Ligands for Corrosion Stability Chromatography

Abstract

The present invention relates to chromatographic ligands having improved corrosion stability, such as ligands based on immunoglobulin binding proteins, such as Staphylococcus protein A, and methods of making and using the same.

Description

[0001] This application is a divisional application of Chinese patent application No. 201810776217.X, filed on December 23, 2009, entitled “Ligand for Corrosion Stability Chromatographic Analysis”.

[0002] Related applications

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 61 / 203,664, filed December 24, 2008, which is hereby incorporated by reference in its entirety into this application. Technical Field

[0004] The present invention relates to chromatographic ligands having improved corrosion stability, for example, ligands based on immunoglobulin binding proteins (eg, Staphylococcal protein A), and chromatographic matrices comprising such ligands. Background Art

[0005] The ligands used in affinity chromatography typically confer high selectivity for the target molecule, resulting in high yields and rapid, economical purification. Reagents and chromatography matrices based on Staphylococcus protein A (SpA) have achieved widespread application in affinity chromatography for the capture and purification of antibodies and are also widely used in antibody detection methods due to their ability to bind IgG without significantly affecting the affinity of immunoglobulins for antibodies.

[0006] Accordingly, a variety of reagents and media containing protein A ligands have been developed and are commercially available, including, for example, -vA High Capacity, vA Ultra and UltraPlus (Millipore) and Protein A Sepharose TM , MabSelect TM , MabSelect Xtra TM and MabSelect (GE Healthcare) and Poros MabCapture A TM (Applied Biosystems).

[0007] In order to maintain the selectivity and binding capacity of the chromatographic ligand (including, for example, a resin comprising an SpA-based chromatographic ligand), the ligand-bound resin (called the chromatography matrix) needs to be cleaned, and is typically cleaned under alkaline conditions, such as with sodium hydroxide. For example, the standard procedure for cleaning and restoring the matrix is ​​an alkaline cleaning-in-place (CIP) protocol, which generally involves treating the matrix with 1 M NaOH at pH 14. However, this less-than-gentle treatment is often undesirable, particularly when the ligand is a protein or protein-based molecule. Summary of the Invention

[0008] The present invention provides alkaline-stable SpA-based chromatographic ligands, for example, capable of withstanding repeated cleaning-in-place (CIP) cycles. More particularly, the ligands of the present invention can withstand traditional alkaline cleaning for extended periods of time, making them attractive candidates, particularly for cost-effective, high-level purification of immunoglobulins.

[0009] In one aspect of the invention, the alkali-stable chromatographic ligand comprises two or more SpA domains. For example, in some embodiments of this aspect, an alkali-stable chromatographic ligand is provided, wherein the ligand comprises two or more B domains of Staphylococcus protein A (SpA) or a functional fragment or variant thereof, or two or more Z domains of SpA or a functional fragment or variant thereof, wherein the two or more B domains or the two or more Z domains are attached to a chromatographic resin, and the attachment sites are more than one site on the resin.

[0010] In some embodiments based on this aspect, the ligand comprises three or more B domains of SpA or a functional fragment or variant thereof or three or more Z domains of SpA or a functional fragment or variant thereof, and the three or more B domains or three or more Z domains are attached to a chromatography resin at more than one site on the resin. In other embodiments, the ligand comprises four or more SpA B domains or four or more SpA Z domains, and the four or more B domains or four or more Z domains are attached to a chromatography resin at more than one site on the resin. In other embodiments, the ligand comprises five or more SpA B domains or five or more SpA Z domains; or comprises six or more SpA B domains or six or more SpA Z domains; or comprises seven or more SpA B domains or seven or more SpA Z domains, and the five or more B domains or five or more Z domains, six or more B domains or six or more Z domains, seven or more B domains or seven or more Z domains are attached to a chromatography resin at more than one site on the resin.

[0011] According to another aspect of the present invention, the alkali-stable chromatographic ligand comprises one or more isolated Staphylococcus aureus protein A E, D, A, B, C or Z domains, wherein the one or more isolated domains comprise one or more amino acid residues at position n+1 that are mutated to any naturally occurring amino acid except cysteine ​​(C), serine (S), alanine (A), glycine (G), asparagine (N), or glutamine (Q). In some embodiments, n represents the asparagine residue at position 23 of the isolated SpA domain. Exemplary ligands having a mutation at position 24 of the isolated SpA domain are shown in Table 1, wherein n represents asparagine.

[0012] Table 1

[0013]

[0014]

[0015]

[0016]

[0017] The one-letter codes for the natural amino acids and their corresponding three-letter codons encoding each amino acid are listed in Table 2. In general, due to the degeneracy of the codons, more than one three-letter codon can encode the same amino acid.

[0018] Table 2

[0019]

[0020] The present invention also includes a chromatography matrix comprising a ligand according to one or more aspects of the present invention coupled to a solid support, such as at least one insoluble carrier.

[0021] Additionally, methods for using such ligands are provided herein. Thus, provided are affinity purification methods for separating one or more target molecules (e.g., immunoglobulins) from a sample, the methods comprising the steps of: (a) providing a sample comprising one or more target molecules (e.g., immunoglobulins); (b) contacting the sample with a matrix according to the present invention under conditions such that the one or more target molecules (e.g., immunoglobulins) bind to the matrix; and (c) recovering the one or more bound target molecules (e.g., immunoglobulins) by elution under suitable conditions, e.g., a suitable pH.

[0022] In some embodiments, the base-stable chromatography ligands of the invention retain at least 95% of their binding capacity after incubation in 0.5 M NaOH for 5 hours, or 10 hours, or 15 hours, or 25 hours, or 30 hours.

[0023] The immunoglobulins described herein that can be bound by multiple ligands include, for example, IgG, IgA and IgM, or any fusion protein comprising an antibody or any fragment of an antibody.

[0024] Also provided herein are nucleic acid molecules encoding a variety of said parts, and host cells comprising these nucleic acid molecules. In some embodiments, the host cell is a prokaryotic cell. In other embodiments, the host cell is a eukaryotic cell.

[0025] In addition, the present invention also includes polypeptide libraries comprising one or more ligands described herein and functional fragments and variants thereof.In another embodiment, the present invention provides a library of nucleic acid molecules encoding one or more ligands of the present invention or encoding functional fragments and variants thereof.

[0026] In some embodiments, the present invention provides SpA-based ligands that exhibit a modified (increased or decreased) ability to bind to the Fab fragment of an immunoglobulin compared to known SpA ligands, but maintain the ability to bind to the Fc fragment of an immunoglobulin. In one embodiment, a SpA-based ligand according to the present invention exhibits a decreased ability to bind to the Fab fragment of an immunoglobulin compared to wild-type SpA. In an exemplary embodiment, the alkaline-stable chromatographic analysis ligand according to the present invention further comprises a substitution of glycine at position 29 with alanine. In another embodiment, the alkaline-stable chromatographic analysis ligand according to the present invention further comprises a substitution of glycine at position 29 with an amino acid other than alanine or tryptophan. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The nucleic acid sequences of the wild-type (wt) SpA IgG binding domains are described as shown in SEQ ID NOs: 1 to 5. SEQ ID NO: 1 represents the nucleic acid sequence of the wt E domain; SEQ ID NO: 2 represents the nucleic acid sequence of the wt D domain; SEQ ID NO: 3 represents the nucleic acid sequence of the wt A domain; SEQ ID NO: 4 represents the nucleic acid sequence of the wt B domain; and SEQ ID NO: 5 represents the nucleic acid sequence of the wt C domain.

[0028] Figure 2 The nucleic acid sequence of the SpA Z domain is described and is shown as SEQ ID NO:6.

[0029] Figure 3 An amino acid sequence alignment of the wild-type (wt) IgG binding domains (E, D, A, B, and C) of SpA is depicted. SEQ ID NO: 7 represents the amino acid sequence of the wt E domain; SEQ ID NO: 8 represents the amino acid sequence of the wt D domain; SEQ ID NO: 9 represents the amino acid sequence of the wt A domain; SEQ ID NO: 10 represents the amino acid sequence of the wt B domain; and SEQ ID NO: 11 represents the amino acid sequence of the wt C domain.

[0030] Figure 4 The amino acid sequence of the SpA Z domain is described and is shown in SEQ ID NO:12.

[0031] Figure 5 A schematic diagram of plasmid pJ56:8620 is depicted.

