Nov. 5, 2018: 10:30 a.m. - 11 a.m.
Lysosomal storage diseases arise from abscence or deficiency of lysosomal enzymes and the accumulation of their substrate within the patient's lysosomes. Currently, recombinant enzymes used for enzyme replacement therapy are mostly produced in mammalian cell-lines. We are creating a platform technology for the production of recombinant human lysosomal enzymes in Pichia pastoris. We engineered the N-glycosylation pathway of glycoproteins secreted by Pichia pastoris (P.p.) to obtain high levels of mannose-6-phosphate (M6P) modifications, which are required for lysosomal targeting1. As a first application, the enzyme alpha-galactosidase A (GLA) was produced. Since the homodimeric GLA is prone to aggregation or dissociation into monomers, a competitive inhibitor such as deoxygalactonojirimycin (DGNJ) or galactose was added during production. This decreases aggregation and enhances stability of the correct fold during purification. The enzyme was purified through nickel affinity chromatography, anion exchange and size exclusion chromatography steps. During purification, acidic buffers (pH 6) are used to keep the enzyme active. These actions result in a homogeneous enzyme with a specific activity in the same range as the current therapeutic standard enzyme.
Purified GLA is being evaluated for uptake efficiency and activity in fibroblasts derived from a Fabry disease patient.
1. Tiels, P. et al. A bacterial glycosidase enables mannose-6-phosphate modification and improved cellular uptake of yeast-produced recombinant human lysosomal enzymes. Nat Biotech 30, 1225–1231 (2012).