CONTINUOUS SEPARATION AND PURIFICATION OF PROTEINS BY RECYCLING ISOTACHOPHORESIS

The continuous separation and purification of proteins by recycling isotachophoresis (RITP) is described. By operating the RITP fractionation process under counterflow conditions with immobilized sample zones, a configuration is available which allows the continuous feed of crude sample and withdrawal of the purified product. The concept is demonstrated by anionic RITP of bovine serum albumin (BSA) and by cationic RITP purification of ovalbumin (OVA) and lysozyme (LYSO) from a commercial OVA product containing LYSO and conalbumin as major proteinaceous impurities. For the OVA and LYSO purifications, continuous RITP provided a higher sample throughput and comparable product purity compared with batch RITP. In the BSA and LYSO separations the compounds to be purified established their isotachophoretic zones at the front of the sample stack, which represents the simplest example of continuous RITP operation. Continuous purification of OVA as a compound located at the rear of the isotachophoretic zone structure is more complex. With prolonged processing time the removal of proteinaceous impurities which accumulate in the front part of the cell, i.e. near the counterflow inlet, is required. Components which do not form their isotachophoretic zone at or near the edges of the zone structures can only be partially purified by continuous RITP. For such compounds a semi-continuous mode of operation in which sample infusion and the withdrawal of purified product are alternated is proposed.