Intact charge variant analysis of ziv-aflibercept by cationic exchange liquid chromatography as a proof of concept: Comparison between volatile and non-volatile salts in the mobile phase.

Ziv-aflibercept (ziv-AFL) is a complex fusion protein which is widely used in hospitals for the treatment of colorectal metastatic cancer. Charge variants are critical attributes for assessing post-transitional modifications (PMTs) that have to be controlled during the development and manufacture of these proteins and until their administration to patients. Cation exchange (CEX) chromatography is a charge-sensitive analytical method that is well suited for analysing charge variants in proteins. The aim of this paper is to analyse the charge variants of ziv-AFL in the medicine (Zaltrap®) when fresh and when degraded. Two CEX chromatographic methods were compared for this purpose. The former was an adaptation of the method used in the first published study in which charge variants were analysed via pH gradient elution using volatile, low ionic strength buffers with direct coupling to high-resolution Orbitrap mass spectrometry. The second method was developed and optimized during our research using the salt-mediated pH gradient mode and classical non-volatile, high ionic strength buffers which were incompatible with direct coupling with mass detection. Fresh and controlled degraded samples of ziv-AFL were used to evaluate the capacity of both CEX chromatographic strategies for detecting charge variants in ziv-AFL. In the controlled degradation study the samples of the medicine were subjected to three stress factors: temperature of 60 °C for three hours, freeze/thaw process -two cycles-, and exposure to light for twelve hours. The CEX chromatographic method with non-volatile salts in the mobile phase enabled better detection of charge variants degraded ziv-AFL samples than the method using volatile salts with lower ionic strength. In addition, the complexity of the mass spectra data generated made it impossible to identify the multicharge variant species of ziv-AFL. Although charge variants were not separated in ziv-AFL fresh sample, our results indicate that the method with non-volatile salts in the mobile phase could be used to characterize and track changes in the charge variant UV chromatographic profile of ziv-AFL in fresh and degraded samples, even though it cannot be coupled to a mass detector and there is therefore no information about mass. The increase of basic protein degraded compounds were the most important degradation pattern detected in ziv-AFL (Zaltrap®).