Integrated SegFlow, µSIA and UPLC for online sialic acid quantitation of
glycoproteins directly from bioreactors
Abstract
Monitoring and controlling of sialic acid contents in glycoproteins such
as erythropoietin (EPO), interferon-γ, Orencia, Enbrel and others are
critical to achieve desired therapeutic benefits. The pharmacokinetics
(PK) profile of asialoglycoprotein is known to impact protein clearance
with its uptake by hepatic asialoglycoprotein receptors (ASGPR) and
subsequent physiological clearance. The ASGPR recognizes and binds to
glycoproteins with exposed terminal galactose or N-acetyl galactosamine
residues to undergo 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 performances including immunogenicity, potency, and
cytotoxicity. Therefore, the level of sialic acid is a critical quality
attribute (CQA) and has become a regulatory requirement to monitor and
regulate sialylation to ensure desired clinical performance. To achieve
consistent levels of sialic acid in certain therapeutics, the harvest
decision as well as the ionic strength of downstream process buffer
composition is dependent upon the sialic acid content. Therefore,
utilization of Process Analytical Technology (PAT) tools for generating
real-time or near-real-time sialic acid content is a business-critical
requirement. The work presented here demonstrating the utility of an
integrated system consisting of a micro-sequential Injection Analyzer
(µSIA) interfaced with SegFlow and a UPLC to enable near-real-time
online sialic acid measurements. The fully automated architecture
exemplifies the execution of online sampling, automatic sample
preparation and subsequent online UPLC analysis. Carefully orchestrated
such framework is in alignment with the requirements of PAT to support
QbD-driven continuous bioprocessing.