Abstract
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.
Highlights
One of the main challenges to produce advanced materials such as enzyme-loaded liposomes is the successful implementation of new manufacturing nanotechnologies at industrial scale
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 Food and Drug Administration (FDA) and the European Medicines Agency (EMA) to develop robust drug manufacturing and control methods, to the preparation of α-galactosidase-loaded nanoliposomes for the treatment of Fabry disease
The optimized intermediate GLA-loaded nanoliposomes (nanoGLA) was diafiltrated and concentrated by Tangential Flow Filtration (TFF) in order to remove the organic solvents and to reach the target enzyme concentration, which cannot be directly achieved in the DELOS-susp step because of the limited solubility of the liposome membrane components in the organic phase, while keeping the nanoformulation quality
Summary
One of the main challenges to produce advanced materials such as enzyme-loaded liposomes is the successful implementation of new manufacturing nanotechnologies at industrial scale. To overcome the main limitations of the current ERTs (e.g. rapid enzyme degradation, high immunogenicity, short circulation half-life, poor biodistribution, and limited efficacy among others [6]) a strategy under development consists in the encapsulation of the GLA enzyme into nanoliposomes. Such encapsulation aims to protect the enzyme, decrease the degradation and immunogenicity of GLA, and increase its plasma half-life. GLA-loaded nanoliposomes (nanoGLA) functionalized with Arginine-Glycine-Aspartic acid (RGD) peptide and prepared by DELOSsusp, have recently been reported to increase enzymatic activity and intracellular penetration, showing the great potential of this CO2-based methodology for the simple production of protein-nanoliposome therapeutic conjugates [4,7].
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