Igor Slivac

Stable expression of chimeric heavy chain antibodies in CHO cells.

Camelid single domain antibodies fused to noncamelid Fc regions, also called chimeric heavy chain antibodies (cHCAb), offer great potential as therapeutic and diagnostic candidates due to their relatively small size (80 kDa) and intact Fc. In this chapter, we describe two approaches, limiting dilution and minipools, for generating nonamplified Chinese hamster ovary cell lines stably expressing cHCAb in suspension and serum-free cultures using a stringent antibiotic selection. Neither of the protocols necessitates the acquisition or implementation of expensive automated infrastructures and thus could be applied in any lab with minimal cell culture setup. The given protocol allows the isolation of stable clones capable of generating up to 100 mg/L of antibody in batch mode performed in shaker flasks.

An omics approach to rational feed: Enhancing growth in CHO cultures with NMR metabolomics and 2D-DIGE proteomics.

Expression of recombinant proteins exerts stress on cell culture systems, affecting the expression of endogenous proteins, and contributing to the depletion of nutrients and accumulation of waste metabolites. In this work, 2D-DIGE proteomics was employed to analyze differential expression of proteins following stable transfection of a Chinese Hamster Ovary (CHO) cell line to constitutively express a heavy-chain monoclonal antibody. Thirty-four proteins of significant differential expression were identified and cross-referenced with cellular functions and metabolic pathways to identify points of cell stress. Subsequently, 1D-(1)H NMR metabolomics experiments analyzed cultures to observe nutrient depletion and waste metabolite accumulations to further examine these cell stresses and pathways. From among fifty metabolites tracked in time-course, eight were observed to be completely depleted from the production media, including: glucose, glutamine, proline, serine, cystine, asparagine, choline, and hypoxanthine, while twenty-three excreted metabolites were also observed to accumulate. The differentially expressed proteins, as well as the nutrient depletion and accumulation of these metabolites corresponded with upregulated pathways and cell systems related to anaplerotic TCA-replenishment, NADH/NADPH replenishment, tetrahydrofolate cycle C1 cofactor conversions, limitations to lipid synthesis, and redox modulation. A nutrient cocktail was assembled to improve the growth medium and alleviate these cell stresses to achieve a ∼75% improvement to peak cell densities.

Toxicity mechanisms of ionic liquids

Abstract Over the past three decades a growing awareness of environmental protection prompted the development of so-called green and sustainable technologies. Therefore, academic and wider community intensively explores new chemicals and safer, more energy efficient processes based on a rational compromise between economic, social, and environmental requirements. Due to low volatility and stability, ionic liquids emerged as a potential replacement for traditional volatile and harmful organic solvents. Various studies have been carried out to validate the green character of ionic liquids, whereby data published suggest that these compounds, due to their relatively high toxicity and poor biodegradability, could have an extremely negative impact on the environment. This paper presents the current knowledge on the toxicity of ionic liquids, with a special emphasis on the mechanisms by which this group of compounds causes changes in the morphology and physiology of organisms at different organisational levels of the ecosystem.