POLYMERIC NANOPARTICLES USED AS PSEUDOSTATIONARY PHASE TO IMPROVE PROTEIN SEPARATION IN A COMPLEX MATRIX

COLLAZO, D; GALLI, M; GRELA D; DABAS, P; VIZIOLI, NM

Mendoza

Simposio; 24th Latin-American Symposium on Biotechnology, Biomedical, Biopharmaceutical, and Industrial Applications of Capillary Electrophoresis and Microchip Technology- LACE 2018; 2018

The use of nano-sized materials in electro-driven separation systems has received special attention during the past decade. Even though nanoparticles (NP) have demonstrated their utility in separation science, they have not yet found regular application in pharmaceutical and other regulated chemistry environments, because complete understanding of the synthesis process along with size control and complete characterization has not yet been realized, so robustness and reliability are the limiting steps in this technology. In this work, the interaction between synthetic bioactive peptides and polymeric NP used as pseudostationary phase in capillary electrochromatography was studied. NP were tested on a P/ACE MDQ system for separation of proteins from a tryptic digest of human serum albumin. The highest number of protein fragments was observed when using the BGE at pH=3.5 with introduction of NP by partial filling technique. The BGE at pH 3.5 was selected to investigate the effect of NP concentration on the separation of protein fragments since it offered good separation conditions. Besides a better peak shape and resolution, increasing NP amount in the capillary resulted in larger peak areas, which would be indicating a concentration effect over protein fragments.The NP showed selectivity toward the protein fragments present in the tryptic digest while the amount of NP in the background electrolyte influenced the separation. The application of polymeric NP as pseudostationary phase showed to be a useful tool to improve peptide separation efficiency by capillary electrochromatography. However, it is necessary to emphasize the importance of thorough knowledge and control of running conditions to ensure successful analytical results. The analytical criteria adopted in this work may be applied to improve the analysis of samples from biological source as well as peptide formulations to be used in biological systems. In all cases, method optimization and assessment must be carried out for the particular application.