A potent, long-lasting form of interferon alpha-2a mono-pegylated with a 40 kilodalton branched poly(ethylene glycol) was designed, synthesized, and characterized. Mono-pegylated interferon alpha-2a was comprised of four major positional isomers involving Lys31, Lys121, Lys131, and Lys134 of interferon. The in vitro anti-viral activity of pegylated interferon alpha-2a was found to be only 7% of the original activity. In contrast, the in vivo antitumor activity was severalfold enhanced compared to interferon alpha-2a. Pegylated interferon alpha-2a showed no immunogenicity in mice. After subcutaneous injection of pegylated interferon alpha-2a, a 70-fold increase in serum half-life and a 50-fold increase in mean plasma residence time concomitant with sustained serum concentrations were observed relative to interferon alpha-2a. These preclinical results suggest a significantly enhanced human pharmacological profile for pegylated interferon alpha-2a. Results of Phase II/III hepatitis C clinical trials in humans confirmed the superior efficacy of pegylated interferon alpha-2a compared to unmodified interferon alpha-2a.

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.