[0032] Figure 6The amino acid sequence of the wt B domain of histidine-tagged SpA and the amino acid sequences of various n+1 mutants of the histidine-tagged SpA are described. SEQ ID NO: 13 represents the amino acid sequence of the wt B domain of histidine-tagged SpA, and SEQ ID NOs: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, and 26 represent the amino acid sequences of the n+1 B domain mutants E24M, E24I, E24F, E24T, E24P, E24W, E24R, E24V, E24L, E24Y, E24H, E24K, and E24D, respectively.

[0033] Figure 7 The nucleotide sequence of the wt B domain of histidine-tagged SpA and the nucleotide sequences encoding various n+1 mutants of the histidine tag are described. SEQ ID NO: 27 represents the nucleotide sequence encoding the wt B domain of histidine-tagged SpA, and SEQ ID NOs: 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40 represent the nucleotide sequences encoding the histidine-tagged n+1 B domain mutants E24M, E24I, E24F, E24T, E24P, E24W, E24R, E24V, E24L, E24Y, E24H, E24K, and E24D, respectively.

[0034] Figure 8 A bar graph summarizes the results of an exemplary experiment measuring the residual IgG binding of various histidine-tagged n+1 B domain mutants, where n represents asparagine at position 23, using the high-throughput ELISA-based assay described herein. The x-axis represents various n+1 B domain mutants, each with a mutation at position 24, and the y-axis represents the percentage of IgG binding retained after 6 hours of exposure to 1N NaOH. As shown in the graph, n+1 B domain mutants with an amino acid containing a bulky side chain at position 24 exhibited improved corrosion stability compared to the wt B domain.

[0035] Figure 9 Depicts a bar graph summarizing the results of an example experiment measuring the percentage of IgG binding capacity retained after multipoint attachment of various SpA domain trimers (i.e., EEE, DDD, AAA, BBB, CCC, and ZZZ) to agarose resin. nPrA refers to wt SpA. The X-axis represents the number of cycles of corrosion exposure, where each cycle consists of a 15-minute exposure to 0.5 N NaOH. The Y-axis represents the IgG binding capacity retained by the bound SpA ligand. Figure 9As shown in , ligands containing C or Z domain trimers have roughly equivalent abilities in terms of corrosion stability, and have higher corrosion stability than ligands containing B domain trimers, which in turn have higher corrosion stability than ligands containing A domain trimers, which in turn have higher corrosion stability than ligands containing E or D domain trimers.

[0036] Figure 10 Described are the results of an example experiment measuring the percentage of IgG binding capacity retained by an attachment ligand of the invention comprising 3 (B3), or 4 (B4), or 5 (B5) B domains attached to multiple sites on an agarose resin. Also used are attachment ligands comprising 5 B domains or 7 B domains and additionally comprising a mutation (G29A) that reduces binding to Fab, referred to as B5-NF and B7-NF, respectively. nPrA refers to wt SpA. The X-axis represents the number of cycles of corrosion exposure, where each cycle consists of a 15 minute exposure to 0.5N NaOH. The Y-axis represents the retained IgG binding capacity of the attachment SpA ligand. As Figure 10 As shown in , the level or extent of corrosion stability is directly proportional to the number of B domains in the ligand and is not altered by the G29A mutant which reduces binding to the Fab. Detailed Description of the Invention

[0037] The present invention provides alkaline-stable chromatographic ligands based on SpA, in particular, ligands based on one or more SpA domains. Exemplary alkaline-stable chromatographic ligands based on SpA are described in the prior art, including, for example, International PCT Patent Application No. WO2008 / 039141, which discusses alkaline-stable chromatographic ligands based on the SpA C domain that bind to the Fab portion of an antibody and are associated with an insoluble support at a single site using a terminal binding group; and U.S. Patent No. 6,831,161, which discusses alkaline-based chromatographic ligands based on SpA in which one or more aspartic acid residues have been modified.

[0038] To make the present disclosure more understandable, certain terms are first defined. Additional definitions are given in the detailed description.

[0039] I. Definition

[0040] As used herein, the term "SpA" or "Staphylococcal protein A" refers to a 42 kDa multidomain protein isolated from Staphylococcus aureus. SpA binds to the bacterial cell wall via a carboxy-terminal cell wall binding region, referred to as the X domain. In the amino-terminal region, it contains five immunoglobulin binding domains, designated E, D, A, B, and C (Sjodhal, Eur J Biochem. Sep 78(2):471-90 (1977); Uhlen et al., J Biol Chem. Feb 259(3):1695-702 (1984). Each of these domains contains approximately 58 amino acid residues, and they share 65-90% amino acid sequence identity. The SpA Z domain is an engineered analog of the SpA B domain that contains an alanine instead of a glycine at position 29 (Nilsson, et al., Protein engineering, Vol. 1, No. 2, 107-113, 1987.). SpA Each of the E, D, A, B, and C domains possesses a distinct Ig-binding site. One site binds to Fcγ (the constant region of the IgG class of Ig), while another binds to the Fab portion of a specific Ig molecule (the portion of Ig responsible for antigen recognition). It has been reported that each domain contains a single Fab-binding site. The non-Ig-binding region of SpA is located at the C-terminus and is designated the X region or X domain.

[0041] The cloning of the gene encoding SpA is described in US Patent No. 5,151,350, the entire contents of which are hereby incorporated by reference herein.

[0042] The present invention provides alkaline-stable chromatographic ligands based on SpA. According to some aspects of the present invention, an alkaline-stable ligand comprises two or more, or three or more, or four or more, or five or more, or six or more, or seven or more isolated SpA wt B or Z domains. In other aspects of the present invention, the alkaline-stable chromatographic ligand comprises one or more isolated SpA E, D, A, B, C, or Z domains, wherein the one or more isolated domains comprise one or more amino acid residues mutated at position n+1 to a residue selected from the group consisting of tryptophan, arginine, threonine, isoleucine, valine, and methionine, wherein n represents asparagine.

[0043] In one specific embodiment, the present invention provides a chromatography ligand comprising two or more SpA B domains attached to a chromatography resin at multiple sites on the resin. In another embodiment, the present invention provides a chromatography ligand comprising two or more SpA Z domains attached to a chromatography resin at multiple sites on the resin. In another embodiment, the present invention provides a chromatography ligand comprising two or more SpA C domains attached to a chromatography resin at multiple sites on the resin.

[0044] The present invention also encompasses amino acid variants of SpA that differ from the parent amino acid sequence from which it is derived by substitution, deletion, and / or addition of one or more amino acids at any position in the parent amino acid sequence, and that exhibit alkaline stability. In some embodiments, the amino acid sequence variants share at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98% sequence identity with the parent sequence (i.e., wt SpA domain or Z domain), and the variants exhibit alkaline stability. In a specific embodiment, the SpA variant further comprises a substitution of the glycine residue at position 29 with an amino acid residue other than alanine or tryptophan, while retaining alkaline stability.

[0045] The term "functional variant" of a protein as used herein refers to a mutant protein whose function, as defined herein, as alkaline stability, is substantially retained. Functional variants include, but are not limited to, SpA variants comprising multiple SpA domains, such as dimers, trimers, and multimers of various SpA domains, and SpA variants comprising one or more deletions, substitutions, and / or additions of one or more amino acids to one or more wild-type SpA domains while retaining alkaline stability as defined herein.

[0046] The term "parent molecule" as used herein refers to the corresponding protein form before modification according to the modification method of the present invention or the corresponding protein form before introduction of the mutation of the present invention.

[0047] The term "sequence identity" refers to two nucleotide or amino acid sequences that, when properly aligned, such as using the GAP or BESTFIT programs with default gap weights, share at least 70% sequence identity, or at least 80% sequence identity, or at least 85% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity or higher. For sequence alignment, typically one sequence serves as a reference sequence (e.g., parent sequence) to which a test sequence is compared. When using a sequence alignment algorithm, the test sequence and the reference sequence are input into a computer, and if necessary, subsequent coordinates are set, and then the parameters of the sequence algorithm program are determined. The sequence alignment algorithm then calculates the percentage of sequence identity between the test sequence and the reference sequence based on the set program parameters.

[0048] For alignment, optimal alignment of sequences can be performed, for example, using the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), using the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), using the finding similarity method of Pearson and Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), using computerized instruments of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or using direct research results (see generally, Ausubel et al., Current Protocols in Molecular Biology). An example of an algorithm suitable for determining the percentage of sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215: 403 (1990). Software for running BLAST analysis is publicly available through the National Center for Biotechnology Information (NCBI, National Center for Health Internet Server). Typically, sequence alignments can be run using default program parameters, although customized parameters can also be used. For amino acid sequences, the default parameters of the BLASTP program are a word length (W) of 3, an expected value (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)).

