Development of a novel, label-free N-glycan method using charged aerosol detection.

Monoclonal antibodies (mAbs) are complex glycoproteins that are developed for treatment of various therapeutic indications such as cancer and autoimmune diseases. MAbs are glycosylated at conserved asparagine residues (N-X-S/T) of the Fc region at amino acid position 297 of the heavy chain. Glycans are important in governing the functions of efficacy and serum half-life of protein therapeutics and are part of the critical quality attribute panel for release testing. Traditionally, N-linked glycans are released from glycoproteins after denaturation and enzymatic digestion with PNGase F, followed by fluorescent labeling of the liberated glycans. The labeled glycans are then separated using hydrophilic liquid chromatography (HILIC) with fluorescence detection to generate chromatographic profile. Despite decades of use, this strenuous process remains unchanged, utilizing toxic reagents and extended sample preparation time. As an intervention, this report showcases a novel, label-free approach to detect and quantify N-glycans without using fluorescent labeling. Separation of glycans using mixed-mode PGC column along with detection of non-derivatized glycans using charged aerosol detector, the overall turnaround time can be greatly reduced with significant cost savings. The label-free method provides similar quantitative results as the conventional fluorescent labeled method, confirming the validity of the method for product release.

Online monitoring and control of upstream cell culture process using 1D and 2D-LC with SegFlow interface.

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 capital investment and operational costs. Continuous bioprocessing, aligned with FDA's quality-by-design 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 bioprocessing, 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 Infinity 2D-LC with a post-flow-splitter in conjunction with the SegFlow autosampler to the bioreactors. With this integrated system, we have established a platform for online measurements of titer and CQAs of monoclonal antibodies as well as amino acid analysis of bioreactor cell culture.