on a carrier with the liquid, (b) adsorbing the immunoglobulin-containing protein to the matrix by interaction with the ligand(s), (c) washing the adsorbed immunoglobulin-containing protein, and (d) recovering the protein by contacting the matrix with an eluent that releases the immunoglobulin-containing protein. This method increases the binding capacity of the ligand to the immunoglobulin molecule, and this increase is brought about by using a ligand that each contains one or more domains (i.e., monomers) of staphylococcal protein A (SpA) (E, D, A, B, C, or Z) or a functional variant thereof.

[0044] In the method described herein, the ligand (i.e., the immunoglobulin-binding protein) is immobilized on a protein-based biomaterial (i.e., the Ubx material), and this immobilization is carried out by fusing the protein ligand to the Ubx protein or a fragment thereof. In the method provided herein, the matrix is composed of the Ubx material as a solid-phase carrier. In all embodiments, the biomaterial can be formed by self-assembly of the Ubx protein, a fusion protein containing the Ubx protein, or a fragment thereof.

[0045] In all embodiments, the ligand in the matrix is present on a solid support. Examples of solid supports include, but are not limited to, activated alumina, powdered cellulose, silicic acid, gels, paper, glass fibers, plastics, agarose, sepharose, silica, and their derivatives, and any other suitable solid support. It has been found that the binding capacity of the ligand increases by about 1,000-fold when supported by the Ubx substance, which was unexpected. The binding capacity can be further increased by adding repeat sequences of the ligand to the Ubx substance via gene fusion (i.e., creating a fusion protein comprising Ubx and one or more ligand repeat sequences). For example, but not limited to, the binding capacity can be increased by using a fusion protein in which an 18-repeat sequence of the SpA domain or the Z domain is immobilized on the Ubx substance (Figure 3).

[0046] The compositions and methods provided herein can also be used to stabilize the ligand via covalent addition to the Ubx substance. In all embodiments, the ligand is added via protein fusion and is generated with the substance-forming Ubx protein, thereby preventing or reducing leakage or dissociation of the ligand from the solid support. As shown in Figure 4, this stability is demonstrated by the consistently high binding capacity of the matrix described herein even after exposure to high heat (Figure 4).

[0047] Methods for isolating immunoglobulins (such as IgG, IgA, and / or IgM) are also provided, using the ligand or matrix according to the present invention. Thus, the present invention encompasses a chromatographic process in which at least one target compound is isolated from a liquid by adsorption to the above-mentioned ligand or matrix. Thus, this aspect of the present invention relates to affinity chromatography, which is a widely used and well-known separation technique. Briefly described, in a first step, a solution containing the target compound (preferably the antibody described above) is passed through the matrix under conditions that allow adsorption of the target compound to the ligand present on the separation matrix. Such conditions are controlled, for example, by pH and / or salt concentration (i.e., ionic strength in the solution). Care should be taken not to exceed the capacity of the matrix, i.e., the flow rate should be slow enough to allow good adsorption and not damage the matrix. In this step, the other components of the solution will, in principle, pass through unhindered. Next, optionally, the matrix is washed to remove retained and / or loosely bound substances (e.g., washed with an aqueous solution). Intermediate washing steps using additives (solvents, salts, or surfactants, or mixtures thereof) are optionally applied to the matrix of the present disclosure. In the next step, a second solution (eluent) is passed through the matrix under conditions that cause desorption (i.e., conditions under which the target compound is released). Such conditions are generally obtained by changing pH, salt concentration (i.e., ionic strength), hydrophobicity, etc. Various elution schemes are known (such as gradient elution and stepwise elution). Elution can also occur with a second solution containing a competing substance, which will replace the desired antibody on the matrix.

[0048] Substances, compositions, and components that can be used in, or in combination with, the disclosed methods and compositions, that can be used in the preparation thereof, or that are products thereof are disclosed. Although substances and other substances are disclosed herein, and combinations, subsets, interactions, groups, etc. of such substances are disclosed, specific mention of each of the various individual and collective combinations and permutations of such compounds may not be explicitly disclosed herein, but it will be understood that each is clearly contemplated and described herein. For example, when a method is disclosed and discussed, and many modifications that can be made to one or more molecules included in the method are discussed, every combination and permutation of the method, as well as possible such modifications, are clearly contemplated unless specifically shown to be inconsistent. Similarly, any subset and combination of such are also clearly contemplated and disclosed. This concept applies to all aspects of the present disclosure (including, but not limited to, steps in methods of using the disclosed compositions). Thus, where there are various additional steps that are possible, each of such additional steps can be carried out in conjunction with any particular method step or combination of method steps of the disclosed method, and it will be understood that each such combination or subset of combinations is clearly contemplated and considered to be disclosed.

[0049] Publications cited herein and the substances to which they refer are hereby expressly incorporated by reference in their entirety.