[0049] The terms "E domain," "SpA E domain," and "Staphylococcal protein A E domain," used interchangeably herein, refer to a polypeptide having the amino acid sequence set forth in SEQ ID NO:7, or a polypeptide encoded by, for example, the nucleic acid sequence set forth in SEQ ID NO:1. The "E domain" is a 51-amino acid polypeptide that folds into a three-helix bundle. It can bind to Fc through residues on the surfaces of helices 1 and 2, or to Fab through residues on the surfaces of helices 2 and 3. In some embodiments, an E domain according to the present invention has at least 70%, at least 80%, at least 90%, or at least 95% or greater sequence identity with the amino acid sequence set forth in SEQ ID NO:7.

[0050] The terms "D domain", "SpA D domain", and "Staphylococcal protein A D domain", which are used interchangeably herein, refer to a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 8, or a polypeptide encoded by a nucleic acid sequence as set forth in, for example, SEQ ID NO: 2. The "D domain" is a 61 amino acid polypeptide that folds into a three-helix bundle structure. It can bind to Fc through residues on the surface of helices 1 and 2, or to Fab through residues on the surface of helices 2 and 3. In some embodiments, the D domain according to the present invention has a sequence identity of at least 70%, or at least 80%, or at least 90%, or at least 95% or more to the amino acid sequence as set forth in SEQ ID NO: 8.

[0051] The terms "A domain," "SpA A domain," and "Staphylococcal protein A A domain," used interchangeably herein, refer to a polypeptide having the amino acid sequence set forth in SEQ ID NO:3, or a polypeptide encoded by, for example, the nucleic acid sequence set forth in SEQ ID NO:9. The "A domain" is a 58-amino acid polypeptide that folds into a three-helix bundle. It can bind to Fc via residues on the surfaces of helices 1 and 2, or to Fab via residues on the surfaces of helices 2 and 3. In some embodiments, an A domain according to the present invention has at least 70%, at least 80%, at least 90%, or at least 95% or greater sequence identity with the amino acid sequence set forth in SEQ ID NO:3.

[0052] The terms "B domain," "SpA B domain," and "Staphylococcal protein A B domain," used interchangeably herein, refer to a polypeptide having the amino acid sequence set forth in SEQ ID NO:10, or a polypeptide encoded by the nucleic acid sequence set forth in, for example, SEQ ID NO:4. The "B domain" is a 58-amino acid polypeptide that folds into a three-helix bundle. It can bind to Fc through residues on the surfaces of helices 1 and 2, or to Fab through residues on the surfaces of helices 2 and 3. In some embodiments, a B domain according to the present invention has at least 70%, at least 80%, at least 90%, or at least 95% or greater sequence identity with the amino acid sequence set forth in SEQ ID NO:10.

[0053] The terms "C domain," "SpA C domain," and "Staphylococcal protein A C domain," used interchangeably herein, refer to a polypeptide having the amino acid sequence set forth in SEQ ID NO:11, or a polypeptide encoded by, for example, the nucleic acid sequence set forth in SEQ ID NO:5. The "C domain" is a 58-amino acid polypeptide that folds into a three-helix bundle. It can bind to Fc through residues on the surfaces of helices 1 and 2, or to Fab through residues on the surfaces of helices 2 and 3. In some embodiments, a C domain according to the present invention has at least 70%, at least 80%, at least 90%, or at least 95% or greater sequence identity with the amino acid sequence set forth in SEQ ID NO:11.

[0054] The terms "Z domain," "SpA Z domain," and "Staphylococcal protein A Z domain," used interchangeably herein, refer to a triple-helical, 59-amino acid polypeptide that is a variant of the B domain of protein A. The amino acid sequence of the Z domain is set forth in SEQ ID NO: 12. Exemplary Z domains are described in Nilsson et al., Protein Engng., 1: 107-113 (1997), the entire contents of which are incorporated herein by reference.

[0055] As used herein, the terms "alkali stable," "alkaline stability," "corrosion stable," or "corrosion stability" generally refer to the ability of a chromatography ligand according to the present invention, either alone or immobilized on a chromatography resin, to withstand repeated cycles of cleaning in place (CIP) using alkaline cleaning without losing its binding capacity. Generally, it is contemplated that a resin, upon immobilization of itself with a ligand of the present invention, exhibits less than a 5% change in stability after soaking in 0.5 M NaOH for up to 30 hours. For example, in some embodiments, chromatography ligands according to the present invention can withstand typical alkaline cleaning for extended periods of time, making the ligands attractive candidates, particularly for cost-effective, large-scale purification of immunoglobulins. In some embodiments, ligands according to the present invention exhibit enhanced chemical stability in alkaline environments, which can be defined, for example, as having an elevated pH, such as above about 10, or up to about 13 or 14. Alternatively, the alkaline environment can be defined by the concentration of the base, such as about 1.0 M NaOH, or about 0.7 M NaOH, or about 0.5 M NaOH. In one embodiment, alkaline stability refers to the ability of a ligand for alkaline stable chromatography according to the present invention to retain at least 80%, at least 85%, at least 90%, or at least 95% of its binding capacity after incubation in 0.5 M NaOH for 5 hours, or 10 hours, or 15 hours, or 20 hours, or 25 hours, or 30 hours. In another embodiment, alkaline stability refers to a decrease in the binding capacity of the ligand by less than 70%, or less than 60%, or less than 50%, or less than 30% after treatment with 0.5 M NaOH for 5 hours, or 7.5 hours, or 10 hours, or 15 hours, or 20 hours, or 25 hours, or 30 hours.

[0056] In some embodiments, the SpA-based chromatography ligands according to the present invention exhibit improved or enhanced alkaline stability compared to wild-type SpA.

[0057] Alkali stability can be readily determined by one of ordinary skill in the art using routine experimentation and / or the methods described herein.

[0058] The term "chromatography," as used herein, refers to a dynamic process that separates an analyte of interest (e.g., an immunoglobulin) from other molecules in a mixture and allows for its isolation. Generally, in chromatography, a mobile phase (liquid or gas) transports a sample containing the analyte of interest through or across a stationary phase (usually a solid) medium. As the mobile phase carries different analytes, differences in separation and affinity for the stationary phase result in different analytes being isolated at different times.

[0059] As used herein, the term "affinity chromatography" refers to a form of chromatography in which an analyte is separated by interaction with a molecule that specifically interacts with the analyte (e.g., an alkaline-stable chromatography ligand). In one embodiment, affinity chromatography involves adding a sample containing the target analyte (e.g., an immunoglobulin) to a solid support containing an alkaline-stable chromatography ligand, as described herein.

[0060] As used herein, the term "Protein A affinity chromatography" refers to the separation or isolation of Protein A or SpA ligand from a substance, where the SpA or Protein A ligand is immobilized, e.g., on a solid support, as described herein. Examples of media / resins for Protein A affinity chromatography known in the art include those that immobilize Protein A to a controlled microporous glass substrate, e.g., PROSEP A. TM and PROSEP vA TM Resins / resins (microporous); those that immobilize Protein A to a polystyrene solid phase, e.g., PIROS 50A TM and Poros MabCapture A TM media / resins (Applied Biosystems, Inc.); and those that immobilize Protein A to an agarose solid support, e.g., rPROTEIN ASEPHAROSE FAST FLOW TM or MABSELECT TM On-column (Amersham Biosciences).

[0061] The terms "immunoglobulin," "Ig," or "antibody" (used interchangeably herein) refer to a protein comprising a basic four-polypeptide chain structure, including two heavy chains and two light chains, which are stabilized, for example, by interchain disulfide bonds, and which have the ability to specifically bind to an antigen. The terms "single-chain immunoglobulin" or "single-chain antibody" (used interchangeably herein) refer to a protein comprising a two-polypeptide chain structure, including one heavy chain and one light chain, which are stabilized, for example, by an interchain peptide linker, and which have the ability to specifically bind to an antigen. The term "domain" refers to a globular region of a heavy or light chain polypeptide that includes stabilized peptide loops (e.g., comprising 3 to 4 peptide loops), for example, in the form of beta-pleated sheets and / or interchain disulfide bonds. Domains are further referred to herein as "constant" or "variable" based on the lack of mutations in the corresponding sequences among the various domain members of the "constant" domains and the significant mutations in the various domain members of the "variable" domains. Antibody or polypeptide "domains" are often referred to interchangeably in the art as antibody or polypeptide "regions." The "constant" domain of an antibody light chain is referred to interchangeably as a "light chain constant region," "light chain constant domain," "CL" region, or "CL domain." The "constant" domain of an antibody heavy chain is referred to interchangeably as a "heavy chain constant region," "heavy chain constant domain," "CH" region, or "CH domain." The "variable" domain of an antibody light chain is referred to interchangeably as a "light chain variable region," "light chain variable domain," "VL" region, or "VL domain." The "variable" domain of an antibody heavy chain is referred to interchangeably as a "heavy chain variable region," "heavy chain variable domain," "VH" region, or "VH domain."

