Integrated Process Analytical Platform for Automated Monitoring of Monoclonal Antibody N-Linked Glycosylation.

Abstract

The biopharmaceutical industry is transitioning toward the adoption of continuous biomanufacturing practices that are often more flexible and efficient than traditional batch processes. Federal regulatory agencies are further urging the use of advanced process analytical technology (PAT) to analyze the design space to increase the process knowledge and enable high-quality biologic production. Post-translational modifications of proteins, such as N-linked glycosylation, are often critical quality attributes that affect biologics' safety and efficacy, requiring close monitoring during manufacturing. Here, we developed an online sequential-injection-based PAT system, called N-GLYcanyzer, which can rapidly monitor mAb glycosylation during upstream biomanufacturing. The key innovation includes the design of an integrated mAb sampling and fully automated sample derivation system for antibody titer and glycoform analysis within 3 h. The N-GLYcanyzer process includes mAb capture, deglycosylation, released glycan labeling with fluorescent dyes, and labeled glycan enrichment for direct injection/analysis on an integrated high-performance liquid chromatography system. Different fluorescent tags and reductants were tested to maximize glycan labeling efficiency under aqueous conditions, while porous graphitized carbon (PGC) was used for optimizing glycan recovery and enrichment. We found that 2-aminobenzamide labeling of glycans with 2-picoline borane as a reducing agent, using the N-GLYcanyzer workflow, shows higher glycan labeling efficiency under aqueous conditions, leading upward to a 5-fold increase in fluorescent product intensity. Finally, we showcase how the N-GLYcanyzer platform can be implemented at-/online in an upstream bioreactor for automated and near-real-time glycosylation monitoring of a Trastuzumab biosimilar produced by Chinese hamster ovary cells.