Affinity Chromatography is undoubtedly the most potent way to separate one active compound from its environment. This strategy is illustrated by the use of Protein A for purification of monoclonal antibodies. The lack of specific enough ligands has up to now hampered the adoption of affinity chromatography for other biologics such as blood products, vaccines and antibody fragments. Nanofitins® bear all the features that could help introduce affinity chromatography as a major step in most downstream processing for these biologics.

Performance comparable with commercial products

Nanofitins® allow capture and elution of selected macromolecules ranging from smallAffinity-Chromato-Image-1 peptides to viruses. Nanofitins® have already been tested in comparison with Protein A with a level of performance comparable to most available commercial products. They could exhibit the same level of performance against virtually any macromolecular target in affinity columns or membranes. They are also particularly amenable to immunoprecipitation processes thanks to their robustness.

In order to enhance the capture capacity of the resins, spacers are introduced between the ligand and the beads composing the resin. In the case of Nanofitins®, it is possible to genetically engineer multimeric Nanofitins®. This allows using strings of Nanofitins® serving as active spacers. A dimer has already demonstrated a binding capacity 1.8 times higher than that of a monomer for the same amount of capture molecules

Custom purification with tailored elution parameters

Usual selection process takes 2 months and yields a number of different monomeric hits with a high affinity down to single-digit picomolar. However, affinity can be tailored in the 10E-5 – 10E-12 range directly from within the selection process for affinity chromatography needs where release of the target is an essential step.

In the case of Protein A capture of antibodies, the usual elution is conducted through a drastic pH shift (pH = 2.5). This can be extremely damaging for some products. The Nanofitin® discovery process allows choosing and engineering ligands that can dissociate from the target under previously selected conditions.

A straightforward conjugation process

The conserved region, common to all Nanofitins®, bears the regions used for conjugatioAffinity-Chromato-Image-2n to resins, beads, membranes,… In particular, regioselective conjugation is possible through insertion of one single Cysteine in the Nanofitin® sequence, thereby offering identical thiol-based covalent coupling. All Nanofitins® can undergo the very same process for incorporation into chromatography media, with similar behavior in terms of association to the material.