[0062] Immunoglobulins or antibodies may be monoclonal or polyclonal and may exist in monomeric or multimeric form. For example, IgM antibodies exist in pentameric form and / or IgA antibodies exist in monomeric, dimeric or multimeric form. The term "fragment" refers to a portion or region of an antibody or antibody chain that contains fewer amino acid residues than a complete or complete antibody or antibody chain. Fragments can be obtained by treating a complete or complete antibody or antibody chain with a chemical or enzyme. Fragments can also be obtained by recombinant methods. Examples of fragments include Fab, Fab', F(ab')2, Fc and / or Fv fragments.

[0063] The term "antibody binding fragment" refers to a polypeptide portion of an immunoglobulin or antibody that binds to an antigen or competes with an intact antibody (i.e., the intact antibody from which it was derived) for antigen binding (i.e., specifically binds). Binding fragments can be prepared by recombinant DNA technology, or by enzymatic or chemical cleavage of intact immunoglobulins. Binding fragments include Fab, Fab', F(ab')2, Fv, single chain, and single-chain antibodies.

[0064] The present invention also discloses fusion proteins comprising an antibody or fragment thereof as part of the fusion protein.

[0065] The terms "polynucleotide" and "nucleic acid molecule" are used interchangeably herein to refer to polymeric forms of ribonucleic or deoxyribonucleic acids of any length. These terms include single-, double-, or triple-stranded DNA, genomic DNA, cDNA, RNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases, or other natural, chemically or biochemically modified, unnatural, or derivatized nucleotide bases. The backbone of a polynucleotide may include sugar and phosphate groups (as commonly found in RNA or DNA), or modified or substituted sugar or phosphate groups. In addition, a double-stranded polynucleotide may be synthesized from a single-stranded polynucleotide product by chemical synthesis or annealing under appropriate conditions, or by de novo synthesis of the complementary strand using a DNA polymerase using appropriate primers. Nucleic acid molecules can take a variety of forms, such as a gene or gene fragment, one or more exons, one or more introns, mRNA, cDNA, a recombinant polynucleotide, a branched polynucleotide, a plasmid, a vector, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may include modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracyl groups, other sugars, linking groups such as fluororibose and thioate, and nucleotide branches. As used herein, "DNA" or "nucleotide sequence" includes, but is not limited to, bases on A, T, C, and G, and also includes any analogs or modified forms of these bases, such as methylated nucleotides, intranucleotide modifications such as uncharged linkages and thioate, the use of sugar analogs, and modifications and / or optional backbone structures, such as polyamides. In a specific embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding an SpA variant.

[0066] The terms "Fc binding", "binding to the Fc portion" or "binding to the Fc portion" refer to the ability of the alkali-stable chromatographic molecule ligands described herein to bind to the crystalline portion (Fc). In some embodiments, the ligands of the present invention bind to at least 10 －7 M, or at least 10 －8 M, or at least 10 －9M binds with affinity to the Fc portion of an antibody (eg, human IgG1, IgG2, or IgG4).

[0067] As used herein, the term "binding to Fab" or "binding to the Fab portion" refers to the ability of the alkaline stable chromatographic analysis ligand described herein to bind to the Fab portion of an antibody or immunoglobulin molecule. The term "reduced binding to the Fab portion" refers to any reduction in the ability of the SpA-based ligand of the present invention to bind to the Fab (or F(ab)2) portion of an immunoglobulin compared to wild-type SpA, the ligand further comprising a mutation in one or more amino acids. In an exemplary embodiment, the ligand based on the present invention further comprises a substitution of glycine at position 29 with alanine. In another embodiment, the ligand based on the present invention further comprises a substitution of glycine at position 29 with an amino acid other than alanine and tryptophan. In one embodiment, binding to the Fab portion of an immunoglobulin molecule cannot be detected using conventional methods in the art and the methods described herein. Binding to immunoglobulin molecules can be detected using various well-known techniques including those described herein, including but not limited to, for example, affinity chromatography and surface plasmon analysis. In some embodiments, the immunoglobulin binding protein disclosed herein binds to at least 10 －10 M binds to immunoglobulin molecules with affinity.

[0068] II. Generation of SpA-based molecules for use as ligands for chromatography

[0069] The SpA-based chromatographic ligand disclosed in the present invention can be prepared using any suitable method known in the art.

[0070] For example, as an initial step, standard genetic engineering techniques, such as those described in the laboratory manual, Molecular Cloning by Sambrook, Fritsch and Maniatis, can be used to prepare nucleic acids expressing the SpA ligand molecules described herein.

[0071] In some embodiments, nucleic acid molecules encoding one or more SpA domains or portions thereof can be cloned into a suitable vector for expression in a suitable host cell. Suitable expression vectors are well known in the art and generally include the necessary elements for transcription and translation of various SpA coding sequences.

[0072] The SpA molecules described herein can also be chemically synthesized to form fragments from amino acid precursors using methods well known in the art, including solid-phase peptide synthesis methods such as the Boc (tert-butyl-butoxycarbazate) or Fmoc (9-fluorenylmethoxycarbonyl) methods (see, e.g., U.S. Patent Nos. 6,060,596; 4,879,378; 5,198,531; 5,240,680).

[0073] Expression of the SpA molecules described herein can be accomplished in cells derived from eukaryotic hosts, such as yeast, insects, or mammals, or in prokaryotic host cells, such as bacteria such as E. coli.

[0074] In some embodiments, SpA molecules or fragments and variants thereof can be expressed on the surface of phage, such that each phage contains a DNA sequence encoding a separate SpA molecule displayed on its surface. The affinity of SpA molecules for immunoglobulins can be readily analyzed using standard techniques in the art, as well as methods described herein, such as ELISA and Biacore. TM 2000 standard apparatus (Biacore AB, Uppsala Sweden). It is expected that the SpA of the present invention will have a binding affinity for immunoglobulins that is at least comparable to that of its parent molecule. Furthermore, it is expected that the alkaline stability of the SpA molecule will be generally improved compared to that of its parent molecule.

[0075] III. Analysis of Alkaline Stability of SpA Molecules

[0076] After constructing and purifying a suitable SpA ligand molecule as described herein, the alkaline stability of the molecule can be tested using standard methods in the art or the methods described herein. For example, the alkaline stability of the SpA molecule according to the present invention can be analyzed by conventional treatment with 0.5M NaOH, e.g., as described in the experimental section below.

[0077] In some embodiments, the alkaline-stable SpA molecule exhibits improved or enhanced alkaline stability, meaning that the molecule is stable after being subjected to alkaline conditions for a longer period of time compared to wild-type SpA. Previously, it has been reported that SpA molecules based on the wild-type SpA C domain or SpA molecules comprising one or more mutations of asparagine residues provide enhanced chemical stability and, thereby, reduced degradation rates in environments wherein the pH is greater than about 10, such as up to about 13 or 14.

[0078] The present invention is based on the surprising and unexpected discovery of novel SpA molecules that exhibit improved alkaline stability, even when they are based on domains other than the C domain or contain mutations other than asparagine. For example, the present invention provides alkaline-stable SpA molecules based on the B or Z domains, as well as SpA molecules containing a mutation at amino acid position n+1, where n represents asparagine (e.g., asparagine at position 23).

[0079] In some embodiments, after constructing the SpA ligands according to the present invention, the alkaline stability of the ligand is evaluated using a novel high-throughput immunological assay, as described in detail in the Examples below. The assay is based on the hypothesis that degradation of SpA under prolonged corrosion exposure conditions can be reflected by loss or reduction of IgG binding. Briefly, soluble SpA-based ligands are treated with water or 1.0 M NaOH for approximately 6 hours. A hydrophobic reaction is used to attach microgram quantities of neutralized candidate ligands to a solid support in the form of an ELISA plate, such as a 96-well plate. Each candidate ligand is then evaluated for IgG binding before and after exposure to 1.0 M NaOH. When the amount of IgG remaining bound to the ligand after exposure to corrosion exceeds that of wild-type SpA or the parent SpA from which it is derived, corrosion stability is enhanced.

