Integrated SegFlow, µSIA, and UPLC for Online Sialic Acid Quantitation of Glycoproteins Directly from Bioreactors

ABSTRACT

This study emphasizes the critical importance of closely monitoring and controlling the sialic acid content in therapeutic glycoproteins, including EPO, interferon‐γ, Orencia, Enbrel, and others, as the level of sialylation directly impacts their pharmacokinetics (PK), immunogenicity, potency, and overall clinical performance due to its influence on protein clearance via hepatic asialoglycoprotein receptors (ASGPR). The ASGPR recognizes and binds to glycoproteins exposed to terminal galactose or N‐acetylgalactosamine residues, leading to receptor‐mediated endocytosis. Recent studies have demonstrated that sialylation of O‐linked glycan plays a role in protecting against macrophage galactose lectin (MGL)‐mediated clearance. In addition to the impact on serum half‐life, sialylation can influence other clinical outcomes, including immunogenicity, potency, and cytotoxicity. Therefore, the level of sialic acid is a critical quality attribute (CQA), and monitoring and regulating sialylation has become a regulatory requirement to ensure desired clinical performance. To achieve consistent levels of sialic acid‐to‐protein ratio, the time of upstream harvest and conductivity of downstream wash buffers must be tightly regulated based on the sialic acid content. Therefore, the utilization of process analytical technology (PAT) tools for generating real‐time or near‐real‐time sialic acid content is a business‐critical requirement. This work demonstrates the utility of an integrated PAT system for near real‐time online sialic acid measurements. The system consists of a micro‐sequential injection analyzer (µSIA) interfaced with SegFlow and an ultra performance liquid chromatography (UPLC). The fully automated architecture exemplifies the execution of online sampling, automatic sample preparation, and subsequent online UPLC analysis. This carefully orchestrated PAT framework effectively supports the requirements of QbD‐driven continuous bioprocessing.