Peptidové mapování bioléčiv pomocí CE

• Main Author: Kosolapov, Dmytro
• Other Authors: Jáč, Pavel
• Format: Dissertation
• Language: Czech
• Published: 2020
• Online Access: http://www.nusl.cz/ntk/nusl-435663

Charles University Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Dmytro Kosolapov Supervisor: PharmDr. Pavel Jáč, PhD. Consultant: PharmDr. Juraj Lenčo, Ph.D. Title of graduation thesis: Peptide Mapping of Biopharmaceuticals Using CE This work focuses on optimization of capillary electrophoresis (CE) method for the peptide mapping. and enzymatic digestion of proteins using trypsin. Myoglobin from equine heart and a reference antibody were used as substrates. Two digestion approaches were compared: a conventional tryptic digestion in solution and digestion with a commercial SMART Digest Trypsin Kit that is based on immobilized trypsin. The digestion was monitored with RP-HPLC. After a short optimization of tryptic digestion, a number of experiments were carried out to determine the optimal conditions for peptide mapping by means of CE. All the measurements were carried out using a PVA-coated capillary of 50 µm internal diameter, effective length of 56 cm and total length of 64.5 cm. Samples were applied hydrodynamically at a pressure of 50 mbar, the separation voltage was 30 kV, the temperature of the capillary was set to 20řC. The analytes were detected with UV-VIS detector at 200 nm. The effect of BGE type and concentration (30, 60, 90, 120, 150 mM formic acid...