[0080] IV. Supports for Preparation of Chromatographic Matrix

[0081] In some embodiments, the alkali-stable SpA ligand disclosed herein is attached to a support, such as a solid support or a soluble support, to construct a chromatography matrix suitable for separating biomolecules, such as immunoglobulins.

[0082] In some embodiments, the ligands of the present invention are attached to a solid support. Without wishing to be bound by theory, any suitable solid support can be used to attach the ligands of the present invention. For example, solid support matrices include, but are not limited to, microporous glass, silica, zirconium oxide, agarose, polymethacrylate, polyacrylate, polyacrylamide, and polystyrene of controlled size.

[0083] It is expected that any porous material that causes less than 5% change in the alkaline stability of attached ligands after soaking in 0.5 M NaOH for about 30 hours can be used as a solid support.

[0084] The porous material used as a solid support may include a hydrophilic compound, a hydrophobic compound, an oleophobic compound, an oleophilic compound, or a combination thereof. The porous material may include a polymer or a copolymer. Examples of suitable porous materials include, but are not limited to, polyethersulfone, polyamides such as nylon, polysaccharides such as agarose and cellulose, polyacrylates, polymethacrylates, polyacrylamides, polymethacrylamides, polytetrafluoroethylene, polysulfone, polyvinylidene fluoride, polypropylene, polyethylene, polycarbonate, fluorocarbons such as poly(tetrafluoroethylene-co-fluoro(alkyl vinyl ether)), glass, silica, zirconia, titanium dioxide, ceramics, and metals.

[0085] The porous material may comprise an organic or inorganic molecule or a combination of organic and inorganic molecules, and may further comprise one or more functional groups, such as hydroxyl, sulfhydryl, amino, carbonyl, or carboxyl groups, suitable for reaction, such as forming covalent bonds for further chemical modification, wherein the purpose of the chemical modification is to add covalent bonds to the protein. In another embodiment, the porous material may not comprise functional groups, but may be coated with a sheet of material that produces functional groups, such as hydroxyl, sulfhydryl, amino, carbonyl, or carboxyl groups.

[0086] In some embodiments, conventional affinity chromatography separation matrices are used, which are, for example, organic and based on polymers that expose a hydrophilic surface to the aqueous medium, i.e., the polymers expose hydroxyl (-OH), carboxyl (-COOH), carbonyl (-CHO, or PCO-R'), carboxyamino (-CONH2, possibly N-substituted), amino (-NH2, possibly substituted), oligo- or polyethylene glycol groups located on their exterior and, if present, on their interior surfaces. In one embodiment, the polymer may, for example, be based on a polysaccharide such as dextran, starch, cellulose, pullulan, agarose, etc., which is preferably cross-linked, for example, using diepoxides, epihalohydrins, or 1,2,3-trihalogenated lower hydrocarbons, to provide suitable porosity and rigidity. In another embodiment, the solid support comprises porous agarose beads. The various supports used in the present invention can be readily prepared by standard methods known in the art, for example, reverse suspension gelation, which is described in, for example, Hjerten, Biochim Biophys Acta 79 (2), 393-398 (1964). Alternatively, the base matrix can be a commercially available product, such as Sepharose. TM FastFlow (GE Healthcare, Uppsala, Sweden) In some embodiments, particularly preferred for large-scale separations, the support is adapted to increase its rigidity, thereby conferring properties on the matrix that are more suitable for high flow rates.

[0087] Alternatively, the solid support may be based on a synthetic polymer, for example, polyvinyl alcohol, polyhydroxyalkyl acrylates, polyhydroxy methacrylates, polyacrylamide, polymethacrylamide, etc. In the case of hydrophobic polymers, such as those based on divinyl and monovinyl substituted benzenes, the surface of the substrate is often hydrophilized so as to expose the hydrophilic groups defined above to the surrounding aqueous liquid. Such polymers can be readily produced according to standard methods, see, for example, Arshady, Chimica e L'Industria 70(9), 70-75 (1988). Alternatively, commercially available products, such as Source TM(GE Healthcare, Uppsala, Sweden) and Poros (Applied BioSystems, Foster City, CA) may be used.

[0088] In other embodiments, the solid support comprises an inorganic support, such as silica, zirconium oxide, etc. The surface of the inorganic substrate is often modified to include suitable reactive groups that can further react with SpA and its variants. Examples include CM Zirconia (Ciphergen-BioSepra (CergyPontoise, France) and (Millipore).

[0089] In some embodiments, the polymer may, for example, be based on zirconium, or silica, or controlled pore glass, which may be modified to contain reactive groups and / or to withstand corrosive immersion for use with the ligand.

[0090] Exemplary solid support formats include, but are not limited to, beads, gels, membranes, cartridges, columns, chips, slides, plates, or monolithic plastics.

[0091] Regarding the form of the matrix, in one embodiment, it is in the form of a porous monolithic plastic. In an optional embodiment, the matrix is ​​in the form of beads or microparticles, which can be porous or non-porous. The matrix in the form of beads or microparticles can be used as a packed bed or suspended form. Suspension forms include those consistent as extended beds and purified suspensions, in which the microparticles or beads can move freely. Regarding monolithic plastics, packed beds and extended beds, the separation step is generally followed by conventional chromatography using a concentration gradient. Regarding purified suspensions, a batch form will be used. In addition, solid supports in the form of planes, chips, capillaries or sheets may be used.

[0092] The matrix can also be in the form of a cylindrical membrane. The membrane can be in the form of a flat sheet, a spiral or a hollow fiber.

[0093] In another embodiment, the ligands according to the present invention are attached to a soluble support, such as a soluble polymer. Exemplary soluble supports include, but are not limited to, biopolymers such as proteins or nucleic acids. In some embodiments, biotin may be used as the soluble polymer, as described in U.S. Patent Publication No. 20080108053. For example, biotin may be conjugated to a ligand, such as the SpA-based corrosion-stabilizing ligands according to the present invention, which, after conjugation to the ligand, can be used to isolate a desired protein, such as an antibody or fragment thereof, present in a crude mixture, and the desired protein can be isolated or separated via precipitation of the biotin-ligand-protein polymer complex, either reversibly or irreversibly. The polymer may also be a synthetic soluble polymer, such as, for example, a polymer containing anionic groups (carboxyl or sulfonyl groups), cationic groups (quaternary, tertiary, secondary, or primary amine groups), hydrophobic groups (phenyl or butyl groups), hydrophilic groups (hydroxyl or amino groups), or combinations thereof. Exemplary synthetic soluble polymers can be found in International PCT Patent Publication No. WO2008091740 and U.S. Patent Publication No. US20080255027, the entire teachings of each of which are incorporated herein by reference. These polymers, under conditions of one or more specific physical changes, such as pH, conductivity, or temperature, can be used to purify desired proteins via reversible or irreversible precipitation processes. Synthetic soluble polymers may be used alone or in combination with corrosion-stabilized ligands according to the present invention to capture / purify desired proteins, such as antibodies or fragments thereof, via reversible or irreversible precipitation processes.

[0094] V. Methods of Attaching Ligands to Supports

[0095] Any suitable method may be used to attach the ligand to the support, including solid supports well known in the art and those described herein. For example, in some embodiments, the ligand may be attached to the support by conventional coupling techniques using, for example, amino and / or carboxyl groups on the ligand. For example, diepoxides, epichlorohydrin, CNBr, N-hydroxysuccinimide (NHS), etc. are known coupling reagents. In some embodiments, a spacer is introduced between the support and the ligand to improve the practicality of the ligand and facilitate chemical coupling of the ligand to the support. Alternatively, the ligand may be attached to the support via non-covalent bonds, such as physical attachment or biospecific attachment.

[0096] In various embodiments disclosed herein, a ligand is attached to a solid support, such as a chromatography resin, at more than one site to form a chromatography matrix.

[0097] Attachment of SpA-based alkali-stable chromatographic ligands to solid supports can be achieved by a number of different methods, most of which are known in the art, as well as those described herein. See, e.g., Hermanson et al., Immobilized Affinity Ligand Techniques, Academic Press, pp. 51-136 (1992).

[0098] For example, the protein ligand can be attached to the solid support via the surface of the solid support or via reactive groups on the protein ligand, such as hydroxyl, sulfhydryl, epoxy, amino, carbonyl, epoxy, or carboxyl groups. Attachment can be achieved using known chemical methods including, but not limited to, cyanogen bromide (CNBr), N-hydroxysuccinimide esters, epoxy (bis(oxirane)) activation, and reductive amination.

