A versatile, quantitative analytical method for pharmaceutical relevant lipids in drug delivery systems.

Over the past few years, liposomal formulations as drug carrier systems have markedly advanced in pharmaceutical research and development. Therefore, analytical methods to characterize liposome-based formulations are required. One particular issue in liposome analysis is the imbalance of lipid ratios within the vesicle formulations and the detectability of degradation products such as lysophospholipids and fatty acids caused by hydrolysis, especially in low molar ranges. Here, a highly sensitive and selective reversed-phase high-performance liquid chromatography (rp-HPLC) method is described by the combination of an organic solvent/trifluoroacetic acid (TFA) triggered gradient and the application of an evaporative light scattering detector (ELSD). Gain setting adjustments of the ELSD were applied to obtain an optimal detection profile of the analyzed substances. This optimization provides simultaneous separation and quantification of 16 components, including different phosphatidylcholines, phosphatidylglycerols and their degradation products, as well as cholesterol. Parameters such as limit of detection (LOD) and limit of quantification (LOQ) were determined for each of the components and had ranges from 0.25-1.00mg/mL (LOD) and 0.50-2.50µg/mL (LOQ), respectively. The intra-day precision for all analytes is less than 3% (RSD) and inter-day precision is about 8%. The applicability of the method was verified by analyzing two different liposome formulations consisting of DSPC:DPPC:DSPG:Chol (35:35:20:10) and DSPC:DPPC:DSPG (38:38:24). For degradation studies, both formulations were stored at 4°C and at ambient temperature. Additionally, forced degradation experiments were performed to determine hydrolysis mass balances. A total recovery of 96-102% for phospholipid compounds was found. Analytical data revealed that the sensitivity, selectivity, accuracy, and resolution are appropriate for the detection and quantification of phospholipids and their hydrolysis products. These results as well as additional preliminary analyses of other relevant components used in liposomal formulations indicate that the developed method is suitable for the development, characterization, and stability testing of liposomal based biopharmaceuticals.

Application of Bio-Layer Interferometry for the analysis of protein/liposome interactions.

The development of biosensor technologies for the investigation of biomolecular interactions has markedly advanced over the last years. One promising biosensor platform, the Bio-Layer Interferometry (BLI), was developed by ForteBio with the main focus to qualify and quantify protein/protein interactions in research and routine applications. Here, a method to characterize protein/liposome binding interactions based on the biophysical principles of this platform is described. Three different liposome formulations and the protein hormone, recombinant human erythropoietin (rh-Epo) were used as models in the test system. Rh-Epo was immobilized on disposable optical fiber streptavidin (SA) biosensor tips and binding of different liposome formulations under certain conditions was measured. The assay performance was evaluated, followed by calculating the kinetic rate and affinity constants. The results showed that all liposome formulations formed extremely stable complexes with the immobilized protein. Nevertheless, liposome specific differences in binding affinities were determined. Furthermore, a liposome concentration dependent binding pattern was demonstrated. The combination of simple sample preparation, the opportunity of automation with high throughput in an acceptable time range and excellent reproducibility, makes this assay suitable for basic research as well as for drug discovery and drug screening to estimate drug/membrane interactions.