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

Abstract

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.

Development of a CE‐MS method to analyze components of the potential biomarker vascular endothelial growth factor 165

“…The analysis of VEGF 165 expressed in E. coli (Fig. 1B) showed the presence of two small shoulders eluting just before and after the main VEGF 165 peak, as it was previously shown in the developed CE-UV method [22]. As VEGF 165 expressed in E. coli is not glycosylated, it is expected that both minor peaks could correspond to other PTMs different to glycosylation.…”

“…The separation voltage was initially set to À205 V/cm. In a prior work [22] we showed the suitability of the use of 40 mM …”

“…1A) like the CE-UV method developed in the previous work [22]. The analysis of VEGF 165 expressed in E. coli (Fig.…”

“…Recently, in a previous paper from this group, the existence of several components in glycosylated VEGF 165 and more than one component in non-glycosylated VEGF 165 has been demonstrated by CE-UV [22]. Nevertheless, the use of detection techniques providing structural information about VEGF 165 is necessary in order to characterize the forms involved in the different components shown by the developed CE-UV methods.…”

Protein glycosylation analysis with capillary-based electromigrative separation techniques

“…pH has a marked influence on glycoprotein separation as shown in all publications dealing with pH optimization (e.g., [55,91,95,106,107,113,186]). In general, in older publications, glycoproteins were predominantly analyzed as anions whereas younger publications seem to prefer cationic separation, especially when using mass spectrometric detection.…”