Purification and characterization of human alpha-galactosidase A expressedin insect cells using a baculovirus vector

Fabry disease is an X-linked inborn error of glycolipid metabolism caused b y deficiency of the lysosomal enzyme alpha -galactosidase A. The enzyme is responsible for the hydrolysis of terminal alpha -galactoside linkages in v arious glycolipids. To perform more extensive biochemical characterization and to develop new approaches for enzyme therapy, a method of producing and purifying recombinant alpha -galactosidase A suitable for scale-up manufac ture for use in humans is needed. Previously, a catalytically active recomb inant human alpha -galactosidase A was expressed using a baculovirus vector and purified using conventional chromatography. However, the level of expr ession was too low to permit economical production and the chromatographic techniques used for enzyme purification were not suitable for enzyme to be used in humans. Therefore, the cDNA of the enzyme was cloned to an improved baculovirus vector and the enzyme was expressed in a 15-liter bioreactor u sing optimized growth conditions. Infection of insect cells by the baculovi rus resulted in a significant fivefold increase in the level of secreted re combinant alpha -galactosidase A activity that is compatible with economic manufacturing. The recombinant alpha -galactosidase A was purified to homog eneity using ion exchange (Poros 20-CM, Poros 20-HQ) and hydrophobic chroma tography (Tosoether, Toso-butyl) using the BioCAD HPLC workstation. These c hromatographic steps are readily scalable to larger volumes and are appropr iate for the purification of the recombinant human alpha -galactosidase A t o be used in clinical trials of enzyme replacement therapy for Fabry diseas e patients. (C) 2000 Academic Press.