[0099] For example, direct thiol protein coupling is described in the literature. See, for example, Ljungquist et al., Eur. J. Biochem. Vol 186, pp. 558-561 (1989). This technique has been used to couple SpA to a solid support. Because wild-type SpA does not contain a thiol group, attachment is achieved by recombinantly inserting a thiol-containing cysteine ​​at the C-terminus of SpA. See, for example, U.S. Patent No. 6,399,750. Some commercial products such as MabSelect TM ,MabSelect TM Xtra and MabSelect TM SuRe is produced by this mechanism. It has been reported that this terminal cysteine ​​reacts only with epoxy groups on the solid surface, thus resulting in a single site of SpA attachment to the solid support. See, e.g., Process Scale Bioseparations for the Biopharmaceutical Industry, CRC Press, 2006, p. 473.

[0100] In certain embodiments of the present invention, SpA-based chromatography ligands comprising two or more SpA domains are attached to multiple sites on a solid support via indiscriminate multi-point attachment. Generally, SpA contains abundant free amino groups from numerous lysine residues within each domain. SpA domains can be attached to multiple sites on a solid support, for example, to a chromatography resin containing epoxy and aldehyde groups, via epoxide ring opening or deamination, respectively, of lysine amino groups on SpA. In certain embodiments, multi-point attachment can be achieved by ring-opening one or more naturally occurring amino acids on SpA containing free hydroxyl groups, such as serine and tyrosine, with an epoxy-containing support. Alternatively, multi-point attachment can be achieved, for example, by reacting naturally occurring amino acids on SpA containing free carboxylic acid groups, such as aspartic acid and glutamic acid, with an amino group-containing support via, for example, N,N'-carbonyldiimidazole. Multi-point attachment of ligands to supports can also be achieved by combining the above mechanisms.

[0101] To achieve alkaline stability using multimers of B and Z domains, the present invention eliminates single cysteine ​​mutations that would result in single-site attachment.

[0102] Ligands for SpA-based chromatography may also be attached to a solid support via a binding mechanism. For example, a binding group may react with a non-covalent ligand containing the ligand of interest, allowing the ligand of interest to be attached to the solid surface via ionic, hydrophobic, or binding reactions. This can promote the effective binding of the ligand to the solid matrix, for example, as described in U.S. Patent Publication No. 20070207500A1, thereby resulting in a higher ligand density than without the binding group. Binding groups suitable for use in the present invention include charged groups, such as ionic groups, and uncharged groups, such as hydrophobic groups. Binding groups can modify the solid support, such as by directly covalently binding to the solid support. Suitable ionic groups may include quaternary amines, tertiary amines, secondary amines, primary amines, sulfonyl groups, carboxylic acids, or any combination thereof. Suitable hydrophobic groups may include phenyl, butyl, propyl, or any combination thereof. It is also contemplated that mixed species may be used. Binding groups may react with protein ligands. Therefore, the reaction between the linker group and the protein ligand may consist of a series of reactions, such as ionic and hydrophobic.

[0103] The group used in conjunction may be covalently bound to the solid support, reacting with a functional group on the conjunction group through a functional group on the solid support. Suitable functional groups include, but are not limited to, amino, hydroxyl, sulfhydryl, carboxyl, imine, aldehyde, ketone, alkene, alkyne, azo, nitrile, epoxy, cyanide, and activated carboxylate. As an example, agarose beads contain hydrophobic groups that may react with the functional epoxy groups of cationic conjunction groups, such as trimethylammonium chloride. Those skilled in the art will appreciate that most conjunction groups may be coupled to the solid support provided, using at least one bifunctional conjunction group. Therefore, the conjunction groups may be coupled in series to the solid support, or they may be directly coupled to the solid support individually.

[0104] In some embodiments, the present invention provides binding groups and / or protein ligands that may be coupled to a solid support via an interfering linker. The linker may comprise at least one functional group coupled to a linker. The linker may comprise any molecule that can be linked to a functional group. For example, the linker comprises any alkyl, alkenyl, or alkynyl group. The linker may comprise a carbon chain ranging from 1 to 30 carbon atoms. In some embodiments, the linker may comprise greater than 30 carbon atoms. The linker may comprise at least one heteroatom such as nitrogen, oxygen, or sulfur. The linker may comprise a branched chain, an unbranched chain, or a cyclic chain. The linker may be replaced with two or more functional regions.

[0105] Those skilled in the art are capable of selecting suitable buffer conditions for coupling protein ligands and solid supports. Suitable buffers include any amino-free buffers, such as carbonate, bicarbonate, phosphate, and acetate buffers. When using a combined chemical, the salt concentration of the buffer will depend on the combined group used. For example, the salt concentration may be in the range of 5nM-100mM. When using charged species, the salt concentration may be at least 5nM, but less than 0.1M, at least 5nM but less than 0.01M, at least 5nM but less than 0.001M. In a specific embodiment, the salt concentration may be 0.01M. When using hydrophobic species, high salt concentrations are generally desired. Therefore, the salt concentration may be greater than 0.001M, greater than 0.01M, or greater than 0.1M.

[0106] In some embodiments, when a combination of compounds is used, the reaction occurs at a temperature ranging from 0°C to 99°C. In some specific embodiments, the reaction method is performed at a temperature below 60°C, below 40°C, below 20°C, or below 10°C. In some embodiments, the method of the present invention is performed at a temperature of about 4°C. In other embodiments, the method of the present invention is performed at a temperature of 20°C.

[0107] VI. Methods for Analyzing Alkaline Stability of Attached Ligands

[0108] The improved alkaline stability of the ligand after attachment to a solid support can be analyzed by techniques known in the art and using the methods described herein. For example, in some embodiments, the alkaline stability of a multimer of SpA domains attached to a resin (e.g., SpA B and Z domains attached to a chromatography resin as described herein) can be determined by treating the resin with 0.5M NaOH. It will be understood that improved stability refers to the retention of an initial IgG binding capacity that is improved over that of a wild-type SpA molecule over an extended period of time. For example, in the case of the present invention, each cycle comprises a 15 min treatment with 0.5N NaOH, and after 100 cycles, the SpA ligand, e.g., comprising multiple B or Z domains, retains a percentage of capacity that is at least 1.5 times greater, 2.0 times greater, 2.5 times greater, or 3 times greater than that of wild-type SpA. In one embodiment, the alkaline stability of a bound ligand is measured as follows, wherein the determination is of the IgG binding capacity retained over a period of time. Binding capacity, referred to as Qd 50%, is determined by obtaining a UV at 50% of the initial IgG concentration. 280nm The volume of IgG loaded at the time of the reading was measured. First, the Qd50% of the initial untreated resin packed on the column was measured. The resin was then exposed to 0.5N NaOH at 0.8 ml / min for 15 minutes, approximately 10 cycles. The Qd50% was measured again. This process was repeated until the resin had been exposed to 0.5N NaOH for approximately 100 cycles. Finally, the Qd50% values ​​were measured again, and the results for the resins with different ligands were compared with those for wild-type SpA.

[0109] In another assay, the corrosion or alkaline stability of the resin was measured by immobilizing the resin of interest. Alkaline stability was determined by the retained binding capacity of the resin by soaking a predetermined amount of resin in 0.5 N NaOH for 25 hours with gentle agitation and measuring the IgG binding capacity before and after the NaOH soak.

[0110] VII. Method for Purifying Target Molecules Using the Chromatographic Ligand of the Present Invention

[0111] In some embodiments, the present invention provides a method for purifying a target molecule from a mixture using the alkaline-stable chromatographic ligands described herein. The target molecule may be any molecule that can be recognized by the alkaline-stable chromatographic ligands provided herein, which are coupled to a solid support. Examples of target molecules include immunoglobulins. The immunoglobulins may be polyclonal or monoclonal antibodies or functional fragments thereof. Functional fragments include any fragment of an immunoglobulin that includes a variable region that can still specifically bind to its antigen while retaining its ability to specifically bind to the protein ligand coupled to the solid support.

[0112] In some embodiments, the method of separating a target molecule of interest using the alkaline-stable chromatographic ligand described herein comprises the steps of: (a) contacting a solid support comprising an attached SpA-based alkaline-stable chromatographic ligand with a mixture comprising a target molecule under conditions such that the target molecule can specifically bind to the ligand; and (b) changing the conditions such that the target molecule is no longer bound to the ligand, thereby separating the target molecule.

