RESUMO
Most commercial media for mammalian cell culture are designed to satisfy the amino acid requirements for cell growth, but not necessarily those for recombinant protein production. In this study, we analyze the amino acid consumption pattern in naïve and recombinant Chinese hamster ovary (CHO) cell cultures. The recombinant model we chose was a CHO-S cell line engineered to produce a monoclonal antibody. We report the cell concentration, product concentration, and amino acid concentration profiles in naïve and recombinant cell cultures growing in CD OptiCHO™ medium with or without amino acid supplementation with a commercial supplement (CHO CD EfficientFeed™ B). We quantify and discuss the amino acid demands due to cell growth and recombinant protein production during long term fed batch cultivation protocols. We confirmed that a group of five amino acids, constituting the highest mass fraction of the product, shows the highest depletion rates and could become limiting for product expression. In our experiments, alanine, a non-important mass constituent of the product, is in high demand during recombinant protein production. Evaluation of specific amino acid demands could be of great help in the design of feeding/supplementation strategies for recombinant mammalian cell cultures.
RESUMO
Several isolates of Bacillus thuringiensis native to Argentina obtained in a nationwide screening program showed atypical crystal morphology. One of these strains, INTA 51-3, was further characterized in order to determine other features like protein composition of its parasporal crystal, plasmid pattern, identification of cry genes and toxicological properties. B. thuringiensis INTA 51-3 (serovar tohokuensis) had an amorphous inclusion containing a major protein component of ca. 130 kDa. After trypsin digestion of solubilized crystals, SDS-PAGE resolved a unique protease-resistant peptide of ca. 90 kDa. The plasmid pattern from INTA 51-3 resembled that of the standard strain HD-1. However, Southern analysis showed no hybridization to fragments of cry1Aa, cry2Aa, cry3A, and cry11A genes. Degenerate primers were used for identification of the cry1 genes by PCR. Nevertheless, the presence of cry1 type gene(s) in B. thuringiensis INTA 51-3 was confirmed. Highly concentrated crystal suspensions showed to be weakly toxic only to lepidopteran species.