Standardization of the Reconstitution Procedure of Protein Lyophilizates as a Key Parameter to Control Product Stability.

Lyophilization of protein formulations is an essential tool for stabilization and is becoming increasingly important for pharmaceutical development. Reconstitution of the lyophilized cakes is crucial to obtain an applicable product. Nowadays, manual reconstitution by patients or medical staff is the common method defined in instructions for marketed lyophilized drug products. Even though this step is influencing the quality of the final solution, it can represent a challenge to develop a standardized manual protocol and the performance is highly dependent on human factors. This study summarizes the implementation and performance of controlled reconstitution studies for protein lyophilizates applying a mechanical reconstitution device. Using automated and standardized protocols, reconstitution time of a bispecific antibody lyophilizate could be reduced effectively from 25 to below 5 min compared to the predeveloped manual protocol. It was shown that the reconstitution protocol is influencing the stability of sensitive proteins. Monomer content as well as formation of subvisible particles differed considerably between the tested protocols emphasizing the relevance of standardized procedures.

Protein-Excipient Interactions Evaluated via Nuclear Magnetic Resonance Studies in Polysorbate-Based Multidose Protein Formulations: Influence on Antimicrobial Efficacy and Potential Study Approach.

Preservatives are excipients essentially needed in pharmaceutical multidose formulations to prevent microbial growth. Among available substances, phenol is widely used for parenterals; however, it is known to interact with nonionic surfactants like polysorbate and potentially with the active pharmaceutical ingredient. Although the need for combinations of surfactants and preservatives is growing, to date possible molecular interactions which can eventually weaken the stability and antimicrobial activity of the formulation are not yet well understood and properly investigated. In the current study, the binding of phenol to a model fusion protein as well as to polysorbate 20 was investigated. For this purpose, the fraction of bound phenol was successfully quantified via diffusion ordered nuclear magnetic resonance spectroscopy. The binding of phenol to the surfactant is negligible in pharmaceutically relevant polysorbate concentrations, but the binding to the employed active pharmaceutical ingredient was relevant and concentration dependent. The resulting consequence of this interaction was the decrease of the antimicrobial efficacy. As a final outcome of this study, nuclear magnetic resonance analysis is proposed as a material saving method to be used in combination with the antimicrobial activity testing described in the Pharmacopeias.

Electrospray Synthesis of Poly(lactide-co-glycolide) Nanoparticles Encapsulating Peptides to Enhance Proliferation of Antigen-Specific CD8+ T Cells.

Polymer nanoparticles (NP) are of escalating interest for their application as immune stimulatory pharmaceutics. The production of nanosized carrier systems is currently being widely investigated, but commonly used techniques, such as the double emulsion technique, are limited by shortcomings of low encapsulation efficiency and poor control over size distribution. In this study, the electrospray technique was successfully implemented and optimized to produce monodisperse 200-nm poly(lactide-co-glycolide) (PLGA) NP. For cytomegalovirus (CMV) pp65 and IE-1 peptides, a consistent encapsulation efficiency of approximately 85% was achieved. In vitro stimulation of peripheral blood mononuclear cells (PBMCs) from CMV+ donors using electrosprayed pp65489-503 peptide-loaded NP revealed a significantly increased proliferation rate and frequency of antigen-specific CD8+ T cells as compared to the soluble peptide. The results of this study demonstrate the suitability of the electrospray technique for production of monodisperse PLGA NP with high drug encapsulation efficiency as promising peptide-based vaccine carriers.