An Integrated Process Analytical Platform for Automated Monitoring of Monoclonal Antibody N-linked Glycosylation

Abstract

The biopharmaceutical industry is transitioning towards adoption of continuous biomanufacturing practices that are often more flexible and efficient than traditional batch processes. Regulatory agencies such as the Food and Drug Administration (FDA) are further urging use of advanced PAT to analyze the design space to increase process knowledge and enable high quality biologics production. Post-translational modification of proteins, such as N-linked glycosylation are often critical quality attributes known to affect biologics safety and efficacy hence requiring close monitoring during manufacturing. Here, we developed an online sequential-injection based PAT system, called N-GLYcanyzer, that can rapidly monitor mAb glycosylation during upstream biomanufacturing. The key innovation includes design of an integrated mAb sampling and derivation system for antibody titer and glycoform analysis in under 2 hours. The N-GLYcanyzer process includes mAb capture, deglycosylation, fluorescent glycan labeling, and glycan enrichment for direct injection and analysis on an integrated high performance liquid chromatography (HPLC) system. Different fluorescent tags and reductants were tested to maximize glycan labeling efficiency under aqueous conditions, while porous graphitized carbon (PGC) was studied for optimum glycan recovery and enrichment. We find that 2-AB labeling of glycans with 2-picoline borane as a reducing agent, using the N-GLYcanyzer workflow, gives higher glycan labeling efficiency under aqueous conditions leading to upwards of a 5-fold increase in fluorescent products intensity. Finally, we showcase how the N-GLYcanyzer platform can be implemented at/on-line to an upstream bioreactor for automated and near real-time glycosylation monitoring of a Trastuzumab biosimilar produced by Chinese Hamster Ovary (CHO) cells. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/468439v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@1fbb698org.highwire.dtl.DTLVardef@90da13org.highwire.dtl.DTLVardef@1dd45e5org.highwire.dtl.DTLVardef@17fadd_HPS_FORMAT_FIGEXP M_FIG N-GLYcanyzer is an automated PAT toolkit for rapid sample processing for mAb N-linked glycans analysis to enable advanced biologics manufacturing C_FIG