Examples

[0050] Antibody binding assay Functional tests of the Z domain fused with the Ubx substance were carried out. Non-modified Ubx fibers, Z-Ubx fibers, and 18Z-Ubx fibers were assembled and placed in a 4-well plate. Each fiber was incubated with an anti-myelin basic protein human antibody (Abcam, Cambridge, UK; 1:300) in 250 μl of phosphate-buffered saline (PBS). After rinsing the fibers three times with PBS, detection was performed using fluorescein isothiocyanate (FITC) (Abcam)-labeled goat anti-human IgG. Since dityrosine binding also emits blue fluorescence, non-modified fibers appear blue under 4’,6-diamidino-2-phenylindole (DAPI) fluorescence. Images were captured under DAPI and FITC using a Nikon Eclipse Ti A1R inverted confocal microscope and analyzed using Nikon Elements Imaging Software. Figure 2A shows that non-modified Ubx substance cannot bind to the antibody, while both Z-Ubx and 18Z-Ubx bind to a significant amount of FITC-labeled antibody. This first test demonstrates that the antibody can specifically bind to the Ubx substance fused with the Z domain, but not to the substance itself.

[0051] Binding capacity The static binding capacities of Z-Ubx and 18Z-Ubx were determined by incubating Z-Ubx fibers and 18Z-Ubx fibers with purified IgG (1 mg / mL) for 15 minutes. After rinsing the fibers three times in PBS, elution was carried out in 100 μl of 2.5 M potassium iodide (pH 7). The eluate was electrophoresed on an 8% SDS-PAGE acrylamide gel. Concentration measurement analysis using NIH Image J image analysis software was performed to measure the binding capacity. Figure 3 shows that the static binding capacity of Z-Ubx fibers is 7.43 g / mL and that of 18Z-Ubx fibers is 67.86 g / mL.

[0052] Durability The same experiment was carried out to examine the durability of both Ubx substances and the stability of the Z domain fused to the Ubx substance. However, as a difference, the fibers were boiled in water for 30 seconds, cooled at room temperature for 1 minute, and then purified IgG was bound to the fibers. After boiling, the static binding capacity of the Z-Ubx fibers was 5.35 g / mL, and that of the 18Z-Ubx fibers was 46.14 g / mL (Figure 4). This demonstrates that although some binding is lost, the Z domain immobilized on the Ubx substance retains its function even after extreme heat exposure. To further test whether a low pH buffer affects the Z-Z-Ubx substance, IgG purification was performed twice using the same membrane. The Z-Z-Ubx membrane was washed with 10 mL of PBS at a flow rate of 0.5 mL / min. Monoclonal IgG was loaded onto the membrane at a flow rate of 0.5 mL / min, and the flow-through fraction was collected. The membrane was thoroughly washed with PBS at a flow rate of 0.5 mL / min. The bound IgG was eluted with 0.1 M glycine (pH 2.5) at a flow rate of 0.5 mL / min to obtain fractions. The membrane was thoroughly washed with PBS and then IgG was loaded onto the membrane again as described. The fractions were electrophoresed on an SDS-PAGE gel (Figure 5). Two findings can be obtained from these gels: (1) The binding of IgG to the membrane in the second purification is at least as good as that in the first purification, if not better, and (2) No Z-Z-Ubx oligomers are observed in the washing steps (lanes 2 and 5) in either purification. These findings suggest that neither the binding capacity of Z-Z-Ubx nor the leakage of the substance occurs even when the pH of the elution buffer is low. Furthermore, the Z-Z-Ubx membrane was subjected to additional washing with 50 mL of the elution buffer, and then the fractions were collected and electrophoresed on a 10% SDS-PAGE gel next to the purified Z-Z-Ubx monomer used as a control. This gel was transferred to a nitrocellulose membrane for Western blot analysis. This nitrocellulose membrane was blocked with 5% milk. To confirm that no leakage occurred, an anti-Z domain antibody was used to search for degradation and leakage from the membrane (Figure 6).It can be seen from such data that even after treatment with a low pH buffer solution, leakage of the Protein A ligand from the membrane does not occur.

