See also multimers of SpA

In General and Definitions

Cross-Link: is a bond that links one polymer chain to another. They can be covalent or ionic bonds.

Many different types of coupling groups can be introduced into a support. Examples include carboxyl grou pactivated by N-hydroxy succinedimide (NHS), a carboxyl group, a cyanogen bromide activating group, a hydroxyl group, an epoxy group, an aldehyde group and a thiol group. Then the ligand which might for example have a primary amino group can be coupled to the support. For example, a carxoyl group activated by NHS, a carboxyl group, a cyanogen bromide active group, an eopxy group and a formyl group are all capable of coupling with a primary amino group. A sapcer may also be inserted between the support and the coupling group. Koguma (US13/996385)

Method of attaching Ligands to the Support

The ligand may be attached to the support via conventional coupling techniques utilising, e.g., amino and/or carboxy groups present in the ligands. Bisepoxides, epichlorohydrin, CNBR, N-hydroxysuccinimide (NHS) etc are well known coupling reagents. Between the support and the ligand, a molecule known as a spacer can be introduced, which imporves the availability of the ligand and facilitates the cheical coupling of the ligand to the support. Alternatively, the ligand may be attached to the support by non-covalent bonding, such as physical adsorption (Ander, US14/385336)

Immobilising ligands to anyone of the above supports is easily performed by the skilled person in the field followng well known methods (Hermanson, “Immobilized affinity ligand techniques”, Academic Press, INC, 1992).

Protein A can be immobilized via a covalent bond to the supporting medium surface by contacting the protein A with a coupling group immoibilized to the surface of the support. Any method may be employed for introducing the coupling group into the support, however, a spacer is generally introduced between the support and the coupling groups. For example, a graft polymer chain having a coupling group at a temminal and or a side chain may be introduced into the supporting medium. The polymer chain may be previously prepared and then coupled wiht the support or the graft chain can be directly polyemrized onto the support via methods such as a “living radical polymerication method” and a “radiation graft polymerization method”. The “radiation graft polymerization method” is preferably since a reaciton initiator does not need to be introduced in advance into the support and the method is applicable to various types of supports. The graft chain is introduced by the “radiation graft polymerization method” by any means for generating radicals from the support such as ionizing radiation like gamma or beta ray or an electron or neutron beam. Examples of monomers having the coupling group to be used in graft polymerization include monomers such as acrylic and methacrylic acid when a carboxyl group is used as the coupling group. When a primary amino group is used as the coupling group, an allyl amine can be used. When an epoxy group is used as the coupling group, glycidyl methacrylate can be used. The monomer having the precursor functional group can be converted into the coupling group is grafted onto the support and then the precurosor funcitonal group grafter may be coverted into the coupling group. The glycidyl methacrylate (GMA) having an eopoxy group can for example be converted into various functional groups by use of various ring-opening reacitons of the eopoxy group. When a carobxyl group is used as the coupling group, ring opening half esterification reaction can be employed where first, GMA is graft polymerized and then the eopoxy group of the GMA is hydrolyzed into a diol, a cyclic acid anhydride is reactive with the hydroxide group derived form diol through a ring opening half esterification reaction to form a carboxyl group derived from the cyclic acid anhydride. The cyclic acid anydride is desirably succinic anhydride or gltaric anhydride. The catalysist used in the ring opening half esterification includes triethylamine, isobutyl ethylamine, pyridine and 4-dimethylaminopyridine. The ring opening half esterificaiton reaction is preferably performed in an inert organic solvent such as toluene. An NHS activation reactions refers to a step of converting the carboxyl group form by the ring opening half esterificaiton reaction into an active ester. The active ester has a high reactivity compared to the carboxyl group and the protien A can be quickly immobilized onto the carrier.  The Protein A which has an amino group reactive with the active ester to form an amide bond and is thereby immobilized ot the support via a covalent bond. (Koguma, US13/996385). 

Methods of attaching protein ligands such as protein A nd G to a solid support have been described. Typically the media is activated with a reactive functional group such as an epoxide (epichlorohydrin), cyanogens (cyanogens bromide (CNBR)), N,N-disuccinimidylcarbonate (DSC), aldehyde or an activated carboxylic acid (e.g., N-hydroxysuccinimide (NHS) esters, carbonyldiimidazole (CDI) activated esters). These activated groups can be attached directly to the base matrix, as in the case of CNBr, or they can be part of a linker or spacer molecule which is typically a linear chain of carbon, oxygen and nitrogen atoms, such as the ten membered chain of carbon and oxygen found in the linker butaenidiol digycidyl ether (a common epoxide coupling agent). (Bian, US 7,833,723, 7,846,682 and US2011/0105730).

