Commercially available IMAC columns:

Profinity IMAC resins: The Profinity IMAC bead is a 60 um particle derivatized with iminodiacetic acid (IDA) which functions as the chelating ligand. The chemical structure of IDA allows hihgly selective binding of recombinant His-tagged prtoeins when charged with Ni2+ or other transition metals. As a result, target proteins can often be purified close to homegeneity in a single step (Protein Purifiaiton Profinity IMAC Resins, “Selective Profinity IMAC resins provide ultrahigh-purifty recombinabt His-Tagged Proteins” BioRad; see aslo US 6,423,666). 

ProPur Affinity spin columns: The Propur metal chelate Mini and midi kits are designed for simple, complete and rpaid purificaiton of His-tagged recombinant prtoein from bacterial cells, inset vectors, mammalian cells and yeast under native or denaturating condition. A highly comprised agarose resin is used in the Nunc spin column. The rapid Mini spin columns can be used to purify up to 24 protiens in a microcentrifuge simultaneously. The ProPur Midi spin columns can be used to purify up to 16 proteins in under 50 min using standard bench centrifuge. When a recombinant protein is expressed in E. coli, the protein elutes as insoluble aggregates called inclusion bodies. Denaturants sucy as urea completely unfold the target protein making the His tag much more accesible for interaction with the matrix. (Brownleader, “ProPur: His-tagged protein purification made easy’ Nature Methods, February 2007). 

Introduction:

In 1975, Porath introduced immboilized metal ion affinity chromatography (IMAC) for fractionating proteins. IMAC consists of derivatizing a resin with iminodiacetic acid (IDA) and chelating metal ions to the IDA derivativzed resin. The proteins are seaprated on the basis of their affinity for metal ions, which have been immobilized by chelation.

One of the most widely used methods for protein purificaiton is immobilized metal ion affintiy chromatogrpahy (IMAC) which allows rapid one-step purificaiton of fusion proteins. For such procedrues, proteins are engineered with affinity tags attached to the t’ or 3′ end of the target gene. Examples of such tags are hesahistidine and an 8-reisude peptide containing alternating histidine. The matrix is attached to chelating groups that immobilize transition metal ions such as Ni2+, Co2+, Cu2+ and Zn2+. Ni2+ is the most widely used metal ion. the simplicity of IMAC is very attractive as it lends itself to the bind-wash-elute mode of oepration if the appropriate buffer is selected. (Brownleader, “ProPur: His-tagged protein purification made easy’ Nature Methods, February 2007). 

Immobilized metal affinity chromatography (IMAC) is a type of affinity chromatography where proteins can be separated according to their affinity for metal ions that have been immobilized by chelation to an insoluble matrix. At pH values around neutal, the amino acids histidine, tryptophan and cystein form complexes with the chelated metal ions (e.g., Zn2+, Cu2+, Cd2+, Hg2+, Co2+, Ni2+ and Fe2+). They can then be eluted by reducing the pH, increasing the mobile phase ionic strenght or adding ethylenediaminetetraacetic acid (0.05M) to the mobile phase. 

IMAC relies on the formation of weak corrdinate bonds between metal ions immobilized on a column and basic groups on proteins, mainly histidine residues. The absorbent can be formed by attaching to the matrix a suitable spacer arm plus a simple metal chelator, usually based on imino diaetate structures. These chelating ligands will bind tightly to metal ions, in particular to the divalent ions of the transition metals Fe, Co, Ni, Cu and Zn. (Vedadi WO03/025156).

IMAC is a versatile separation procedure that exploits differences in the affinities exhibited by many biopolymers for metals ions. The technique involves the chelation of a suitable metal ion onto a solid support matrix whose surface has previously been modified with a polydentate ligand. The resulting immobilized metal ion chelating complex then has the potential to coordinate with one or mroe elecggron donor groups residing on the surface of the interacting proteins. Separation selectivity is acheived on the basis of differences in the thermodynamic staiblities of the immobilized metal ion complexes with the vairous adsorbed proteins. Proteins whose adsorption complexes are the least stable will be eluted first, while proteins that form more stable omcplexes will be eluted later. The greater the diference in the equilibrium association constands (i.e., the larger the differences in the dissociation constants (KD) of the respective protein/immobilied metal ion coordination complexes, the higher the resolution obtained. (Hearn (US 2009/0143529). 

