Cell uptake evaluation of human recombinant lysosomal enzymes produced in Pichia pastoris

Lysosomal enzymes for enzyme replacement therapy (ERT) have been traditionally produced in mammal-derived cells. During the last years alternative hosts have been proposed, such as plant cells, transgenic hens, or microorganisms. Among them, microorganisms represent an alternative to reduce the cost of the ERT or to produce proteins with improved stability, pharmacokinetics, or pharmacodynamics profiles. We have reported the production of active human iduronate-2-sulfatase (IDS), N-acetylgalactosamine-6-sulfate sulfatase (GALNS), and β-N-acetylhexosaminidases (Hex-A, Hex-B, and Hex-S) in the yeast Pichia pastoris. Although these enzymes have similar stability profiles to those reported for the mammal-derived recombinant proteins, the cell uptake and intracellular traffic have not been evaluated. Here, we report the results of cell uptake and intracellular traffic for recombinant IDS, GALNS, and Hex (A, B, and S) produced in P. pastoris. Recombinant enzymes were produced at bioreactor scale (1,7 L) and purified from the extracellular fraction through ion-exchange and size-exclusion chromatography. Purified proteins were added to the media of human skin fibroblasts and HEK293 cells cultures, at concentrations of 10, 50, and 100 nM. Cell uptake assay was carried out at 37 and 4 °C, and in the absence or presence of mannose or mannose-6-phosphate (M6P). For all the studied lysosomal enzymes it was observed a cell uptake in a doses-dependent manner, without any additional protein or host modification. Cellular capture of the recombinant enzymes was reduced when the assay was carried out at 4 °C, suggesting that this is a process potentially and partially mediated by an endocytosis pathway. Inhibition assays showed that each enzyme has a different profile of mannose or M6P inhibition, suggesting a different glycosylation profile for each enzyme. These results confirm the potential of P. pastoris as a host for the production of human lysosomal enzymes towards the development of alternative ERT for these disorders.