Fluidized‐bed receptor‐affinity chromatography

Abstract

A multipurpose fluidized-bed receptor-affinity purification system based upon the biological recognition between an immobilized receptor and its soluble protein ligands is described. The fluidized affinity sorbent consists of a soluble form of interleukin-2 receptor chemically bonded to an aldehyde derivative of controlled pore glass beads, which have a pore diameter of 1000 A and a particle density of 1.2-1.3 g/mL. The fluidized-bed separation device used in this study consists of a specially designed column fitted at the inlet end with a perforated distributor plate covered with a screen and the top outlet with an adjustable piston. The fluidized-bed consisting of a loose gel matrix permits the unimpeded passage of cell debris and particulate matter, while the target protein is captured by the affinity beads. Purification of the humanized-anti-Tac monoclonal antibody is used as a model system to determine the operational parameters. Also, fluidized-bed receptor-affinity chromatography has been successfully employed in the purification of recombinant interleukin-2 and single chain anti-Tac(Fv)-Pseudomonas exotoxin immunotoxin from unclarified inclusion body extracts. Overall, fluidized-bed receptor-affinity chromatography is found to be a productive affinity method suitable for the purification of recombinant human interleukin-2 and related molecules.

Measurement of Total and Bioactive Interleukin-2 in Tissue Samples by Immunoaffinity-Receptor Affinity Chromatography

“…In the present report, HPIAC has been shown to efficiently isolate bioactive IL-2 from a complex biological matrix, such as whole tissue extract, and concentrate this material prior to detection by a specific biological ligand. The incorporation of an immobilized receptor as the capture ligand in the detection cartridge greatly enhanced the selectivity of this detector and similar techniques that have been used in fluidized beds for isolating specific forms of recombinant molecules (Spence et al, 1994). In analytical applications, incorporation of such materials enables the measurement of the bioactivity of the analyte (Scheller and Schubert, 1992) which can be extremely useful when used to measure active biological molecules such as cytokines.…”