Majima (US 14/916,316, published as US 2016/0215027) teaches a multimeric immunoglobulin binding protein having 6-10 total domains from SpA such as the C domain having the general structure (R1)n-(R2m) or (R2m-(R1)n where the R1 domain is an amino acid sequence in which a non-lysine amino acid has been replaced such as the lysine being substituted with a non-lysine amino acid at 1-3 of positions 4, 7, and 35 R2 is an immunoglbulin binding domain coccuring at the N-temrinus or the C-terminus of the protein and includes an amino acid residue that covalently bonds to an insoluble support and the R2 domain is based on an amino acid sequence in which the lysine residue(s) are substituted with a non-lysine amino acid only at position 35, or at posiiton 35 and one or more positions 4, 7 and 35. In one embbodiment the R2 domain includes substitutions of 1-6 amino acid reisdues with lysine at positions 40, 43, 46, 53, 54, and 56. Because only the (R2) domains are selectively immobilized on the support via a covalent bond, it is possible to acheive a highly selective immobilization reaction trhough for example a lysine or a cysteine residue. The R1 in trun has an amino acid sequence that does not contain an amino acid that is active to the chemical reaction used for immobilization. When the immobilization reaction used to immobilzie the protoien on the support takes place via an amino group, an R2 domain can be produce by substituting the lysine residues contained in the amino acid sequence with non-lysine aino acids only in lysine residues occurring at positiion(s) that interfere with binding to an immunoglobulin upon immobilizing the protein on the support and by substituting some of the non-lysine amino acids not involved in bidning to an immunoglobulin with lysine. In the case of immobilization via a thiol group, a new cysteine residue can be added to the R2 domain. A support immobilization reaction using a disulfide bond or a maleimide group that is highly selective to the thiol group may be used for said immobilization.

Using a disulfide or a thioester bond:

A cystein can be introduced into the carboxy end of a protein A moelcule and the protein A immobilized to a support via a covalent bond at a single site by specifically using a sultheydryl (SH) group which is a side chain of the cystein residue. In a first method of using a dislufide bond, a surface having a sulfhydryl group exposed to the suface such as for example activated Thiol SEPHAROSE 4 B is used as the support and a disulfide bond is formed by the condensation reaction between sulfhydryl grups in both the recombinant protein A and the support.  In a second method of using a thioether bond, a glycopolymer support such as agarose is preliminarily activated with an active epoxy group introducing reagent such as epichlorohydrin and a thioether bond is subsequently formed together with the sulfhydryl group of the protein A. In both methods, protein A can be immobilized at a single site at the carboxy end, which brings about advantages such that the binding stability due to the covalent bond can be ensured and the moelecualr orientation can be uniformed while sustaining the binding sites of the protein A. Iwakura (US 2008/0051555) 

Methods of coupling protein A to a solid matrix such as crosslinked, uncharged agarose (Sepharose, freed from charged fraction of natural agarose) are commonly known in the art and include coupling via primary amino functions of the protein to a CNBr-activated matrix. Recombinant protein A can also be attached by a thiether sulfur bond to the support as is described in US 6399750 from Pharmacia and commercially available as Streamline™ or MabSelect™ from Amersham-biosciences. The thiether can be a thieoether bridge generated by reacting the sulfhydryl group of a cysteine residue of the protein A with an epoxide group on the activated support (WO 2004/076485).  With MabSelect, the ligands comprise recombinant Protein A coupled to a cross-linked agarose support via a C-terminal cysteine. The median particle diameter is 85 um (Berg, US2006/0134805).

Attachment of recombinent Protein A to the column matrix via a single thioester bond allows for higher capacity protein A column. Such single-point attachment by means of suitable reactive residues which further are ideally placed at an exposed amino acid position, namely in a loop, close to the N- or C-temrinus or elsewhere on the outer cirucumference on the protein fold. Suitable reactive groups are sulfihydryl or amino functions. More preferabley, such recombinant protein A comprises a cysteine in its amino acid sequence. Most preferably, the cysteine is comprised in a segment that consists of at least 30 amino acids of the C terminus of the amino acid sequence of the recombinant proteiin A, preferably the protein A is attached by at least 50% via thioether sulfphur bond to the matrix. An example of such an embodiment is described in US 6,399,750 from Pharmacia and is commercially availabe under the tradenames MabSelect™ from Amersham Biosciences. However, the leakae rate of such Protein A matrices if often drastically increased in contrast to tradition, multi-point attached natural Protein A matrices obtained by CNBr coupling (US2008/0312425).