[0053] Purity and activity The ability of the Z-Z-Ubx membrane was measured in the purification of monoclonal human anti-insulin antibody (Abcam) from hybridoma medium. The Z-Z-Ubx membrane was washed with PBS at a flow rate of 0.5 ml / min. Next, the hybridoma medium supplemented with anti-insulin antibody was passed through the membrane at a flow rate of 0.5 ml / min. After thoroughly washing the membrane with PBS, the antibody was eluted using 0.1 M glycine (pH 2.5). Fractions from the starting solution and eluate 2 were collected and electrophoresed on a 10% SDS-PAGE gel (Figure 7A). The fractions derived from the starting solution and eluate 2 were transferred to a nitrocellulose membrane for Western blot analysis as well. This nitrocellulose membrane was blocked with 5% milk. A goat anti-human secondary antibody was used to confirm the presence of IgG (Figure 7B). It can be seen from such data that the purity of the antibody is over 90%. Finally, the antibody retains its activity. The potency comparison between the untreated human anti-insulin antibody (Abcam) (Figure 7C) and the antibody purified using the Z-Z-Ubx membrane (Figure 7D) was performed using Western blot analysis. Insulin was electrophoresed on a 20% acrylamide gel and transferred to a nitrocellulose membrane. The nitrocellulose membrane was blocked with 5% milk. Insulin was detected using the antibody that had not been passed through the membrane and the antibody eluted from the Z-Z-Ubx membrane as primary antibodies. A goat anti-human secondary antibody (abcam) was used for the detection of the primary antibody, and the nitrocellulose membrane was imaged with a Biorad Imager. The purified antibody exhibits an activity similar to that of the wild type. In embodiments of the present invention, for example, the following items are provided. (Item 1) A fusion protein comprising at least one Ubx protein and an immunoglobulin-binding protein. (Item 2) The fusion protein according to Item 1, wherein the immunoglobulin-binding protein comprises a single domain of staphylococcal protein A (SpA), domain Z, or a functional variant thereof. (Item 3) The fusion protein according to Item 2, wherein the domain of the staphylococcal protein A (SpA) is the E domain, D domain, A domain, B domain, or C domain. (Item 4) The fusion protein according to Item 2, wherein the functional variant is protein G, protein L, or protein A / G. (Item 5) The fusion protein according to Item 1 or Item 2, wherein the immunoglobulin-binding protein comprises two or more different monomeric domains of staphylococcal protein A (SpA), domain Z, or a functional variant thereof. (Item 6) The fusion protein according to Item 5, wherein the two or more different monomeric domains of the staphylococcal protein A (SpA) are the E domain, D domain, A domain, B domain, or C domain. (Item 7) The fusion protein according to Item 1 or Item 2, wherein the immunoglobulin-binding protein comprises two or more monomeric domains of staphylococcal protein A (SpA), domain Z, or a functional variant thereof, and the monomeric domains are the same. (Item 8) The fusion protein according to Item 7, wherein the two or more different monomeric domains of the staphylococcal protein A (SpA) are the E domain, D domain, A domain, B domain, C domain, or Z domain. (Item 9) The fusion protein according to any one of Items 1 to 8, wherein the Ubx protein comprises the sequence of SEQ ID NO: 20 or a fragment thereof, and the immunoglobulin-binding protein comprises the sequence of SEQ ID NO: 1 or a fragment thereof. (Item 10) A solid-phase matrix comprising the fusion protein according to any one of Items 1 to 9. (Item 11) The matrix according to Item 9, wherein leakage of the immunoglobulin-binding protein from the solid-phase matrix is prevented or reduced. (Item 12) The matrix according to item 9 or item 10, wherein the binding capacity of 1 mL of the substance exceeds 1 g / mL. (Item 13) A method for isolating an immunoglobulin from an immunoglobulin-containing solution, the method comprising: a) contacting the immunoglobulin-containing solution with a solid-phase matrix according to any one of items 10 to 12 under conditions such that the immunoglobulin contained in the solution adsorbs to the matrix; b) washing the adsorbed immunoglobulin; and c) eluting the immunoglobulin from the matrix. The method as described above.

Claims

1. A fusion protein comprising at least one Ubx protein and an immunoglobulin-binding protein, wherein the Ubx protein is a full-length Ubx protein comprising SEQ ID NO: 20, and wherein the immunoglobulin-binding protein comprises one or more monomeric domains of domain Z, and wherein each domain comprises a polypeptide having at least 90% identity to SEQ ID NO:

1.

2. The fusion protein according to claim 1, wherein the immunoglobulin-binding protein comprises two or more monomeric domains of domain Z, and each domain comprises a polypeptide having at least 90% identity to SEQ ID NO:

1.

3. The fusion protein according to any one of claims 1 or 2, wherein the Ubx protein comprises the sequence of SEQ ID NO: 20, and each monomeric domain of domain Z comprises the sequence of SEQ ID NO:

1.

4. A solid-phase matrix comprising the fusion protein according to any one of claims 1 to 3.

5. The matrix according to claim 4, wherein leakage of the immunoglobulin-binding protein from the solid-phase matrix is prevented or reduced.

6. The matrix according to claim 4 or 5, wherein the binding capacity for 1 mL of the substance exceeds 1 g / mL.

7. A method for isolating an immunoglobulin from an immunoglobulin-containing solution, the method comprising: a) contacting the immunoglobulin-containing solution with the solid-phase matrix according to any one of claims 4 to 6 under conditions such that the immunoglobulin contained in the solution adsorbs to the matrix; b) washing the adsorbed immunoglobulin; and c) eluting the immunoglobulin from the matrix. The method comprising the above steps.

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