Affinity chromatography technique capitalizes on the property of the biomolecules to selectively bind to functional group, like the antibody for its antigen, the hormone for its receptor, and the enzyme for its substrate.

We commonly agree that affinity chromatography was first used in the isolation of enzymes in 1953. But a paper was already published in 1910 on the first experimental demonstration of the biospecific adsorption of an enzyme onto a solid substrate. In 1951, a successful isolation of rabbit anti-bovine serum albumin by affinity purification was also described.

So let’s go back a the beginning of the 20th century and see how a natural concept was translated into practice.

Affinity purification employs the principle of interaction between two different and complementary phases ; a stationary phase and a mobile phase. You can figure the column as a wall (called « matrix ») covered with a series of the same padlock, reproduced again and again (called « specific affinity ligand » or « affinant »). Your mobile phase is composed with a mix of different keys, but only one key is able to specifically recognized the padlock : your compound of interest.

From this complex moving phase, only the desired molecule (or key) will be retained by the stationary phase (locked into a padlock), while the rest of the solution is passing through (flow through), unaffected and unretarded by the column. The compound of interest is then released from the complex with the immobilized ligand by changing operational parameters.

Back in the 20th century, it seems that Lerman, Starkensteinin and Campbell perceived early enough the many virtues of this technique.

But which advantages have pushed so extensively the use of affinity purification ?

Biospecific affinity chromatography approach enables high selective capture and circumvents the multistep purification processes. With a single step chromatography process, you can retain and purify your compound of interest with high yield, saving precious time.

As genius Leonardo da Vinci loved to repeat “Simplicity is the ultimate sophistication”. Conceptually, the affinity purification technique represents scientist Graal thanks to unparalleled simplicity of a ‘load-wash-elute’ approach.

Finally, affinity chromatography can be used for multiple purposes : to purify and concentrate a substance from a mixture, to reduce the amount of unwanted substances, to identify a specific biological compound, to prepare a sample before a more complex analysis.

Too good to be true, huh ?

Maybe. Decades of use and multiplicity of applications have shown some penalties to pay to use affinity purification.

• Due to steric hindrance, the molecule of interest would not be able to bind the allosteric binding site on the ligand. Consequently spacer arms may need to be present to create space between the ligand and the matrix.

• The best is the enemy of good. The ligand should bind the molecule of interest in reversible fashion. If the molecule binds to the ligand with a very high affinity, then the molecules will not be eluted out at the end of the process.

• The matrix should be inert and stable. Its property should not change due to change in pH, ionic strength and temperature during the elution step. Sepharose, agarose, cellulose, silica, polymethacrylate are commonly used porous supports with different pore sizes offering larger surface areas.

• The ligand should have a minimum of two binding sites. One to bind with the matrix and the other to bind with the molecule of interest. 

So which affinity ligands can fulfill the previous requirements ?

With a growing demand for recombinant proteins, and in search of the best potential ligand, a new technology emerged : epitope tagging, allowing a high flexibility in terms of purified compounds.

Epitope tagging is a technique that employs genetic engineering to fuse a known epitope - called an affinity tag - to either the C or N terminus of a recombinant protein to facilitate affinity purification (and detection by blot methods). 

A new matrix has been developed using metal ions grafting. This idea has given birth to Immobilized Metal ion Affinity Chromatography technique a.k.a IMAC technology. In fact, Ni2+ or Co2+ can form complexes with 4 or 6 bonds by affinity interaction. These metals can be immobilized on stationary phases grafted with chelating agent that can bind it with 4 coordinations.

Within the expression vector, and just upstream the transgene, it’s possible to insert six histidine codons encoding the peptide tag (invented by Roche). Imidazole competes with the his-tag for binding to the metal-charged resin and thus is used for elution step.

In order to purify proteins containing exposed His tags, companies have developed antibodies targeting N-terminal and C-terminal ends with strong specificity. Immobilized anti-His antibody can then be used to affinity purification.

But is His-tag really « THE » answer to the purification of any recombinant protein ?

In fact, reasons for using another solution over a 6X or  even a 10X His tag, are the fact that His tag may interfere with the proteins structure, functions, or binding characteristics.

For instance, IMAC is not always efficient for purification of difficult-to-express proteins like GPCRs, especially for targets that are expressed at low levels.

Even if Strep-tag/StrepTactin system has showed good outcomes when it comes to GPCR, it remains limited by the high necessary concentrations of biotin and the slowness of the elution step.

