Development of the Glycoengineered CHO Cell Panel

Mammalian cells have many glycosyltransferases synthesizing the glycan structures on secreted proteins, and a few dozen enzymes that are responsible for adding the abundant N-linked glycans on recombinant proteins. CHO cells express most glycosyltransferases involved in mammalian N-linked glycosylation, with the key enzymes for making complex and hybrid N-glycans. However, the enzymes synthesize the glycans through complex pathways. Thus, for a given glycoprotein, often a diverse set glycoforms are made, with subtle differences in the glycan structures decorating the protein. Unfortunately, this heterogeneity leads to two problems. First, when glycosylation effects function, heterogeneity dilutes the efficacy of the protein product and this can also lead to safety issues. Second, heterogeneity results in more glycans whose abundance must be controlled, if possible, during scale-up and manufacturing once the product is approved. Thus, there is a need to identify which glycoforms possess the most desirable properties and then to develop cell lines producing the optimal glycoform as homogeneously as possible.

To achieve these goals, we deeply characterized glycosylation in CHO cells (Fig. 1). First, we sequenced and annotated the CHO-K1 and Chinese hamster genomes 1,2,3 and quantified glycosyltransferase mRNA expression. Furthermore, we and others linked each glycosyltransferase to their functions by knocking out (or knocking in) each gene individually and in combination 4,5,6,7 , and then quantified the glycome and transcriptome. Further using specialized systems biology modeling techniques, we were able to assign their role in glycosylation, including primary enzyme specificities and off-target effects. Using this information, over the course of 10 years we developed a panel of 30 glycoengineered CHO cells (geCHO) each expressing a unique human glycoform. These cells were derived from CHO-S cells (Thermo Fisher), which can grow to industrial-level cell densities in suspension on serum-free media. We use these cells to produce diverse, but more homogeneous glycoforms of various recombinant proteins of interest.

A decade of pioneering systems glycoengineering in CHO

A history of the last decade of pioneering systems glycoengineering in CHO.
Figure 1. Glycoengineered CHO panel development. In the last decade, we have spearheaded and collaborated on pioneering research in CHO cell engineering with a primary focus on glycosylation, having attracted >$50M in research funding. Highlights of this process are shown above.

References

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