Reduced glutamine concentration improves protein production in growth-arrested CHO-DG44 and HEK-293E cells

For most cultivated mammalian cells, glutamine is an essential medium component. However, glutamine consumption results in the production of ammonia, a cytotoxic byproduct. Here we investigated the effect of glutamine reduction on recombinant protein production and ammonia accumulation in transiently transfected CHO and HEK-293E cells maintained under conditions of growth arrest. Maximum transient recombinant protein yields were observed in HEK-293E cultures without glutamine and in CHO cultures with 2 mM glutamine. The initial concentration of glutamine correlated with the level of ammonia accumulation in each culture. For both a stable CHO-derived cell line and a polyclonal population of recombinant CHO cells grown under conditions of mild hypothermia, the highest volumetric protein productivity was observed in cultures without glutamine. Here, the level of ammonia accumulation also corresponded to the initial glutamine concentration. Our data demonstrate that reduction of glutamine in the medium is an effective approach to improve protein production in both transiently and stably transfected mammalian cells when applying conditions that reduce or arrest the growth of these cells.     

The effect of yeast extract and methyl jasmonate on rosmarinic acid accumulation in <Emphasis Type="Italic">Coleus blumei</Emphasis> hairy roots

The leaves of axenically grown Coleus blumei were inoculated with Agrobacterium rhizogenes strain A4 and hairy root were established. PCR and Southern hybridization analysis confirmed transgenic nature of hairy root clones. Cultures of normal roots, induced by α-naphthaleneacetic acid on leaf explants, and hairy roots were evaluated for growth and rosmarinic acid content. Significantly better growth and up to 2.8 higher amount of rosmarinic acid was detected by HPLC analysis in hairy root clones. Methyl jasmonate stimulated rosmarinic acid accumulation in 6 out of 11 tested clones, while yeast extract induced RA accumulation in two and diminished it in 5 out of 11 tested hairy root clones.     

A simple high-yielding process for transient gene expression in CHO cells

Here we describe a simplified method for transient gene expression (TGE) in suspension-adapted Chinese hamster ovary (CHO) cells using polyethylenimine (PEI) for DNA delivery. Both the transfection and production phases of the bioprocess were performed at a density of 4 × 10? cells/mL at 31 °C. In addition, the amounts of both PEI and plasmid DNA were reduced up to 50% on a per cell basis compared to previously published protocols from this laboratory, resulting in higher cell viability after transfection and higher volumetric recombinant protein yields. In batch cultures of up to 14 days, reproducible recombinant antibody yields up to 300 mg/L were achieved at small scale (5 mL) and up to 250 mg/L at large scale (500 mL). The simplicity and improved yields are expected to increase the utility of CHO cells for the rapid production of recombinant proteins at larger scales by TGE.