Several favorable characteristics for pharmaceutical drugs have been achieved by their conjugation with biodegradable polymers such as PEG. Proteins are commonly PEGylated to enhance in vivo stability and reduce immunogenic responses.
The composition and molecular characteristics of a 10 KDa protein conjugated with 5 KDa and 10 KDa PEGs was measured by tetra-detection SEC.
Compositional analysis was calculated from the UV absorbance of the protein (PEG does not absorb UV at 280 nm) and the total concentration of the PEGylated-proteins measured by RI detector.
Absolute Mw, IV, and Rh were also determined by LS and viscometer measurements. Furthermore, aggregates were detected in the PEGylated-proteins.
Figure 1. Overlay chromatogram obtained from the RI detector of virgin protein (black) and PEGylated protein A (red) and B (green).
Advanced detection SEC/GPC is a powerful tool for analyzing pharmaceutical protein conjugates. Detailed information about protein conjugation ratios and aggregation can be readily obtained.