Bridging the gap from lab- to industrial-scalability: Microfluidic optimization for a NP-based Enzyme Replacement Therapy against Lysosomal Storage Disorders.

Lamzede®, containing the recombinant enzyme Velmanase-α, is the first enzyme replacement therapy (ERT) approved for the treatment of the lysosomal storage disorder (LSD) α-Mannosidosis. Since Lamzede® can not cross the blood brain barrier (BBB), it can only ameliorate the peripheral manifestations without addressing those of the central nervous system (CNS). Moreover, when administered in its free-form, the enzyme is sensitive to the biological milieu, requires weekly intravenous administration, and has the possibility to trigger adverse effects linked to its high immunogenicity. Nanomedicine has emerged as a promising strategy to overcome these pitfalls and enable targeted brain delivery of the therapeutic enzyme. Due to the high-cost of Lamzede®, conventional bench-top formulative methods are economically unsustainable: emerging microfluidic (MF) technologies offer a promising alternative, enabling small-scale optimization with minimal waste of reagents. In this view, this thesis work - part of the N2ERT project, which also involved Chiesi Pharmaceuticals - focused on the optimization of a scalable MF protocol for the encapsulation of Lamzede® in BBB-targeted poly-lactic-co-glycolic acid (PLGA) nanoparticles (NPs). The NanotechLab Te.Far.T.I. group already demonstrated the capability of PLGA modified with the peptide g7 to promote CNS internalization; thus small aliquots of PLGA-g7 were added during formulation. Moreover, based on a previously optimized double emulsion protocol for enzyme encapsulation, bovine serum albumin (BSA) was used as a stabilizer. Firstly, a screening with a lab-scale instrument, named syringe pump (SP), was performed showing that the highest encapsulation efficiency (EE%) (around 15%) and the best dimensional stability over time was reached with a flow rate ratio (FRR) o/w of 1:3, a total flow rate (TFR) of 3 mL/min and an enzyme:BSA molar ratio of 1:50. BSA was fundamental to increase the EE%. No significant decrease in EE% was observed when PLGA-g7 was added during the formulation: this trend was maintained during the scaling-up with the automated nanoparticles system (ANP), an industrial scale MF device, where the best EE% was achieved with the same parameters. In both cases, the NPs were under 300nm, had a PDI < 0.3, and a Zeta potential around -25mV, all features desiderable for parenteral administration. Dimensional stability was maintained both at +4°C and, with the addition of trehalose, at -20°C for up to 1 month, 4 cycles of freeze-thawing, and lyophilization. The dimensional stability to lyophilization, assessed with dynamic light scattering and corroborated through SEM images taken before and after the process, is a critical factor to facilitate future product commercialization. Thanks to the collaborators, the maintenance of the enzymatic activity after formulation, and NPs cell-internalization and localization in the same region of lysosomes were assessed. This thesis shows promise for the development of a tunable, BBB-targeted, enzyme-loaded nanoplatform capable of addressing central manifestations of alpha-mannosidosis. Moreover, it will be scalable to industrial production thanks to the ANP system and its GMP-certified counterpart. This paves the way for the development of new versatile therapeutic solutions, suitable for different hard-to-treat diseases that require biological administration and/or CNS targeting.