The development of a scalable bioreactor process for the production in P. pastoris of recombinant human α -Glucocerebrosidase (rhGC), aiming to implement a reproducible process to obtain significant amounts of the enzyme. One of the main advantages of the use of P. pastoris in protein production is the extraordinary cell densities that can be reached in bioreactor cultures by the yeast. Indeed, >10 g/L of product titers have been documented in P. pastoris for certain proteins. Methanol-limited fed batch (MLFB) has been one the most extended strategies for the protein production in P. pastoris. Unlike MLFB, non-limited cultures require a complex set-up for the control of cultivation parameters, representing technical and economic constraints. The methanol-limited strategy can be divided in three major phases. The first two (glycerol batch and fed-batch) have as a clear objective the fast increase of cell biomass. The process was not only useful in terms of productivity, but also allowed to decrease dramatically the consumption of pure O2 and improved notably the stability and reproducibility of the cultures. This novel strategy was then used to compare the bioreactor productivity of 2 different strains that showed a marked differences in specific productivity at the 10 mL-scale of the clone screening. Performing in vitro and in vivo studies often require high amounts of the protein to be tested, ranging usually from milligrams to a few grams. Finally, methods were defined for the characterization of the purified enzyme, with the aim to describe its properties when produced in a wild type P. pastoris strain (i.e. NRRLY-11430).