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.

The biopharmaceutical industry is transitioning from currently deployed batch-mode bioprocessing to a highly efficient and agile next generation bioprocessing with the adaptation of continuous bioprocessing, which reduces the capital investment and operational costs. Continuous bioprocessing, aligned with FDA’s quality-by-design (QbD) platform, is designed to develop robust processes to deliver safe and effective drugs. With the deployment of knowledge based operations, product quality can be built into the process to achieve desired critical quality attributes (CQAs) with reduced variability. To facilitate next generation continuous bio-processing, it is essential to embrace a fundamental shift-in-paradigm from “quality-by-testing” to “quality-by-design”, which requires the deployment of process analytical technologies (PAT). With the adaptation of PAT, a systematic approach of process and product understanding and timely process control are feasible. Deployment of PAT tools for real-time monitoring of CQAs and feedback control is critical for continuous bioprocessing. Given the current deficiency in PAT tools to support continuous bioprocessing, we have integrated Agilent 2D-LC with a post-flow-splitter in conjunction with the SegFlow automated sampler to the bioreactors. With this integrated system, we have established a platform for online measurements of titer and CQAs of monoclonal antibodies (mAbs) as well as amino acid concentrations of bioreactor cell culture.

Process analytical technology (PAT) has been defined by the Food and Drug Administration (FDA) as a system for designing, analyzing, and controlling manufacturing through timely measurements to ensure final product quality. Based on quality-by-design (QbD) principles, real-time or near-real-time data monitoring is essential for timely control of critical quality attributes (CQAs) to keep the process in a state of control. To facilitate next-generation continuous bioprocessing, deployment of PAT tools for real-time monitoring is integral for process understanding and control. Real-time monitoring and control of CQAs is essential to keep the process within the design space and align with the guiding principles of QbD. The contents of this manuscript are pertinent to the online/at-line monitoring of upstream titer and downstream product quality with timely process control. We demonstrated that a UPLC system interfaced with a process sample manager (UPLC-PSM) can be utilized to measure titer and CQAs directly from bioreactors and downstream unit operations, respectively. We established online titer measurements from fed-batch and perfusion-based alternating tangential flow (ATF) bioreactors as well as product quality assessments of downstream operations for real-time peak collection. This integrated, fully automated system for online data monitoring with feedback control is designed to achieve desired product quality.

Process analytical technology (PAT) has been defined by the Food and Drug Administration (FDA) as a system for designing, analyzing, and controlling manufacturing through timely measurements to ensure final product quality. Based on quality-by-design (QbD) principles, real-time data monitoring is essential for timely control of critical quality attributes (CQAs) to keep the process in a state of control. To facilitate next-generation continuous bioprocessing, deployment of PAT tools for real-time monitoring is integral for process understanding and control. Real-time monitoring and control of CQAs is essential to keep the process within the design space, which is in alignment with the guiding principles of QbD concepts. The contents of this manuscript are pertinent to the online/at-line monitoring of upstream titer and downstream product quality. We have demonstrated that Waters’ PATROL system can be utilized to measure product titer and CQAs directly from bioreactors and downstream unit operations, respectively. We have established that online titer assay results from fed-batch and perfusion-based alternating tangential flow (ATF) bioreactors are comparable to at-line and conventional offline results. The utility of the PATROL system for online product quality measurements of down-steam unit operations for real-time peak pooling has been demonstrated.