12 research outputs found

    Lyophilization of synthetic gene carriers

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    Lyophilization, also known as freeze drying, is a widely used method for stabilization, improvement of long-term storage stability, and simplification of handling of drugs and/or carrier systems. Lyophilization is time-consuming and energy-consuming, and hence optimized processes are required to avoid time loss and higher costs without compromising product stability. Beginning from the last decade, nonviral, synthetic carriers for gene delivery have been of increasing interest. However, these systems suffer from poor physical stability in aqueous solution or suspension. Hence, to ensure long-term storage stability lyophilization of the gene carrier systems is favored. This chapter gives an overview of the basic steps and troubleshooting for successful lyophilization of synthetic gene carriers. Furthermore, the required excipients and their mechanism of action are summarized

    Characterizing the Freeze–Drying Behavior of Model Protein Formulations

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    The freeze–drying behavior of three model proteins, namely, lysozyme, BSA, and IgG, has been studied using a variety of techniques under two different primary drying conditions (shelf temperatures of −25°C and +25°C, respectively) in an amorphous formulation. Manometric temperature measurements were used to characterize product temperature (Tpr), sublimation rates, and product resistance (Rp) during primary drying. Biophysical techniques such as circular dichroism, fluorescence, and Fourier transform infrared spectroscopy were used to study protein conformation. Size exclusion chromatography was used to monitor the formation of high-molecular-weight species (HMWS) over time on storage, and cake morphology was studied using scanning electron microscopy. The differences in the freeze–drying behavior of the three proteins were more evident at higher protein concentrations, where the protein significantly influences the behavior of the formulation matrix. However, these differences were minimized in the aggressive mode and were insignificant at lower protein concentrations where excipients dominated the freeze–drying behavior. Differences in cake morphology were observed between the two drying conditions employed as well as between the three proteins studied. The stability and the protein structure, however, were equivalent for the protein cakes generated using the two different primary drying conditions

    Long-Term Stability of a Vaccine Formulated with the Amphipol-Trapped Major Outer Membrane Protein from Chlamydia trachomatis

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    Chlamydia trachomatis is a major bacterial pathogen throughout the world. Although antibiotic therapy can be implemented in the case of early detection, a majority of the infections are asymptomatic, requiring the development of preventive measures. Efforts have focused on the production of a vaccine using the C. trachomatis major outer membrane protein (MOMP). MOMP is purified in its native (n) trimeric form using the zwitterionic detergent Z3–14, but its stability in detergent solutions is limited. Amphipols (APols) are synthetic polymers that can stabilize membrane proteins (MPs) in detergent-free aqueous solutions. Preservation of protein structure and optimization of exposure of the most effective antigenic regions can avoid vaccination with misfolded, poorly protective protein. Previously, we showed that APols maintain nMOMP secondary structure and that nMOMP/APol vaccine formulations elicit better protection than formulations using either recombinant or nMOMP solubilized in Z3–14. To achieve a greater understanding of the structural behavior and stability of nMOMP in APols, we have used several spectroscopic techniques to characterize its secondary structure (circular dichroism), tertiary and quaternary structures (immunochemistry and gel electrophoresis) and aggregation state (light scattering) as a function of temperature and time. We have also recorded NMR spectra of (15)N-labeled nMOMP and find that the exposed loops are detectable in APols but not in detergent. Our analyses show that APols protect nMOMP much better than Z3–14 against denaturation due to continuous heating, repeated freeze/thaw cycles, or extended storage at room temperature. These results indicate that APols can help improve MP-based vaccine formulations
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