Development of CE methods to analyze potential components of the angiogenic glycoprotein vascular endothelial growth factor 165

The vascular endothelial growth factor 165 (VEGF165) is the predominant form of the complex VEGF family. This glycoprotein has, among others, an angiogenic effect in many physiological and pathological events. For this reason, its roles as a biomarker and as a therapeutic drug have been considered. However, very little is known about the existence of different forms of VEGF165 arising from glycosylation and other potential PTMs. This aspect is crucial because it is known that for other glycoproteins the ratio between these isoforms actually acts as a biomarker for certain diseases and other physiological states. In addition, for therapeutic use of glycoproteins it is known that the biological activity may differ for the various isoforms. In this work CE methods to separate up to seven peaks without baseline resolution containing various forms of VEGF165 are developed. Using a computer program previously developed in-house peak assignment could be performed with accuracy close to 100%. In this way, comparison between recombinant human VEGF165 expressed in insect cells, which is a glycosylating system, and in Escherichia coli cells, which are unable of performing glycosylation of proteins, has been possible. The methods developed, besides providing information about the existence of several forms of VEGF165, mean a starting point that permits the study of the role of VEGF165 as a potential biomarker of different diseases and physiological processes and to perform quality control of the recombinant drug during manufacturing. To the best of our knowledge this is the first time that CE methods for VEGF165 have been developed.