Hearn (US 2009/0143529) disloses a polyer substrate such as polysaccharides (e.g., agaroses, destrans, celluloses, etc) functionalized with at least one cyclic, metal ion coordinating ligand group which includes at least 3 nitrogen donoar atoms in the ring of the cyclic group, at lone of the nitrogen atoms having an optionaly substituted carboxy (lower alkyl) or optionally substituted phosphono (lower alkyl) group covalently attached thereto. It is advantageous that the functionality in a functionalized polymer substrate is covalently attached to the polymer substrate by means of a liner or spacer group. The IMA based systems sacheive a mixed modality of interaction with their target molecules that is based on a combination of coordination (electron donor/electron acceptor) and electrostatic (ion-exhcange) processes. The functionalized polmer substrates can be used to purify a desired protein of interest by virue of the presence of a metal ion such as divalent and trivalent metal ions such as Ca2+, Mg2+, Zon2+ and Fe3+ which iself is bound coordinatively to the cyclic ligand group in the functionality and which is in turn capable of binding coordinatively to donor atoms in the amino acid residues of a tag part of a fusion protein. 

His-tags:

The most important applicaiton of IMAC is purificaiton of recombinant proteins expressed in fusion with an epitope containing six or more histidine residues, the His tag. Due to the relatively high affinity and specificity of the His tag a single IMAC purificaiton step in most cases leads to a degree of purity of the target protein preparation taht is sufficient for many applicaitons. Block “Immobilized-metal affinity chromatography (IMAC): a review” Methods in Enzymology, 463 2009). The His-tag (also called 6xHis-tag) is one of the simplest and most widely used purificaiton tags, with six or more consecutive histidine residues. These residues readily coordinate with transition metal ions such as Ni2+ or Co2+ immobilized on beads or a resin for purificaiton. IMAC is the preferred choice as a first step during purificaiton of His-tagged proteins., although small batch reactions or spin columns with IMAC beads can be used for expression tests or small scale prepations. Metal ions are immobilized using linkages such as Ni(II)-nitrilotriacetic acid (Ni-NTA) or Co2+ carboxymethyl-asparatate on resinds and beads available from many commercial sources. Malhotra “Tagging for protein expression” chapter 16m Methods in Enzymology, volumen 463, 2009). 

The use of short histidine stretches or his tags, typically placed as an affinity tag at either the N-temrinus or C-terminus, enables the purification of the desired protein from the crude extract of the host cells in a single step. Different chromatographic supports and strategies are available of IMAC. The most widespread IMAC supports use either nitrilotriacetic acid (NTA) as a ligand for immobilizationg metals like nickel in affinity chromatogaphy (Ni-NTA) or different chelating Sepharose matrices. Although unviersally applicable, the use of his-tags and IMAC purificaiton is not recommended for proteins containing metals iones. Similarly, other aa like cysteine and nturally occurring histidine rich regions in host proteins may result in unwanted prtoein binding during IMAC purification. Arnau (Protein Expression and Purification 48 (2006) 1-13). 

cleavage:

The use of fusion tags for teh purificaiton of recombinant proteins is attractive for a number of reasons including effective capture of target proteins present in complex. In some circumstances, the removal of the tag is a crucial step partticularly in cases wehn the target protein is inteded for pharmaceutial applications in addition to cyrstallization and structural determination studies. Cleavage of fusion tags can be acheived by using either chemical or enzymatic methods. (Abdullah, Biotechnology and Bioengineerig, 92(4), 2005). 

Particular Types of Proteins Purified by IMAC

 IgG:

Histidine ligand affinity chromatography has been used to recover IgG. The antibodies were eluted from the adsorbents under very mild conditions, which ensures the structural integrity of the Abs (Vuayalakshmi “Antibody purification methods”, Applied Biochemistry & Biotechnology, 75, 1998.

Pentameric IgM including J chain and Dimeric IgA including J chain:

Brown & Simon (US Patent Application No: 14/476,559, published as US Patent 10385117; see also US Patent Application No: 16/401,322, published as US 2019/0256577) discloses purfication of pentamieric IgM including J chain as well as a dimeric IgA including J chain by adding secretory component which is tagged with an affinity or eipitope tage such as polyhistidine, psoing the mixture to a binding moeity immobilized on a solid phase resin such that the secretory IgA binds to the solid phage resin, washing and then eluting the secretory IgA therapeutic. 

