Rapid baseline hump-free analysis of therapeutic proteins in a wide molecular weight range by SDS - capillary agarose gel electrophoresis

Abstract

Background: Sodium dodecyl sulfate capillary gel electrophoresis traditionally employs entangled polymer networks (CE-SDS) or borate cross-linked gels (SDS-CGE) for size-based protein analysis. However, the separation of SDS proteins in these transiently cross-linked sieving matrices requires long analysis times and in addition, baseline humps/waves frequently occur making peak identification and quantification challenging. The analytical biopharma community has been trying to solve this problem for a decade, making it clear that a novel gel composition was required to provide baseline hump-free separation of SDS-proteins by capillary electrophoresis of higher MW biopharmaceuticals.

Results: Using a transiently cross-linked agarose matrix shown in this paper enabled rapid separation of therapeutic proteins with excellent resolution, but more importantly, eliminated the commonly observed baseline disturbances of dextran-based gel formulations. Using capillaries as short as 10 cm effective length, the tetrahydroxyborate cross-linked agarose matrix supported fast analysis (∼5 min) of an intact anti-SARS-CoV-2 therapeutic antibody and its subunits with excellent run-to-run migration time (RSD <0.3 %) and peak area (RSD <5 %) reproducibility. High resolution between the closely migrating non-glycosylated heavy chain and the heavy chain fragments was also obtained (RS = 1.65). The high molecular weight thyroglobulin (660 kDa), and the highly glycosylated fusion protein of etanercept were also successfully analyzed with borate-agarose-based gels, exploiting the stable baseline at the upper molecular weight range of the separation trace.

Significance: This paper reports for the first time on a baseline hump-free approach for rapid analysis of therapeutic proteins in a wide MW range by SDS capillary agarose gel electrophoresis. The unique potential of tetrahydroxyborate-stabilized agarose gels revolutionizes capillary SDS gel electrophoresis analysis of mAbs and more complex new modalities, offering a robust and efficient platform for high-resolution protein separation, paving the way for advancements in baseline hump-free therapeutic protein characterization and consequently good quantification.

Keywords: Agarose; Baseline hump; Capillary gel electrophoresis; Sodium dodecyl sulfate; Therapeutic proteins.