Monoclonal antibodies
are large, complex biomolecules that can be difficult to characterize.
Characterization is important because of the various post translational
modifications that can occur during manufacturing, processing, and storage.
Some modifications can lead to efficacy and safety issues and therefore are
heavily monitored. A leading way to monitor various modifications is by using
liquid chromatography. The high sensitivity, reproducibility, and ability to
quantitate analytes makes it very attractive for monoclonal antibody
characterization. The large molecular size of monoclonal antibodies (150 kDa)
makes them challenging to separate efficiently and with high enough resolution
to be helpful. New column technologies that would help improve protein
separation efficiencies and slectivities would greatly help in this challenging
process. In this thesis, three novel bonded phases are developed for the
separation of monoclonal antibodies including a weak anion and cation exchanger
(WAX, CEX) for the separation of charged species as well as a novel hydrophilic
interaction chromatography (HILIC) for the separation of glycoforms. Column
develop is achieved by optimizing selectivity and improving efficiency of
separations by altering particle surface chemistry.