Sequential Injection Based on-Line PAT for Real-Time Glycosylation Monitoring of Monoclonal Antibodies

Protein glycosylation is an important post translational modification that needs to be monitored during upstream bioprocessing as this critical quality attribute dictates the efficacy of the final biologic. As glycosylation patterns are highly heterogenous there is need to monitor and control this attribute in real-time. Current techniques for monitoring rely on off-line sampling and assays that are expensive and have long turn around times. Typically, glycan profiles are only measured during the end of a batch-culture for quality assurance, which does not allow for proper understanding of the process. Here, we look to create a PAT tool that can monitor glycosylation to be used to advance continuous biomanufacturing. We look to use a sequential injection based technique to removing cell-free permeate, concentrate and purify mAb using Protein A, denaturing the protein with detergents and heat, removing glycans using a glycanase, using reductive amination to fluorescently label glycans, enrich the samples using PGC and then injecting the sample to a HILIC based column on an HPLC. We look to use Trastuzumab as our model glycoprotein cultured from CHO Cells on a bench-scale bioreactor. This automated proof of concept design will be able to monitor glycan patterns in real-time, and hold promise to study glycosylation in real-time to allow for automated decision making during an upstream bioprocess.