Fabry disease is a lysosomal storage disease arising from a deficiency of the enzyme α-galactosidase A (GLA). The enzyme deficiency results in an accumulation of glycolipids, which over time, leads to cardiovascular, cerebrovascular, and renal disease, ultimately leading to death in the fourth or fifth decade of life. Currently, lysosomal storage disorders are treated by enzyme replacement therapy (ERT) through the direct administration of the missing enzyme to the patients. In view of their advantages as drug delivery systems, liposomes are increasingly being researched and utilized in the pharmaceutical, food and cosmetic industries, but one of the main barriers to market is their scalability. Depressurization of an Expanded Liquid Organic Solution into aqueous solution (DELOS-susp) is a compressed fluid-based method that allows the reproducible and scalable production of nanovesicular systems with remarkable physicochemical characteristics, in terms of homogeneity, morphology, and particle size. The objective of this work was to optimize and reach a suitable formulation for in vivo preclinical studies by implementing a Quality by Design (QbD) approach, a methodology recommended by the FDA and the EMA to develop robust drug manufacturing and control methods, to the preparation of α-galactosidase-loaded nanoliposomes (nanoGLA) for the treatment of Fabry disease. Through a risk analysis and a Design of Experiments (DoE), we obtained the Design Space in which GLA concentration and lipid concentration were found as critical parameters for achieving a stable nanoformulation. This Design Space allowed the optimization of the process to produce a nanoformulation suitable for in vivo preclinical testing.
Keywords: BCA, Bicinchoninic acid assay; CMA, Critical Material Attributes; CO2, Carbon dioxide; CPP, Critical Process Parameters; CQA, Critical Quality Attribute; Chol, Cholesterol; Chol-PEG400-RGD, Cholesterol pegylated with arginyl–glycyl–aspartic (RGD) acid peptide; CoA, Certificate of Analysis; Cryo-TEM, Cryogenic Transmission Electron Microscopy; DELOS; DELOS-susp, Depressurization of an Expanded Liquid Organic Solution into aqueous solution; DLS, Dynamic Light Scattering; DMSO, Dimethyl sulfoxide; DPPC, 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine; DoE, Design of Experiments; EA, Enzymatic Activity; EE, Entrapment Efficiency; EHS, Environment, Health and Safety; EMA, European Medicines Agency; ERT, Enzyme Replacement Therapy; EtOH, Ethanol; FDA, Food and Drug Administration; Fabry disease; GLA, α-galactosidase A enzyme; H2O, Water; HPLC, High Performance Liquid Chromatography; ICH, Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use; LSD, Lysosomal storage disorders; MKC, Myristalkonium chloride; N2, Nitrogen; NTA, Nanoparticle Tracking Analysis; PEG, Polyethylene Glycol; PIC, Pressure Indicator Controller; PLS, Partial Least Squares; PdI, Polydispersity Index; Protein-loaded liposomes; Pw, Working pressure; QbD, Quality by Design; Quality by Design; RGD, Arginine-Glycine-Aspartic acid; S-MLS, Static Multiple Light Scattering; SAXS, Small-Angle X-ray Scattering; SDS-PAGE, Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis; SbD, Safe by Design; Scale-up; TFF, Tangential Flow Filtration; TGX, Trys-Glycine eXtended; TIC, Temperature Indicator Controller; TSI, Turbiscan Stability Index; Tw, Working temperature; USP, United States Pharmacopeia; XCO2, Carbon dioxide molar fraction; fsingle, Ratio of monolayered liposomes; nanoGLA, GLA-loaded nanoliposomes; α-galactosidase.
© 2021 The Authors.