A genetic model to study GlcNAc cycling in immortalized mouse embryonic fibroblasts.

-GlcNAcylation is an abundant posttranslational protein modification in which the monosaccharide O-GlcNAc is added to Ser/Thr residues by GlcNAc transferase and removed by GlcNAcase. Analyses of GlcNAc-mediated signaling and metabolic phenomena are complicated by factors including unsatisfactory inhibitors and loss-of-function cell lines lacking identical genetic backgrounds. In this work, we generated immortalized WT, knockout, and floxed allele ( floxed) mouse embryonic fibroblast (MEF) cell lines with similar genetic backgrounds.

Identifying a robust design space for glycosylation during monoclonal antibody production.

Glycan distribution has been identified as a "critical quality attribute" for many biopharmaceutical products, including monoclonal antibodies. Consequently, determining quantitatively how process variables affect glycan distribution is important during process development to control antibody glycosylation. In this work, we assess the effect of six bioreactor process variables on the glycan distribution of an IgG1 produced in CHO cells.

A comparison of strategies for immortalizing mouse embryonic fibroblasts.

The genetically amenable mouse model has led to a large collection of genetically defined lines from which mouse embryonic fibroblasts (MEFs) have been derived. Despite their widespread use, MEFs are time consuming to generate and have a limited lifespan. Immortalizing primary MEFs with the desired genetic manipulations greatly reduces culture maintenance time, enables the generation of near limitless amounts of protein lysate, and facilitates biological replicates during experimentation.

Identification of manipulated variables for a glycosylation control strategy.

N-linked glycan distribution affects important end-use characteristics such as the bioactivity and efficacy of many therapeutic proteins, (including monoclonal antibodies), in vivo. Yet, obtaining desired glycan distributions consistently during batch-to-batch production can be challenging for biopharmaceutical manufacturers. While an appropriately implemented on-line glycosylation control strategy during production can help to ensure a consistent glycan distribution, to date no such strategies have been reported.

Controllability analysis of protein glycosylation in CHO cells.

To function as intended in vivo, a majority of biopharmaceuticals require specific glycan distributions. However, achieving a precise glycan distribution during manufacturing can be challenging because glycosylation is a non-template driven cellular process, with the potential for significant uncontrolled variability in glycan distributions. As important as the glycan distribution is to the end-use performance of biopharmaceuticals, to date, no strategy exists for controlling glycosylation on-line.