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

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.