N-linked glycosylation and other post-translational protein modifications require close monitoring during biomanufacturing, especially in continuous biomanufacturing environments. Researchers at Rutgers, The State University of New Jersey, have devised an online, sequential-injection-based, process analytical technology (PAT) monitoring system for near-real-time sampling and analysis, thus helping biomanufacturers minimize the production of out-of-spec lots or batches.
The system, called N-GLYcanyzer, uses “integrated mAb sampling and a fully-automated sample derivation system for antibody titer and glycoform analysis,” Aron Gyorgypal, PhD candidate, and Shishir P.S. Chundawat, PhD, wrote in a recent paper. The fully-integrated process was validated using Chinese hamster ovary (CHO) cells to produce a trastuzumab biosimilar.
N-GLYcanyzer monitors monoclonal antibody (mAb) glycosylation in upstream biomanufacturing, enabling at- and on-line bioprocess monitoring of mAb titer within minutes of drawing a sample, and glycosylation monitoring within one to three hours of drawing a sample. It offers scalable, end-to-end integration and can be configured for batch and fed-batch bioprocessing models as well as continuous perfusion models for cell culturing.
“Being able to monitor the mAb glycan patterns every few hours help manufacturers understand the drift in mAb glycan quality, so automated mitigation strategies may be implemented before the drug being produced falls outside desired quality specifications,” Chundawat tells GEN. “In principle, the system can be built and deployed by commercial manufacturers now, but it still needs further validation with multiple biologics and glycan profiles.”
Chundawat and Gyorgypal are working with researchers at the FDA’s Center for Drug Evaluation and Research (CDER) to do just that, using multiple CHO cell-lines. “This will allow researchers to use this system to characterize different mAbs that may have different glycosylation patterns, including truncation, high sialylation, and complex branched glycans,” continues Chundawat.
The team also is developing process control strategies to incorporate into the N-GLYcanyzer to control mAb N-glycosylation in real-time–“something that has not been readily achieved before,” Gyorgypal says. They aim to streamline the N-glycan labeling chemistry workflow to enable much faster monitoring by moving from traditional Schiff-base reductive amination chemistry towards instant-labeling chemistry workflows, which will decrease process times and increase scalability.
Chundawat says automatically monitoring N-glycosylation in near-real time could save weeks of time by eliminating the need to hand off product to quality control departments to ascertain N-glycosylation.
“This also will help researchers implement process control strategies with real-time decision-making during continuous processing campaigns to maintain the targeted quality of mAb glycosylation as identified during risk-based assessment,” he adds.