Category: Manufacturing and Bioprocessing
Purpose: Liposomal formulations hold significant promise in developing state of the art drug products. Because of the versatility of these nanoparticles, they can be used in an abundance of applications including anti-cancer, anti-fungal, nucleic-acid delivery and immune therapy. One challenge is to meet strict specifications of quality attributes post-liposomal processing. Our approach was to turn to continuous processing, where we leveraged process analytical technology and continuous flow characteristics to maintain liposomal physicochemical properties throughout processing. In this work, we outline a design of experiments optimization strategy and sterile processing for 20-liter lots.
Methods: Liposomal Formation Process
The nanoparticles were formed using an ethanol injection method with a custom-built continuous processing system that is controlled using a program written in National Instruments LabVIEW™. An injector forming a turbulent jet in co-flow was used to form the nanoparticles. At-line particle size analysis was performed using a Malvern Zetasizer™ Nano.
Design of Experiments: HSPC/Cholesterol Liposomes
A full factorial DOE was run to relate processing parameters and material attributes for a lipid formulation of HSPC/Cholesterol at a molar ratio of 59.7/40.3. Factors included liposomal formation temperature, aqueous phase flow rate, water percentage and ethanol temperature. Responses included the Z-Average particle size, polydispersity index (PDI) and zeta-potential.
Optimization was performed on the DOE study above to establish a fixed particle size (~80 d.nm) and a low PDI (< 0.1). A 20-Liter lot of liposomes was collected and sterile filtered through a Millipore Opticap® XL 300 filter.
Results: The DOE study established relationships between the processing conditions for this formulation. For the Z-Average particle size, flow rate and formation temperature were statistically significant terms, whereas ethanol temperature was not. Z-average particle size ranged from ~45 d.nm up to 240 d.nm for this formulation. For PDI, ethanol temperature did not significantly reduce the PDI, but higher temperatures did reduce PDI variability. In many cases, the PDI was controlled to less than 0.1 (indicating monodispersed liposomes). Post-optimization of the DOE analysis was used to establish processing conditions for test lots of 20-Liter liposomal collections. The at-line particle size data is shown in Figure 1 and includes 95% confidence and prediction bands. For the sterile filtration, liposomes were sterile filtered at 800 mL/min and achieved a high Vmax of up to 9620 L/m2. In addition, particle size data was non-significant (one-way ANOVA, alpha=0.05) for before and after sterile filtration.
Conclusion: Using our liposomal continuous processing system, processing conditions were optimized based on DOE experiments to predictably form monodispersed liposomes at 80-90 d.nm. We successfully sterile filtered to 20-liter lots and are able to demonstrate that much greater volumes can be sterile filtered (e.g. up to 278 Liters from a single, 0.029m2 filter). At-line particle size measurements demonstrated the importance of process analytical technology that may be implemented into a process control strategy. Lastly, this continuous processing system allows for rapid analysis of relationships between process parameters and material attributes, enabling faster development of a design space for drug candidates.
FDA Grant# 1U01FD005773-01.
This article reflects the views of the authors and should not be construed to represent FDA’s views or policies.
Xiaoming Xu– Senior Staff Fellow, U. S. Food and Drug Administration, Silver Spring, Maryland
Su-Lin Lee– Science Staff, USFDA, Silver Spring, Maryland
Celia Cruz– Division Director, FDA/CDER/OPQ/OTR/DPQR, Silver Spring, Maryland
Bodhi Chaudhuri– Associate Professor, University of Connecticut, Storrs, Connecticut
Diane Burgess– Distinguished Professor of Pharmaceutics, University of Connecticut, Storrs Mansfield, Connecticut