Secretory Component:

Secrectory component (SC) is a protein that specifically binds to J-chain containing immunoglobulin. SC in its natural form is the extracelllar portion of the polymeric immunoglobulin receptor (pIgR) which usually gets associated during secretion with dimeric or polyemric IgA or pentameric IgM comprising a J chain. J chain containig IgA/IgM ibinds to the polyemric immunoglboulin receptor at the basolateral surface of eptithelial cells and is taken up into the cells by transcytosis. This receprtor complex then transits through the cellular compartments before being transported to the luminal surface of the peithelial cells. The transcytosed IgA/IgM-pIgR complex is then released through proteolysis, and part of the polymeric immunoglboulin receptor (pIgR) referred to as the natural secretory component, stays assocaited with the J chain contianing IgA/IgM, relasing secretory IgA/IgM. (Corthesy, WO 2013/132052). 

Corthesy, (WO 2013/132052) discloses adding a tag, such as a hexa-Histidine tag, which can aid in the purificaiton of the resulting prtoein. If such a tag is attached via a cleavalbe linker, the tag may be cleaved off prior to use in the invention. 

Nanobodies:

Beirnaert (Wo 2006/122786A2) discloses purificstion of His tagged anti-TNFalpha nanobodies using IMAC and elution using citric acid. The nanobodies were further purified on CEX. 

Rossi (WO2005/049649) teaches purification of IL-18 binding protein from a fluid by subjecting the fluid to immobilized metal ion affinity chromatography (IMAC), subjecting the eluate to HCIC, subjecting this eluate to CEX and subjecting the flow through to HIC and subjecting the eluate the eluate to reverse phase chromatography.

Particular Metal Binding – Chelating Agents

Iminodiacetic aicd

IMAC is based on the metal ion mediated interaction with protins. To acheive this interaction on solid phase, metal ions are adsorbed on a chelating resin and the resulting solid phase is used for protein adsorption. Chelation of metal ions occurs iva the intervention of selected ligands chemically attached on the matrix. The most popular chelating ligand is iminodiacetic acid. (Boschetti, Chapt 4, “Approaches to devise antiboy purificaiton processes by chromatography” Antibodies, volume 2, novel Technolgies and Thepaturic Use, 2004 

TREN chelated with copper:

The nobiological ligand TREN (Tris(2-aminoethyl)amine) is a quadridentate chelating ligand used in IMAC with four nitogen atoms, three of which are primary in nature adn the fourth one is tertiary. TREN chelated with copper and nickel ions has been employed in protein purificaiton. Due to its high amine residue content, TREN (without chelated metal ion) can serve as an anion exchanger. At a pH lower than 10, TREN is positvely charged and can adsorb negatively charged molecules, so this ligand could be an excellent candidate for the purificaiton of IgG from serum proteins. Bresolin (J of Chromatography B, 877 (2009) 17-23)

Boden (J Immunological Methods 181 (1995 225-232) discloses a rpaid, single step purificaiton of immunoglobulins from goat serum using immobilized metal ion affintiy chromatography (IMAC) on a high capacity gel, Novarose couple to tris(2-aminoethylamine (TREN) chelated with copper.

Bresolin (J of Chromatography B, 877 (2009) 17-23) discloses using Tris(2-aminoethyl)amine (TREN) as a chelating agent in IMAC for the purificaiton of IgG from human serum. 

Ribeiro (J Chromatography B, 861 (2008) 64-73) discloses purificaiton of IgG from human plasma using an affinity membrane complexed with Ni(II) prepared by coupling iminodiacetic acid (IDA) and Tris(2-ainoethyl)aine (TREN) to poly(ethylenevinyl alcohol), PEVA, hollow fiber membranes. 

PEG chelated with a variety of metals

Arnold (US 5,283,339) discloses that certain PEG compounds are capable of chelating a variety of metals such as Zn2+, Ni2+, Fe3+, Fe2+, Co2+ and Ca2+ and can be used to precipitate proteins form solution.