CHO‐K1 host cells adapted to growth in glutamine‐free medium by FACS‐assisted evolution

Abstract

During the process of recombinant cell line optimisation for production of biopharmaceuticals, multiple cellular properties like robustness against stress, the attainment of high cell concentrations and maintenance of high viability must be considered to maximize protein yield. To improve growth and viability, glutamine is supplemented as an alternative energy source for rapidly dividing cells that oxidize glucose inefficiently. However, the resulting by‐product ammonia is toxic at high concentrations and has a negative impact on protein glycosylation, a major quality‐determining parameter of biopharmaceuticals. In this work, the CHO‐K1 cell line was adapted to a chemically defined medium and suspension growth within 3 weeks. Subsequently, the glutamine concentration was stepwise reduced from 8 to 4 and 2 mM. After each reduction, both the final cell concentration in the batch and the viability decreased. To force a rapid evolution of cells to achieve high final cell concentrations, cells were seeded at high densities (107 cells/mL) and surviving cells were sorted by FACS or MACS when viability declined to 10% (typically after 24 h). Sorted cells were grown in batch until viability declined to 10% and viable cells recovered again. The final sorted population was able to reach comparable or even better viable cell concentrations and showed a significantly improved viability compared to their ancestors. The 2 mM glutamine‐adapted cell line was directly transferred into glutamine‐free medium and was able to grow at comparable rates without requiring further adaptation. Cells compensated the lack of glutamine by increasing their consumption of glutamate and aspartate.

Towards dynamic metabolic flux analysis in CHO cell cultures

“…The decomposition rate is dependent on the pH [81,82], temperature [80] and phosphate concentration [83]. For example, Ozturk and Palsson [81] showed that the actual uptake of glutamine in hybridoma cell culture was three-fold lower than the apparent uptake.Ammonium that is released via glutaminolysis and glutamine decomposition is a toxic by-product in mammalian cell cultures [84]. Ammonium can accumulate to levels up to 2-10 mM in batch cultures [11], and can negatively affect cell growth [85][86][87][88], protein production [89] and protein glycosylation [9,[90][91][92][93].…”

Reduced quenching and extraction time for mammalian cells using filtration and syringe extraction

“…CHO-K1 suspension cell lines adapted to growth in glutamine free media (Bort et al, 2010) were cultivated in 500 ml spinner flasks using CD CHO media (Gibco, Invitrogen, Carlsbad, CA, USA) under 7% CO 2 and incubated at 37 °C with constant stirring at 50 rpm. Viability and cell counts were measured on a Vi-Cell analyzer (Beckman Coulter Inc., Fullerton, CA) based on the trypan-blue dye exclusion method.…”

Glutamine substitution: the role it can play to enhance therapeutic protein production

“…CHO-K1 cells that can grow in the glutamine-free medium were selected using fluorescence-activated cell sorting FACS or magnetic activated cell sorting [87]. The selected cells showed similar or even better growth and viability profiles compared with parental CHO-K1 cells.…”