Optimization of layering technique and secondary structure analysis during the formulation of nanoparticles containing lysozyme by quality by design approach.

In our study, core-shell nanoparticles containing lysozyme were formulated with precipitation and layering self-assembly. Factorial design (DoE) was applied by setting the process parameters during the preparation with Quality by Design (QbD) approach. The factors were the concentration of lysozyme and sodium alginate, and pH. Our aim was to understand the effect of process parameters through the determination of mathematical equations, based on which the optimization parameters can be predicted under different process parameters. The optimization parameters were encapsulation efficiency, particle size, enzyme activity and the amount of α-helix structure. The nanoparticles were analysed with transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FTIR) and circular dichroism (CD) spectroscopy. Based on our results, we found that pH was the most important factor and pH 10 was recommended during the formulation. Enzyme activity and α-helix content correlated with each other very well, and particle size and encapsulation efficiency also showed very good correlation with each other. The results of the α-helix content of FTIR and CD measurements were very similar for the precipitated lysozyme due to the solid state of lysozyme. The mixing time had the best influence on the encapsulation efficiency and the particle size, which leads to the conclusion that a mixing time of 1 h is recommended. The novelty in our study is the presentation of a mathematical model with which the secondary structure of the protein and other optimization parameters can be controlled in the future during development of nanoparticle based on the process parameters.

Pegylation and formulation strategy of Anti-Microbial Peptide (AMP) according to the quality by design approach.

Antimicrobial resistance is one of the main global threats according to the World Health Organization's (WHO) report (World Health Organization 2014), therefore there is a need for the development of other agents, such as antimicrobial peptides (AMPs). Although AMPs are considered as major candidates for next-generation antibiotics, several challenges including low bioavailability, high manufacturing cost and toxicity are still to be solved for their practical use in therapeutic applications. Novel chemical modification approaches as well as strategies for their delivery offer several opportunities to overcome these barriers and develop more stable and cost-effective synthetic peptides with efficient delivery to the target site. The integration of the Quality by Design (QbD) approach in the early pharmaceutical developments supports researchers in optimizing the targeted product by a risk based manner. Peptide modifications and formulation of peptide delivery systems are challenging tasks and hide several risks. Understanding and evaluating the cause - effect relations within the initial Risk Assessment (RA) step in case of all attributes give the basis for the experimental design as the next step, and aids the formulation development in order to get the final product in the targeted quality range. This study presents a Quality by Design based antimicrobial peptide modification and formulation design. Analyses the potential risks in the AMP PEGylation process through the example of PGLa. The QbD based initial RA screened and evaluated the risk factors in this AMP modification procedure. The critical quality and process related factors were defined and their ranking was performed due to their estimated critical effect on the PEGylated AMP. This pre-formulation design study highlights the critical risk factors as decision points for the further steps.