In these particular cases, it would be possible to develop a new type of ligand…

In order to circumvent previous mentioned issues, epitope-tagged proteins can be purified using immobilized primary antibodies as ligands.

Short epitope tags such as FLAG, hemagglutinin (HA), c-myc, SUMO and T7 among others, are used for the detection of fusion proteins.

Sometimes, it can also be of interest to generate a Fc-X fusion protein format, rather than recombinant protein X alone. IgG-Fc tag is the constant region of immunoglobulin heavy-chain, and the Fc-tag is about 25 kDa.

• The use of murine Fc facilitates the use of Fc-X to immunize mice for the generation of antibodies, since the murine Fc region should not be immunogenic in the host animal.

• You can confer novel properties to your hybrid molecule. This includes Fc receptor binding, complement fixation, increasing the half-life and crossing the blood–brain barrier.

• Gene fusion construct consisting of signal peptide, the Fc fragment, followed by the protein of interest, facilitates efficient secretion of many different categories of proteins.

• For non-secretory proteins, the over-expression of which may be toxic to the host cell, this technology enhances expression because the protein product is secreted.

• Fc, which is glycosylated and highly charged at neutral pH, helps to solubilize hydrophobic proteins. 

• Protein of interest X in the fusion protein format retains its native conformation and biological activities

• Finally, the use of Fc-X proteins offers an added advantage for ELISA assay. Fc portion of the molecule - with an abundance of positive charged amino acid residues - binds preferentially to the ELISA plate, leaving the X group free to interact with the ligand in the subsequent binding step.

But what about antibodies to purify?

Can we apply immuno-affinity technique like any other protein ?

Antibodies are glycoproteins that specifically recognize antigen. They’re produced by cells of the immune system - called B cells or plasmocytes - in order to block and neutralize foreign agents while infection occurs. In case of auto-immunity diseases (e.g. diabetes, lupus…) antibodies are secreted against host molecules, and are consequently named auto-antibodies.

Antibodies are part of the immunoglobulin family, composed by 4 chains of amino-acids : two light chains and two heavy chains, shaping a Y letter. Each light chain consists of a constant domain and a variable domain; heavy chains are composed of a variable fragment and three or four constant fragments according to the isotype. For a given antibody, the two heavy chains are identical, likewise for the two light chains. 

The association between a variable domain carried by a heavy chain (VH) and the adjacent variable domain carried by a light chain (VL) constitutes the site of recognition of the antigen, called paratope. As for the portion of the antigen that is specifically recognized is named epitope. Thus, an immunoglobulin molecule has two antigen binding sites, one at the end of each arm. These two sites are identical (but intended for different epitopes), hence the ability to bind two molecules of antigen by antibody.   

Affinity purification of monoclonal antibodies has been largely confined to the use of Protein A and Protein G chromatography.

But as new antibody formats are raising, new ligands are appearing…

Staphylococcus aureus protein A is a 42 kDa cell wall constituent characterized by its binding capacity to the Fc portion of immunoglobulins.

Protein A resins are commonly used to purify monoclonal antibodies as a robust capture step.

Protein G was first isolated from Streptococcal bacteria strains C and G. It is a 65-kDa (G148 protein G) and a 58 kDa (C40 protein G) cell surface protein that has found application through its binding to antibodies Fc region.

Protein L is an immunoglobulin-binding protein purified from the bacteria Peptostreptococcus magnus. It is a 76 to 106 kDa protein containing four or five highly homologous, consecutive extracellular immunoglobulin (Ig) binding, or B domains.

Protein L has the unique ability to bind through kappa light chain interactions without interfering with an antibody's antigen-binding site.

In order to specifically purify monoclonal and polyclonal antibodies from rat, MARK ligand has been developed. Rat antibodies are now representing approximately 15% of the entire market of antibodies with an increasing interest as strong binders of small molecules.

• MARK recognizes all rat isotypes (IgG1, IgG2a, IgG2b, IgG2c, IgM)
• No serum contamination, MARK antibodies don’t cross-react with bovine Ig
• Glycin elution, keep the entirety of your antibodies intact 

Complementary to this antibody, MARG recognizes the heavy chain of the rat immunoglobulins.

When researchers are working in serum cell culture conditions for mouse hybridoma cell lines, it is sometimes not possible to opt for serum-free conditions. One clone is not another, and can be very difficult to adapt. This is why we have developed immuno-affinity columns grafted with our rat anti-mouse antibodies. Thanks to their very high specificity, they allow a very high purification yield without catching the bovine antibodies present in the serum.