[0113] In some embodiments, the step of changing comprises changing the pH so that the target molecule no longer binds to the ligand. In a specific embodiment, the pH is changed in a manner such that it is more acidic than the pH conditions of step (a). For example, in one embodiment, step (a) may be performed at a neutral pH, or a pH in the range of about 6 to about 8, while step (b) may be performed at an acidic pH, such as, in the range of about 1 to about 5 pH values.

[0114] In another embodiment, step (b) comprises changing the salt concentration in the buffer used so that the target molecule no longer binds to the ligand. For example, in one embodiment, a high salt concentration, such as >0.1 M, may be used in step (a), while a low salt concentration, such as <0.1 M may be used in step (b). Conversely, in some embodiments, a low salt concentration, such as <0.1 M may be used in step (a), while a high salt concentration may be used in step (b). In other embodiments, the pH and salt concentration of the buffer solution may be changed between steps (a) and (b).

[0115] One skilled in the art can readily determine conditions suitable for binding a target molecule to a ligand and can therefore alter the conditions to disrupt binding of the molecule to the ligand.

[0116] The present invention is further illustrated by the following examples, which should not be considered limiting. The contents of the references, patents, and published patent applications cited in this application, as well as the drawings thereof, are hereby incorporated by reference. Example

[0117] Example 1: Construction of SpAB domain variants containing n+1 mutations, where n represents an asparagine

[0118] In an exemplary experiment, a synthetic gene encoding the "B domain" of protein A was obtained from DNA 2.0 (Menlo Park, CA). The 5' end of the gene includes a codon for an initial methionine and the 3' end of the gene includes six codons for histidine. The gene is present in plasmid pJ56:8620 from DNA 2.0. The parent plasmid pJ56 confers resistance to ampicillin, kanamycin, chloramphenicol, and gentamicin. For subsequent cloning of the gene into an expression vector, appropriate restriction endonuclease sites were introduced at the 5' and 3' ends of the gene. A map of the plasmid is shown in FIG. Figure 5 shown.

[0119] The glutamic acid at position 24 was subsequently mutated to other naturally occurring amino acids except cysteine ​​(C), serine (S), alanine (A), glycine (G), aspartic acid (N), and glutamine (Q) using a PCR-based method using Phusion High-Fidelity DNA polymerase (New England Biolabs, Ipswich, MA). Primers were purchased from IDT DNA (Coralville, IA) and stored in Tris EDTA buffer at 100 μM. The mutagenic primer contains the sequence CTGCCGAACCTGAACNNSGAACAACGCAACGG (SEQ ID NO: 41), where NNS represents the 3 bases encoding the 24 amino acid. PCR was performed in a 50 μL reaction system containing dNTPs (0.2 mM each), 125 ng of each primer, 50 ng of template plasmid, and 1 U of Phusion enzyme. PCR was performed according to the protocol in Table III.

[0120] Table III

[0121]

[0122] PCR reactions were treated with the restriction endonuclease DpnI (New England Biolabs, Ipswich, MA) to reduce wild-type background. Approximately 1 μL of DpnI enzyme was added to each 50 μL PCR reaction, and the samples were incubated at 37°C for approximately 1 hour.

[0123] The PCR reaction of 2 μL of DpnI was used to transform Escherichia coli NEB5α competent cells (New England Biolabs, Ipswich, MA). The cells were thawed on ice and 2 μL of PCR reaction was added to 25 μL cells. After incubation on ice for about 30 minutes, the cells were heat shocked at about 42°C for 30 seconds. The cells recovered on ice for about 5 minutes and then 125 μL of SOC culture medium (New England Biolabs) was added. The cells were incubated at 37°C for about 1 hour, and then 100 μL were plated on LB plates containing 100 μg / mL of ampicillin (Northeast Laboratory Services, Winslow, ME) and grown overnight at 37°C. Positive clones were determined by detecting the expressed proteins in the total cell lysate using SDS PAGE.

[0124] To obtain purified DNA, individual clones were selected and cultured overnight in LB containing 100 μg / mL ampicillin. DNA was purified using a Qiagen (Valencia, CA) spin mini-prep kit.

[0125] Mini-prepped DNA was sequenced to confirm the identity of each clone (MWG, Biotech, Huntsville AL). The constructed plasmids were used to transform E. coli NEB5α competent cells as described above.

[0126] After positive clones were identified, 35 ml of overnight culture was grown in Terrific Broth's medium containing 100 μg / mL ampicillin and pelleted by centrifugation at 13,500 g for 10 minutes. The pellet was resuspended in 10 ml of phosphate-buffered saline (PBS) containing 20 mM imidazole, disrupted by sonication, and centrifuged at 13,500 g for 30 minutes to pellet insoluble material. The lysate was then applied to 750 μl of Ni-NTA resin pre-equilibrated with 10 column volumes of PBS containing 20 mM imidazole. After washing with 20 column volumes of PBS containing 20 mM imidazole, the sample was eluted from the resin with PBS containing 200 mM imidazole. The purified protein was dialyzed against PBS overnight using a Pierce Slide-A-Lyzer 3.5 MWCO dialysis cassette. After dialysis, total protein was quantified using the Pierce MicroBCA assay, and samples were stored at -30°C.

[0127] The amino acid and nucleotide sequences of the wild-type histidine-tagged B domain and various n+1 mutants are shown in Figure 6 and Figure 7 shown.

[0128] Example 2: Detection of alkaline stability of expressed protein

[0129] Affinity-purified wild-type and mutant SpA histidine-tagged constructs as described in Example 1 were diluted to a final concentration of 1 N with MilliQ water or NaOH and incubated at room temperature for 6 hours. A Biorad Micro Biospin 6 gel column was used to neutralize and buffer-exchange 50 μL of each sample into PBS. Quantification of total protein was performed using the Pierce MicroBCA assay, and samples were diluted to 10 μg / ml in PBS for loading on the ELISA plate. Approximately 200 μl (2 μg) of treated PrA was attached to the wells of the ELISA plate at 37°C for 2-24 hours. The plate was blocked in Pierce Superblock Blocking buffer in PBS (Superblock-PBS) for approximately 2 hours at room temperature. Approximately 200 μl of Sigma human gamma globulin diluted to 0.05 mg / ml in Superblock-PBS (10 μg) was added to each well of the plate, and the binding reaction was allowed to proceed for approximately 1 hour at room temperature. The plates were then washed three times with PBS containing 0.05% Tween 20 (PBS-T), and then incubated with 200 μl of a 1:10,000 dilution of chicken IgY-HRP conjugate against human IgG for approximately one hour at room temperature. After a final three washes with PBS-T, the plates were developed with 100 μl of a Pierce 1-Step Slow TMB ELISA for 30 minutes at room temperature. The reaction was stopped by adding 100 μl of 1N HCl, and primary IgG binding was quantified by reading absorbance at 450 nm. All samples were assayed in triplicate, and data were analyzed for differences in primary IgG binding before and after corrosion treatment.

[0130] Figure 8 The bar graph summarizes the results of an example experiment analyzing the alkaline stability of various n+1B domain mutants. The various constructs are plotted on the X-axis, and the corresponding percentage of IgG binding retained after 6 hours of exposure to 1M NaOH is plotted on the Y-axis. Figure 8 As shown in the bar graph, some n+1 mutants containing a large amino acid at position 24 of the B domain showed improved corrosion stability.

[0131] In another embodiment, the above method is used to analyze the alkaline stability of an SpA ligand comprising two or more SpA B domains or two or more SpA Z domains. In another embodiment, the above method is used to analyze the alkaline stability of an SpA ligand comprising three B domains (B3), four B domains (B4), five B domains (B5), six B domains (B6), or seven B domains (B7).

[0132] In one exemplary experiment, the percentage of residual IgG binding capacity for ligands B3, B4, and B5, as measured by the ELISA-based method described herein, is as follows. The B3 ligand retained approximately 79% of its residual IgG binding capacity after six hours of exposure to 1 M NaOH; the B4 ligand retained approximately 86% of its residual IgG binding capacity after six hours of exposure to 1 M NaOH; and the B5 ligand retained approximately 83% of its residual IgG binding capacity after six hours of exposure to 1 M NaOH. The wild-type SpA ligand (nPrA) retained only approximately 49% of its residual IgG binding capacity after six hours of exposure to 1 M NaOH.

