Purification of antibodies through convective chromatography

Abstract

Downstream processing of immunoglobulin G is conventionally performed by bead-based column chromatography; affinity chromatography with Protein A is the most selective process in biotechnological industry for the purification of antibodies. However, this method suffers from several limitations, such as high pressure drops across the column, compaction of the granular porous bed and low diffusivity between the particles. In addition to fluid dynamic problems, the traditional process is affected by limitations related to the use of Protein A as affinity ligand. Materials activated with Protein A are very expensive, since the production cost of this recombinant protein is very high. Finally, ligand leakage causes contamination of the final product. The aim of the present work was to overcome the limitations that affect the traditional process through the development of new convective chromatographic supports, like membranes and monoliths, functionalized with synthetic affinity ligands to be used instead of columns packed with Protein A beads. The performance of regenerated cellulose membranes functionalized with two affinity ligands were studied; the commercial A2P ligand and the new recently synthesized HPTA were used to replace Protein A. Static and dynamic binding capacity and elution recoveries, were determined for the affinity membranes prepared, using pure solutions of IgG. Mixtures and complex solutions containing IgG were tested to determine ligands selectivity. A processing method for the preparation of porous cellular ceramic monoliths was developed. Cellular Al2TiO5 and Al2TiO5-Al2O3 composites ceramics were obtained by emulsification of liquid paraffin in aqueous suspensions of mixed Al2O3 and TiO2 powders, with subsequent burnout of the organic phase and two-step reactive firing. The ceramic monolith were fully characterized to assess their use as novel chromatographic stationary phases, measuring porosity, axial dispersion coefficient and permeability; the surface was chemically modified to obtain functional groups to be used for ligand or direct protein immobilization.