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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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

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., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Currently, one of the most used techniques for the determination of isoform pattern analysis is isoelectric focusing. Routinely, this is performed by immunoblotting. Blotting of proteins after isoelectric focusing on IPG gels may cause several problems, such as protein loss by the blotting itself and band broadening, in some cases the immunostaining with antibodies might be problematic. In the present study, an alternative isoform prestaining method with CyDye fluors is presented. For this approach, a highly glycosylated fusion protein, Epo-Fc, was used consisting of two recombinant human erythropoietin attached to the Fc part of a human IgG(1) molecule. By using CyDye fluors, up to three samples can be focused on the same lane under identical electrophoretic conditions. A fundamental benefit of this technique is the ability to perform quantitative isoform pattern analysis directly from serum-free culture supernatant.

A computer-based technique using a 2((5-2)) fractional factorial design was applied for screening the factors affecting the penetration effectiveness of liposomal recombinant human-Cu/Zn-superoxide dismutase. Unilamellar liposomes, containing recombinant rh-Cu/Zn-SOD in the aqueous core, are aimed at enhancing the penetration of the drug applied topically. Factors that mainly influence the chemical and physical characteristics of liposomes such as charge, molar content of cholesterol, size, surfactant and lipid were evaluated at two levels. In vitro skin penetration studies with pigskin were carried out in Franz-type diffusion cells over a period of 4 and 8h. The response variables, namely the amounts of rh-Cu/Zn-SOD penetrated into the different skin layers, were analyzed by ELISA (enzyme linked immunosorbent assay). Analysis of variance showed that the size and the cholesterol content of liposomes as well as the duration of the penetration studies have a statistically significant influence on the amount of protein found in deeper skin layers.