–Thiol groups of arginine or cysteine (e.g., C-terminal cysteine -Thioether bridge coupling):

The protein can be coupled via a C-terminal cysteine provided on the protein. This allows for efficient coupling of the cysteine thiol to electrophilic groups e.g., epoxide groups, halohydrin groups etc. on a support, resulting in a thioether bridge coupling. (Ander, US14/385336)

Hall (WO2008/039141) discloses that mehtods are readily available for coupling of protein ligands via certain amino acids preferably amino acids that contain nitogen and/or sulphur atoms such as arginine or cysteine. In one embodiment, coupling group is in the C terminal region.

Johansson (US 6,399,750) disclsoes a Protein A based matrix which is attached to the column matrix via a single thioester bond and/or a secondary amin (–NH–)

Ljungquist (Eur. J. Biochem. 186, 557-561 (1989) discloses that a single cysteine residue introduced into the C terminal part of different recombinant protein A molecules can be used for immobilization on a thiol containing solid matrix.

Amino groups of an N-terminal amino acid and lysine: One can use a carrier having an amino group or a carboxyl group and subjecting the carrier to react with a carboxyl group or an amino group of a protein ligand in the presence of a dehydration condesning agent such as a water soluble carbodiimide, to thereby form an amide bond. Tamori (US13/636410

carboxyl grups of a C-terminal amino acid, glutamic acid, and aspartic acid: Protein A can also be immobilized in a C-terminal carboxyl group selective manner via an amide bond on a carrier where a polymer compound having a primary amino group has been introduced (JP 2005-112827A).Using a carrier having a carboxyl group and activating this carboxyl group with N-hydroxysuccinic acid imide to react with an amino group of a protein ligand Tamori (US13/636410).

Epoxy Group – Amino group:

One can also introduce an epoxy group to a carreir by means of bisepoxide, epichlorohydrin or the like and subject the carrier to react with an amino group, a hydroxyl group or a thiol group of a protein ligand. Tamori (US13/636410).

Tamori (US2007/0224424A1) teaches magnetic particles which may contain an epoxy group and reacting substances having a biotin bonding site with the epoxy group.

–ring-opening epoxy group:

The affinity particles can include a ring-opening epoxy group produced by ring-opening of the epoxy group.  The ring-opening epoxy group is produced by ring-opening of an epoxy group as a result of reacting the epoxy group with a nucleophile compound that includes a hydroxide ion, a chloride ion, a mercapto group, an amino group, or the like.  The ring-opening epoxy group may be a substituted or unsubstituted 2,3-dihydroxypropyl group.  An unsubstituted 2,3-dihydroxypropyl group may be produced by ring-opening of a glycidyl group via hydrolysis, as an example. A substituted 2,3-dihydroxypropyl group may be produced by ring-opening of a glycidyl group using a mercapto group-containing blocking agent (e.g., mercaptoethanol) or an amino group–containing blocking agent (e.g., monoethanolamine. (Tamori, US2011/0262748A1) (see also Tamori, US13/636410).

Hydroxyl group on carrier – Amino group:

One can use a carrier having a hydroxyl group and activating the carrier with a cyan halide such as cyan bromide to react with an amino group of a protein ligand. One can also tosylate or tresylate a hydroxyl group of a carrier and subject the carrier to react with an aino group of a protein ligand. Tamori (US13/636410)

Multi-point Attachments of Multi-Domains

Bian (US 12/653888 and EP2202310) discloses chromatography ligands comprising 2 or more B or Z domains of SpA attached to a chromatography resin at more than one site on the resin. In some embodiments the ligands comprising 2 or more domains of SpA are attached to a solid support at more than one site via non-discrimbinate, multipoint attachment. SpA contains abundant free amino groups from numerous lysines in each domain. Thus the attachemt of an SpA domain to more than one site on a solid support can be achieved by reacting the amino group of lysine on SpA, via epoxide ring-opening or reductive amination, respectively. In certain embodiments, multipoint attchment can be acheived by the reaction of one or more naturally occurring amino acids on Sp having free hdyroxyl groups, such as, for example, serine and typosine, with a support containing an epoxide group via a ring opening reaction. Alternatively, multipoint attachment can be acheived, for example, by the reaction of naturally occurring amino acids SpA having free carboxylic acid groups, such as aspartic acid and glutamic acid, with a support containing amino groups via, for example, N, N’-carbonyldiimidazole.