[0133] Example 3: Attachment of wild-type SpA and SpA variants to a solid support

[0134] After constructing various SpA variants and determining which corrosion-stable variants are identified using one or more analytical methods known in the art as described in Example 2, the SpA variants are attached to a support, such as a solid support. In an exemplary experiment, agarose beads (Sepharose 4B) (GE Healthcare, Piscataway, NJ) are cross-linked with epichlorohydrin using a method previously described (Porath and Fornstedt, J. Chromatography, 51:479 (1979)). The agarose beads are reacted with positively charged associating groups, such as cations, by adding 50 mL of beads to 40 g of 75% by weight glycidyl trimethylammonium chloride (GTMAC), 10 mL of milli- Water (Millipore Corp., Billerica, MA) and 1.67 g of 50% by weight sodium hydroxide were added. The reaction was shaken vigorously (>100 rpm) on a shaker at room temperature overnight. The beads were then filtered and eluted using three 100 mL volumes of Millipore. Rinse with water (Millipore Corp, Billerica, MA).

[0135] The beads (50 mL, filter cake) were added to a bottle containing 15 mL of 4.6 M NaOH. The mixture was homogenized and then 19.5 mL of butanediol diglycidyl ether (BUDGE) was added. The mixture was shaken at 35°C for 2 hours. The beads were then washed with 750 mL of Milli- The tube was rinsed with water (Millipore Corp, Billerica, MA) and equilibrated with 250 mL of 10 mM NaHCO 3 .

[0136] Following the BUDGE activation step, 10 mL of filtered beads were added to 10 mL of a 10 mM NaHCO3 solution containing either wild-type SpA or a SpA ligand of the present invention at a concentration of 15 g / L. The mixture was capped and placed in a glass vial and shaken in a hybridization oven at 37°C for 2 hours. After two hours, 30 mL of Milli- The beads were rinsed with water (Millipore Corp, Billerica, MA). The filtered bead mass (10 mL) was added to a bottle containing 10 mL of a solution containing 1 mL of monothioglycerol and 9 mL of a buffer solution containing 0.2 M NaHCO and 0.5 M NaCl. The mixture was homogenized and then shaken overnight at room temperature. The beads were subsequently washed with 20 mL of the following buffer: 0.1 M Tris buffer (pH 8), 0.1 M Tris buffer (pH 8) supplemented with 0.15 M NaCl, 50 mM acetic acid (pH 4.5), and PBS (pH 7.4) supplemented with 0.002% sodium azide.

[0137] Resin samples bound to different SpA variants were labeled as follows: nPrA represents wild-type SpA; Z3 represents a SpA ligand containing three Z domains; E3 represents a SpA ligand containing three E domains; D3 represents a SpA ligand containing three D domains; A3 represents a SpA ligand containing three A domains; C3 represents a SpA ligand containing three C domains; B3 represents a SpA ligand containing three B domains; B4 represents a SpA ligand containing four B domains; B5 represents a SpA ligand containing five B domains; B5NF and B7NF represent SpA ligands containing five or seven B domains, respectively, with an additional G29A mutation at position 29. After attachment to the resin, the alkaline stability of the various SpA variants was tested.

[0138] Example 4: Detection of Resin IgG Binding Capacity (Qd50%) Before and After Corrosion Cycles

[0139] In an exemplary experiment, the IgG binding capacity of various SpA constructs according to the present invention was measured after being attached to a support. In an exemplary experiment, a standard method for detecting the dynamic capacity of the resin / medium using commercial polyclonal IgG was used. Briefly, the resin was combined with an SpA ligand according to the present invention and filled in an Omnifit column (6.6 mm × 70 mm) in PBS, pH 7.4, with a flow rate set to 300 cm / hr. The packed column was equilibrated with 10 column volumes (CV) of PBS. Polyclonal IgG (Sigma-Aldrich, 2 mg / mL in PBS at pH 7.4) was loaded into the column until UV 280nm A concentration of more than 50% of the starting IgG was achieved. After washing with equilibration buffer, IgG was eluted with 0.1 M citric acid, pH 3.0. After each run, the medium was sterilized with 6 M Guanidinine hydroxide. Qd50% was calculated based on the UV 280nm The amount of IgG injected to achieve 50% of the initial IgG concentration.

[0140] After the initial dynamic capacity measurement, the medium was exposed to 0.5N NaOH (flow rate 100 cm / hr) for 15 minutes, 10 cycles, and then another dynamic capacity measurement of IgG was performed. The medium was then exposed to 0.5N NaOH for 15 minutes, 10 cycles, and then another dynamic binding capacity measurement was performed. Dynamic binding capacity measurements were performed after each sample was exposed to 0.5N NaOH for 15 minutes and 100 cycles.

[0141] In an example experiment, the results are as follows Figure 9 As shown, trimers of the E, D, A, B, C, and Z domains of the linker containing the native B domain were attached to an agarose resin, and their corrosion stability was measured by dynamic binding capacity assays after 100 corrosion exposure cycles, each cycle consisting of a 15-minute exposure to 0.5 N NaOH. Figure 9 As summarized in the graph, the number of cycles is plotted on the X-axis and the IgG binding capacity is plotted on the Y-axis. The C and Z domain trimers show roughly the same level of corrosion stability. The B domain trimer is more corrosion stable than the A domain trimer, which in turn is more corrosion stable than the E and D domain trimers. Therefore, the order of corrosion stability can be summarized as follows: C and Z > B > A > E and D.

[0142] In another exemplary experiment, agarose resins coupled to B domain variants: BBB (B3), BBBB (B4), BBBBB (B5), BBBBB-NF (B5-NF), and BBBBBBB-NF (B7-NF) were tested for their dynamic binding capacity to IgG compared to wtSpA (nPrA) after 100 cycles of corrosion exposure, each cycle comprising a 15 minute exposure to 0.5N NaOH, using the above assay. Figure 10 As summarized in the graphs in Figure 3, the degree of corrosion stability is directly proportional to the number of domains, and further, the G29A mutation, which reduces binding to the Fab, does not affect corrosion stability.

[0143] The teachings of the detailed descriptions incorporated herein by reference can help to more thoroughly understand the detailed description of the present invention. In the embodiments in the detailed description, diagrams of embodiments of the present invention are provided, but should not be construed as limiting the scope thereof. Those skilled in the art will readily appreciate that the present invention may also include many other embodiments. All publications and inventions are hereby incorporated herein by reference in their entirety. In terms of scope, if the various materials incorporated by reference exist in conflict or inconsistency with the detailed description of the present invention, the detailed description of the present invention will abandon any such materials. Any reference cited herein is not considered to be prior art to the present invention.

[0144] Unless otherwise indicated, all numerical values ​​expressing quantities of ingredients, cell culture media, and processing conditions set forth in this detailed description, including the claims, are to be understood as modified in any instance by the term "about." Accordingly, unless specifically indicated to the contrary, the numerical parameters are approximate and may vary depending upon the desired properties to be obtained by the present invention. Unless otherwise indicated, the term "at least" preceding a list of components is to be understood to refer to every component in that list. Those skilled in the art will know, or be able to ascertain, various equivalents to the specific embodiments of the invention described herein. Such equivalents are also intended to be encompassed by the following claims.

[0145] Many modifications and variations of the present invention may be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described are intended to be illustrative only and are not intended to be limiting in any way. It is intended that the detailed description and examples be considered merely as examples, while the following claims are intended to indicate the definitive scope and spirit of the invention.

Claims

1. A ligand for alkaline-stable chromatography analysis, comprising 3 to 5 isolated Staphylococcal protein A B domains, wherein the amino acid residue at position n+1 of the isolated domains is mutated to an amino acid residue selected from the group consisting of arginine, tryptophan, proline, tyrosine, and valine, wherein n represents asparagine at position 23; wherein the B domain consists of the amino acid sequence shown in SEQ ID NO:

10.

2. A method for identifying the ligand for alkaline-stable chromatographic analysis according to claim 1, comprising the following steps: (a) Treatment of Staphylococcus protein A-based ligand with NaOH; (b) NaOH-treated Staphylococcus protein A ligand was added to the multiwell plate; (c) contacting the Staphylococcal protein A-based ligand with a saturating amount of a first immunoglobulin such that the first immunoglobulin binds to the ligand; (d) contacting the bound first immunoglobulin with a molecule capable of binding to the first immunoglobulin, wherein the molecule is coupled to a detectable substance; and (e) detecting the amount of a signal derived from the substance, The signal amount is proportional to the alkaline stability of the ligand, so it can be determined that the ligand based on Staphylococcus protein A is alkaline stable.

3. The method of claim 2, wherein the molecule is a second immunoglobulin.

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