Linkers
Attachment of Protein A to a solid support, Generally
Methods of attaching protein ligands sucha s protein A and G to a solid support include ativating a media with a functional group (“activated group”) such as epoxide (epichlorohydran), cyanogen (cynogens bromide (CNB4), N,N-discuccinimidylacronate (DSC), aldehyde or an activated carboxylic acid (e.g., N-hydroxysuccinimide (NHS) esters), carbonyldiimidazole (CDI) activated esters). These activated groups can be attached directly ot the base matrix, as in the case of CNBr, or they can be part of a “oinker” or spacer molecule which is typically a liner chain of carbon, oxygen and nitrogen atoms, such as the ten membbered chain of carbon and oxygen found in the linker butane0diol diglycidyl ether (a common epxide coupling agent). The activated media is then equilibrated with the protein ligand under coupling cnoditions. For Protien A, typically 4-6 mg of progen (lgiand density) may be loaded per ml of media reusulting in an IgG maxim static capacity of 40 g/L. (Gian, US 7,833,723 and US 2007/0207500).
Support-Arginine Linker – (amide bond) -ProteinA:
Profy (US5,260,373) disclsoes covalently joining an immobilization support to an arginine containking linker and then joining the linker through an amide bond to Protein A.
Support- amino group –amide bond –linker –(carboxyl end) Protein A: NH2 –R1 (protein A) – CO – NH –R2 (linker) — NH – Y (support)
Iwakura (US 2008/0051555) discloses a support having affinity for an antiboy that includes a peptide capable of binding to an antibody such as Protein A that is immobilzied at a carboxy end to an insoluble support haivng a primary amino group such as one contained in a lysine via an amide bond mediated by a linker sequence.
An affinity separation matrix is often of organic nature and based on polymers that expose a hydrophilic surface to the aqueous media used, i.e., expose hydroxy (-OH) carboxy (-COOH) carboxamido (-CONH2), amino (-NH2), oligo or polyethylenoxy groups on their external and, if present also on internal surfaces). (Ander, US14/385336)
Cross-Linking
The support can be corsslinked, such as with hydroxyalkyl ether crosslinks. Crosslinker reagents producing such crosslinks can be e.g., epihalohydrins like epichlorohydrin diepoxides like butanediol diglycidyl ether, allylating reagents like ally halides or allyl glycidyl ether. Crosslinking is beneficial for the rigidity of the support and improves the chemical stability. Hydroxyalkyl ether corsslinks are alkli stable and do not cause significant nonspecific adsportion. (Ander, US14/385336)
Berg (US6,602,990) discloses a process for the production of a porous cross-linked polysaccharide gel by (a) preparing a solution of the polysacharide, (5) adding a bifunctional cross-linking agent have one active site and one inactive site (c) reacting hydroxylgroups of the polysaccharide with the active site of the cross linking agent (3) forming a polysaccharide gel (e) activating the inactive site of the cross-linking agent (4) reacting the active site from (e) with hydroxylgroups of the polysaccharide gel.
Arginine Linkers:
Profy (EP0282308) describes an improved immobilized protein A resin which has high binding capacity for IgG1. The immobilization support material can be any support used in immunoassays such as filter paper, plastic beads, test tubes made from polyethylene, polystyrene, polypropylene or other suitable material. The linker consists of arginine coupled to the support directly or through a chemical chain of any lenght. The chemical chain can be another protein. The immunoglobulin binding prtoein is preferably protein A which can be joined to the linker through an amide bond.
Johansson 9US6,399,750) discloses a separation medium of the formula –B–X–Protein A-cys where B is a bridge which binds to the base matrix, X contains a heteroatom N or S (X is a thioether sulfur (-S-) and/or a secondary amine (-NH-) originating from Protein A-cys and Protein A-cys is recombinantly produced Protein A which contains cysteine in its amino acid sequence. The optimal molar ratio between the total IgG binding capacity and the amount of Protein A on the matrix may vary depending on the number of IgG bidning doamins that are present in the Protein A of the adsorbent.
