Automated Workflow for Instant Labeling and Real-Time Monitoring of Monoclonal Antibody N-Glycosylation

Abstract

With the transition toward continuous bioprocessing, process analytical technology (PAT) is becoming necessary for rapid and reliable in-process monitoring during biotherapeutics manufacturing. Bioprocess 4.0 is looking to build an end-to-end bioprocesses that includes PAT-enabled real-time process control. This is especially important for drug product quality attributes that can change during bioprocessing, such as protein N-glycosylation, a critical quality attribute for most monoclonal antibody (mAb) therapeutics. Glycosylation of mAbs is known to influence their efficacy as therapeutics and is regulated for a majority of mAb products on the market today. Currently, there is no method to truly measure N-glycosylation using on-line PAT, hence making it impractical to design upstream process control strategies. We recently described the N-GLYcanyzer: an integrated PAT unit that measures mAb N-glycosylation within 3 hours of automated sampling from a bioreactor. Here, we integrated Agilents Instant PC (IPC) based chemistry workflow into the N-GLYcanzyer PAT unit to allow for nearly 10x faster mAb glycoforms analysis. Our methodology is explained in detail to allow for replication of the PAT workflow as well as present a case study demonstrating use of this PAT to autonomously monitor a mammalian cell perfusion process at the bench-scale to gain increased knowledge of mAb glycosylation dynamics during continuous biomanufacturing of biologics using Chinese Hamster Ovary (CHO) cells. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=169 SRC="FIGDIR/small/521623v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@16cf139org.highwire.dtl.DTLVardef@150db0borg.highwire.dtl.DTLVardef@15cbe36org.highwire.dtl.DTLVardef@1cbdf6a_HPS_FORMAT_FIGEXP M_FIG C_FIG