Detection and quantification of single chain rFVII impurity in final drug products by SE-UPLC and CE-SDS as an alternative to SDS-PAGE.

Recombinant factor VII, produced in recombinant BHK cell line, is secreted as a single chain zymogen form (rFVII, non-activated) in cell culture supernatant and subsequently converts to its active form during anion exchange chromatography step in the downstream purification process, with the aid of calcium ion. Single chain rFVII impurity (non-activated form) in final drug products should not exceed more than 3.0 % of total rFVIIa content. Therefore, one of the most essential quality control tests in pharmaceutical companies is to precisely quantify and report this impurity. SDS-PAGE, as a traditional method in quality control laboratories to quantify single chain rFVII, is a laborious, time-consuming, low output, and semi-quantitative method for quantification of non-activated form impurity which utilizes a densitometer to scan the gel and calculate the non-activated form band density. In this work, we developed two novel instrumental-based techniques (SE-UPLC and CE-SDS) with superior precision, accuracy, sensitivity, and efficiency that overcome SDS-PAGE shortcomings. The results of both methods were comparable to SDS-PAGE and showed an even higher correlation with expected values. Finally, we concluded that these two methods could be used as a high throughput routine method in quality control laboratories as an alternative choice to manual SDS-PAGE.