PolyHistidine Linkers
Tamori (US 2013/0041135) discloses filler for affinity chromatography represented by the formula R-R2 where R represetns an amino acid sequence consisting of 4-300 amino acid residues containing a region consisting of 4-20 contiguous histidine residues and R2 represents an immunoglobulin binding sequence of 50-500 amino acids containing Z domain of Protein A.
PolyProline Linkers
IChii (US 15/745,855, published as US 2018/0222939) discloses an affinity support which contains a ligand ofr the geenral formula R-R1 wherein R represents a linker that contains a polyproline bound to the solid support and R2 is an antibody bidning protein like Protein A.
Fusion Proteins as Linkers
Avidin-Avidin Fusion Proteins: Nordlund (WO2005/047317) discloses fusion peptides where two avidin molecules are fused together via a linker to form a single polypeptide with four binding sites for biotin or other ligand. The fusion protieins can be used for separations. For example, the avidin fusion protein can be bound to a biotinylated substrate and the biological activity of the secondary molecule can then be used to capture and separate a particular molecule out of an impure solution. The purified molecule is then dissocaited from the fusion protein. The lwoer biotin affinity of the fusion protein as compared to that of wild type avidin allows release of the fusion protein from the substrate and resue of the fusion protein. Nordlumnd exemplify avidin mutants which were more reversilbe and exhibited reduced affinity as compared to the wt avidin.
Streptavidin-Protein A chimeric Proteins:
Streptavidin is a protein very closely related to avidin which provides a very stable noncovalent complex with vitamin D-biotin. Avidin itself is a very highly specialized protein that is only rarely expressed. Streptavidin, on the other hand, is readily expressed in a species of Streptomyces, in Streptomyces avidinii. Streptavidin specifically binds a water soluble vitamin D biotin (vitamin H). Similarly to avidin, it also binds rapidly and almost irreversible and with a remarkably high affinity to any molecule which contains an unhindered biotin. Sano (US 5328985)
Sano (US 5328985) discloses a streptavidin-Protein A chimeric protein in which the streptavidin binds biotin and the Protein A binds an antibody. Accordingly, one side specificity is caused by high affinity binding of the Protein A side to antibody molecules and the second side specificity is caused by high affinity binding of the streptavidin side to any target biological material which contains biotin or which is capable of being biotinylated. The fusion protein can be used to purify target molecules onto solid supports.
Kyhse-Andersen (US14086621) discloses a process for purification of a target molecule using a dual affinity polypeptide (DAP) which serves as a linker molecule and a solid support comprising a catching ligand. The DAP as at least two binding sites, one of which has affinity for the ligand and the other which has affinity for the target molecule. Binding of the DAP to the catching ligand on the solid support is provided by cleavage of a para-substituted benzyl guanine resulting in a thioether bond. The binding of DAP to the target is in preferred embodiments weaker than binding of DAP to the ligand. For example, the equilibrium dissociaition constant towards the target (Kdt) is in the range of 10-4 to 10-13 M and the equilibration dissociation constant towards the catching ligand (Kds) is in the range of 10-9 to 10-16M. In one embodiment, the DAP comprises at least one binding domain of a protein A fused to at least one biotin binding domain of avidin, streptavidin or neutravidin. In another embodiment, the DAP comprises at least one binding domain of an affibody fused to at least one biotin binding domain of avidin, streptavidin or neutravidin. In another embodiment the DAP comprises at least one binding domain of an antibody fused to at least one biotin binding domain of avidin, streptavidin or neutravidin.
Biotin-Protein A chimeric Proteins:
Patchornik (US20080108053) also discloses an antibody binding moiety such as protein A which is attached to at least one corrdinating moeity such as biotin. In order to initiate purification, the ligand attached to the coordinating moeity is added to the sample, allowing binding of the target molecule to the ligand and then a coordinator ion or molecule such as a metal is added to allow precipitation.