Understanding of altered N‐glycosylation‐related gene expression in recombinant Chinese hamster ovary cells subjected to elevated ammonium concentration by digital mRNA counting

Abstract

To understand the effects of ammonium on N-glycosylation, recombinant Chinese hamster ovary (rCHO) cells that produce the Fc-fusion protein were cultivated in serum-free suspension cultures with 10 mM ammonium addition. The addition of ammonium to the cultures reduced the relative proportion of acidic isoforms and sialic acid content of an Fc-fusion protein. Fifty two N-glycosylation-related gene expressions were assessed by the NanoString nCounter system, which provides a digital readout using custom-designed color-coded probes. Among these queried genes, thirteen genes (gale, nans, gpi, man2a1, b4galt5, b4galt7, st3gal2, st3gal5, glb1, hexa, hexb, neu1, and neu3) were up-regulated over 1.5 times in the culture with ammonium addition after 5 days of culture; however, none of the 54 genes were significantly different after 3 days of culture. In particular, the expression level of neu1 (sialidase-1) and neu3 (sialidase-3), which play a role in reduction of sialylation, increased over 2 times. Likewise, the protein expression levels of sialidase-1 and sialidase-3 determined by Western blot analysis were also increased significantly in the culture with ammonium addition. Transient transfection of neu-1 or neu3-targeted siRNAs significantly improved the sialic acid content of the Fc-fusion protein in the culture with ammonium addition, indicating that the decreased sialic acid content was in part due to the increased expression level of sialidase. Taken together, the results obtained in this study provide a better understanding of the detrimental effect of ammonium on N-glycosylation, especially sialylation, in rCHO cells.

Multiplexed Digital mRNA Expression Analysis Profiles System‐Wide Changes in mRNA Abundance and Responsiveness of UPR‐Specific Gene Expression Changes During Batch Culture of Recombinant Chinese Hamster Ovary Cells

“…While the NanoString nCounter has the capacity to quantify a large subset of mRNAs belonging to varied and linked pathways, we have used the current studies to define mRNAs that are most likely to provide an informative fingerprint of cellular state. The smaller subset offers the potential for exploitation to interrogate a range of cellular challenges such as sodium butyrate treatment, hyperosmotic conditions, or elevated ammonium concentrations . Despite the profiling observed in this study, further work will be needed to refine the association between mRNA expression patterns and cellular phenotypes (and to provide deeper understanding of molecular descriptions of cellular phenotype).…”

Understanding of decreased sialylation of Fc‐fusion protein in hyperosmotic recombinant Chinese hamster ovary cell culture: N‐glycosylation gene expression and N‐linked glycan antennary profile

“…Analysis of 52 N-glycosylated gene expressions by the NanoString nCounter system, which can provide a direct digital readout using customdesigned color-coded probes, revealed that hyperosmolality, unlike ammonia (Ha et al, 2015) and NaBu treatment , did not increase sialidase-1 and sialidase-3 gene (neu1 and neu3) expression. Analysis of 52 N-glycosylated gene expressions by the NanoString nCounter system, which can provide a direct digital readout using customdesigned color-coded probes, revealed that hyperosmolality, unlike ammonia (Ha et al, 2015) and NaBu treatment , did not increase sialidase-1 and sialidase-3 gene (neu1 and neu3) expression.…”

“…The DUKX-Fc was established as described previously (Ha et al, 2015). The Fc-fusion protein is a homodimer with two glycosylation sites in the head protein and one in the Fc region of human IgG4.…”

“…The Z value, which is the hypothetical N-linked glycan charge value of a glycoprotein, enables the evaluation of the protein glycosylation in a simple and efficient manner (Hermentin et al, 1996). Previously, adding ammonia to cultures reduced the sialylation of the Fc-fusion proteins, though it did not significantly affect the q Fc (Ha et al, 2015). qRT-PCR analysis of the N-glycan branching/antennary genes (mgat1/2/3/4b/5a) showed that decreases in mRNA levels of mgat2 and mgat4b in hyperosmotic culture were rescued by adding betaine, supporting the idea that downregulated expression of these genes is in part responsible for the reduced sialylation of Fc-fusion protein in hyperosmolar conditions ( Supplementary Fig.…”

Effect of Bcl‐xL overexpression on sialylation of Fc‐fusion protein in recombinant Chinese hamster ovary cell cultures

“…Alternatively, culture condition can also be optimized to reduce the expression level of glycosidases including sialidase. Recently, it was reported that sodium butyrate concentration and ammonium concentration significantly affected the expression level of sialidase …”