Optimization and Comparation of Hidrophobic Interaction Chromatography, Strong Cation Exchange Chromatography and Capillary Isoelectric Focusing to Purify Anti-TNFα Fragments obtained After Papain Digestion

Antibodies are powerful tools used in therapy nowadays. A number of antibodies have been used to the treatment of inflamatory diseases. Several chimeric, humanized and full human antibodies have been used to inibit the tumor necrosis factor-α (TNFα), responsible for the inflammation, but in some applications the use of fragments could be a useful alternative to full length antibodies because their small size which could lead to a fast clearance from the circulation and to a low immunogenicity.

When and antibody fragment is employed as a therapeutic molecule, the application of appropriate tools for their purification is an important requirement. Strong Cation Exchange Chromatography (SCX) and Hydrophobic Interaction Chromatography (HIC) have been described in the literature as efficient techniques for the purification of biomolecules, mostly because of their ability to reduce the sample pretreatment. Capillary isoelectric focusing (cIEF) is also used for the separation of monoclonal antibodies presenting advantages such as the potential for automation and shorter analysis time.

This work shows the results of the application of HIC, SCX and cIEF to analyse the fragments of full-human Anti-TNFα antibody after papain digestion. A comparison in terms of efficiency, accuracy and resolution was carried out and the advantages and disadvantages of using the mentioned approaches for the purification and identification of these fragments are also discussed.