(200g) Continuous Processing of Doxorubicin-Loaded Liposomes

Complex dosage forms such as liposomes have tremendous potential to revolutionize the pharmaceutical industry and provide significant health benefits. One challenge is to process large quantities of these nanoparticles while also maintaining physicochemical properties to ensure a high-quality formulation. One approach to overcome challenges was to turn to continuous processing. Continuous processing can achieve improved process control (via process analytical technology), while avoiding inherent issues associated with batch processing to ultimately improve product quality and reduce manufacturing cost. Accordingly, we developed a continuous process for liposomes. In this work, we outline important steps in the particle formation process, in-line/ at-line measurements, in-line concentrating and drug-loading of doxorubicin-HCl.

METHODS

Risk Assessment

Ishikawa diagrams were formed to outline process parameters and material attributes that had the likelihood to effect quality attributes. Some common critical quality attributes (CQAs) for nanoparticle formulations include particle size distribution (z-average particle size and polydispersity index), zeta-potential, nanoparticle concentration and encapsulation efficiency. A custom-built system was designed to incorporate process control strategies to ensure CQAs were within user specifications.

Nanoparticle 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 using National Instruments LabVIEW. Briefly, the continuous processing system consists of multiple tanks that contain lipid dissolved in ethanol and another set of tanks for the aqueous components (e.g. DI water or buffer salts). The system further consists of an injection port such that a turbulent jet is formed under controlled flow conditions with downstream conditioning modules and concentrating modules.

Active-loading Doxorubicin-HCl into Liposomes

Hydrogenated soy phosphatidyl-choline (HSPC), cholesterol and DSPE-mPEG2000 or DSPG were prepared at a molar ratio of 5.6:3.8:0.52 and were dissolved in ethanol. The lipid solution was subsequently injected into the aqueous phase (250 mM Ammonium Sulfate) via the co-flow injection port. Liposomes were then concentrated downstream and doxorubicin-HCl was injected into the pre-formed liposomes under controlled conditions. Offline UV/HPLC measurements were performed to confirm loading percentage.

RESULTS

HSPC:Chol:DSPE-mPEG2000 and HSPC:Chol:DSPG liposomes were successfully formed under different processing conditions. The z-average particle size could be controlled from approximately 40.0 to >150 d.nm with a PDI of less than 0.10 under most conditions. For the doxorubicin loading studies, high encapsulation efficiency was achieved using the liposomes prepared on our continuous processing system.

CONCLUSION

Continuous processing of liposomes that incorporates a turbulent jet in co-flow enables a fine-control of nanoparticles. On-line/ at-line measurements techniques are important to characterize and provide feedback during the continuous process and can easily be implemented into a process control strategy. Lastly, we have provided groundwork on continuous processing of liposomes that can be used to make doxorubicin-loaded liposomes with similar quality attributes of existing drug products.

ACKNOWLEDGEMENTS

This work was supported by the FDA (HHSF223201610121C and 1U01FD005773-01).

DISCLAIMER

This article reflects the views of the authors and should not be construed to represent FDA’s views or policies.