Science & Research

Critical Process Parameters for the Preparation for Amphotericin B Liposomes

Principal Investigator: Alex Nivorozhkin, Ph.D.

Performer: Neo-Advent Technologies LLC

Project Duration: 09/23/2016 – 09/22/2017

Regulatory Science Challenge:

AmBisome® is a commercially produced liposomal formulation of Amphotericin B approved by the FDA in 1997 to treat fungal infections. The drug is made in the form of a freeze-dried powder consisting of single bilayer liposomes of less than 100 nm in diameter, with Amphotericin B intercalated into the liposomal membrane. Ambisome® is administered intravenously through a controlled infusion device, and it works by binding to the fungal cell membrane, leading to the rupture of the cell wall, and consequently cell death.

As the AmBisome® patent (currently owned and licensed by Gilead) is due to expire, it is predicted that various pharmaceutical companies will produce generic versions of AmBisome®. The prospect of this presents a regulatory challenge for the FDA as 1) the manufacturing process for liposomal drug products (LDP) is highly complex, and 2) information on the overall development pathways specific to LDP is very limited. Developing approaches to properly evaluate Amphotericin B generics, as well as other liposomal drug platforms manufactured under different processes and methodologies will guide the generics industry in their development of bioequivalent amphotericin B liposome products, as well as support the FDA in understanding how variability in product manufacture can affect overall product quality.

Project Description & Goals:

The goals of the project were to 1) evaluate manufacturing effects on the critical quality attributes (CQAs) of liposomal amphotericin B products and 2) identify which analytical methods are most sensitive to changes in the CQAs.

For the first goal, the researchers from Neo-Advent Technologies, LLC surveyed a range of current liposomal manufacturing techniques, and applied these techniques using the Quality by Design approach in the production of Amphotericin B. The first explored areas were commercial manufacturing techniques for large-scale production. Processes such as homogenization, sonication, alternate shear force method (using a microfluidizer), and some of the latest experimental liposome production technologies (for example, focused acoustics) were evaluated. In addition, manufacturing conditions such as temperature, pH, shear forces, and product recovery techniques were also tuned for method optimization (to obtain maximum product yield). For the second goal, which is to properly characterize the products made from each manufacturing process, the following physicochemical properties of the liposomes were measured:

  • Drug and lipid content
  • Drug-to-lipid ratio
  • In vitro release profile
  • Particle size – mean and distribution
  • Percentage of drug encapsulation
  • pH
  • Surface charge
  • Phase transition temperature


  • Critical Process Parameters for the Preparation of Amphotericin B Liposomes (Internal presentation, FDA Workshop: Demonstrating Equivalence of Generic Complex Drug Substances and Formulations, October 6, 2017)

Page Last Updated: 11/16/2017
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