(W1030-01-04) Preparation and Scale-Up of Liposomal Amphotericin B Formulation and a Comparison with Commercially Available Counterpart, Ambisome®

Purpose: The study investigates the development of an AmBisome formulation using a Good Manufacturing Practice (GMP) flow machine and micro-vortex mixing for large-scale production. AmBisomes® are liposome-based drugs commonly used for treating various diseases, and the development of scalable production methods is crucial for clinical applications, commercialization, cost-effectiveness, and quality control. The FlowGMP machine can produce small batch sizes of 40mL and larger volumes of up to 50L with advanced technology that ensures quality and consistency. The machine allows for studying different parameters to determine their effect on particle size and drug entrapment efficiency. The optimized AmBisome formulation has a mean particle size of 80 nm, a positive zeta potential of -20 mV, and an encapsulation efficiency of over 80%, indicating good stability. The study demonstrates the potential of the FlowGMP machine in the large-scale production of liposome-based drugs and the use of micro-vortex mixing as a scalable production method. The results provide valuable insights into the physical and chemical properties of the optimized AmBisome formulation and its potential for clinical use.

Methods: The manufacturing process involves two primary phases: the lipid phase, containing a specific molar ratio of HSPC, Cholesterol, DSPG-Na, Alpha Tocopherol, and Amphotericin B, and the aqueous phase, containing sucrose and di-Sodium succinate hexahydrate. These two phases are mixed through micro-vortex mixing at specific flow rates and temperatures, forming liposomes. Then using a Vivaflow 200 filter the liposomes are purified by removing ethanol and DMSO from the liposome mixture with the Tangential Flow Filtration (TFF) process. The liposomes are then characterized by determining particle size distribution, mean particle size, peak summary, Polydispersity Index (PDI), and zeta potential using a DLS analyzer. The encapsulation efficiency of Amphotericin B is quantified using UV-visible spectrophotometry. Further visualization of the bilayer is achieved through Transmission Electron Microscopy (TEM). The liposomes are lyophilized for long-term storage to remove water content, converting them into a dry cake form. The dry cake's redispersibility and drug entrapment are then assessed. Stability studies are performed using Slide-A-Lyzer Dialysis Cassettes to examine drug release rates at room temperature and 4°C over 5 days.

Results: The manufacturing process of Liposomal AmBisomes was successfully following the outlined procedures. The formation of the liposomes was achieved through the microfluidization of the lipid phase and the aqueous phase. This was proved by the characterization results showing a mean particle size distribution predominantly within the desired range of 60-80 nm, validating the effectiveness of the mixing process. The Polydispersity Index (PDI) values were consistently below 0.2 across samples, indicating a high uniformity in size distribution. Furthermore, zeta potential measurements, which are critical for predicting the storage stability of the liposomes, were within the expected range, suggesting that the Ambisomes would maintain their integrity over time. The encapsulation efficiency of Amphotericin B in the liposomes was quantified using UV-visible spectrophotometry showing a high efficiency rate of 93%. This indicates that a significant portion of the drug was successfully encapsulated within the liposomes, which is vital for the therapeutic efficacy of the Ambisomes. Additionally, Transmission Electron Microscopy (TEM) was utilized to visualize the bilayer structure of the liposomes. This analysis confirmed the expected structural characteristics of the liposomes, with no apparent anomalies detected. Stability studies conducted over 5 days at room temperature and 4°C showed that the liposomes maintained their size, structure, and drug content, indicating good physical and chemical stability. This stability is critical for the practical application of the Ambisomes in a clinical setting, ensuring that they remain effective over their intended shelf life. Finally, the liposomes underwent a lyophilization process for long-term storage, resulting in a dry cake form. The redispersibility of this dry cake was assessed, confirming that upon rehydration, the liposomes reverted to their initial homogeneous state, retaining their particle size, drug entrapment, and stability. This aspect is particularly important for the transportation and storage of the Ambisomes, as it ensures that they can be effectively reconstituted before use.

Conclusion: The study successfully demonstrated the efficient and reproducible manufacturing of Liposomal AmBisomes with desirable characteristics. The Ambisomes exhibited a uniform size distribution, high encapsulation efficiency of Amphotericin B, and maintained stability under various conditions. The lyophilization process proved effective in extending the shelf life of the liposomes without compromising their integrity upon rehydration. These findings suggest that the developed Ambisomes have significant potential for clinical application, providing a stable and effective delivery system for Amphotericin B. Further studies and clinical trials may be required to fully establish the therapeutic efficacy and safety of these liposomal formulations.

Figure 1: Liposome manufacturing process step-by-step overview.

Figure 2: Microfluidic vortex mixer setup with lipid and aqueous Solutions: Pumps with attached tubes deliver the two solutions to the mixer, where they are combined to form a liposome solution. The arrows indicate the direction of fluid flow.

Figure 3: Filtration of Liposomal AmBisomes with a Tangential Flow Filtration System (TFF). The arrows indicate the direction of fluid flow.