Affinity Perfusion Chromatography

Applications of affinity chromatography for quantitative analysis (1 –10 ), purification at laboratory scale (11 –16 ), and for large-scale manufacture of recombinant DNA technology derived therapeutics (17 –23 ), have been continually expanding since the first application introduced by Cuatrecasas in 1968 (24 ). Although there have been some examples of the use of rigid high-performance liquid chromatography (HPLC)-based supports for affinity chromatography, the majority of applications are found on agarose-based particles. The historic utility of agarose as an affinity support stems from attributes such as a well-developed base of activated chemistries, relatively large pore volume for immobilization of proeteinaceous ligands, chemical inertness, and charge neutrality (25 –29 ). However, the particle porosity is defined by the degree of swelling and therefore, the holdup of solvent acts as a stagnant pool inside the particle. This solvent pool leads to slow mass transport to interior binding sites, necessitating relatively low operating flow rates and long cycle times (30 –33 ). For many years, these attractive features, overcame the inherently slow speed of operation dictated by these soft gel columns.