Application of Quality by Design to the robust preparation of a liposomal GLA formulation by DELOS-susp method

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.

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.

Graphical abstract

Fig. 1

Schematic representation of the DELOS-susp method for the preparation of GLA loaded nanoliposomes (nanoGLA). The procedure includes: a) Loading of an organic solution containing the liposome membrane components (Cholesterol, DPPC, Cholesterol-PEG₄₀₀-RGD, MKC) into the high pressure vessel; b) Addition of compressed CO₂ until a certain pressure to produce a CO₂-expanded solution, where all membrane components remain dissolved in a single liquid phase; and c) Depressurization of the expanded solution over an aqueous solution containing the GLA enzyme at atmospheric pressure, obtaining the protein-liposomes nanoconjugates.

Fig. 2

Process Flow Diagram to produce the nanoGLA. Raw materials and the materials leaving the process are also represented. In red, the intermediate nanoGLA formulation obtained by DELOS-susp that has been studied in the QbD implementation. Diafiltration and concentration processes are necessary to remove the remaining free GLA and organic solvents from the nanoformulation and to maximize the enzyme concentration in the final nanoformulation for preclinical studies, respectively.

Fig. 3

Representative cryo-TEM images of nanoGLA DoE samples at 1 week after production. Scale bar 200 nm.

Fig. 4

Left side: scaled and centered coefficients of the regression equations describing the influence of formulation parameters X1-GLA concentration, X2-lipid concentration, X3-Chol-PEG₄₀₀-RGD molar ratio, X4-EtOH concentration, on the liposomes size, ζ-potential, and uni-lamellarity of nanoGLA intermediate dispersion. Right side: contour plots for the same CQAs of the nanoGLA intermediate dispersion. Molar ratio of chol-PEG₄₀₀-RGD to lipid and EtOH concentration were kept constant at 2% mol and 7.5% v/v, respectively.

Fig. 5

The Design Space for intermediate nanoGLA liposomal dispersion that meets the specifications in terms of CQAs, expressed as the probability of failure (%). Molar ratio of chol-PEG₄₀₀-RGD to lipid and EtOH concentration have been optimized in the run at 1.16% mol and 6.2% v/v, respectively.

Fig. 6

Cryo-TEM images of optimized nanoGLA prototype after concentration. The images were acquired 2 weeks after production.

Fig. C.1

Ishikawa diagram illustrating the summary of CPPs and CMAs that may impact on each selected CQA of intermediate nanoGLA dispersion.

Fig. D.1

Evolution of a) mean particle size, b) PdI, and c) ζ-potential over time during 14 days after the production of the samples.