Engineering CHO cells galactose metabolism to reduce lactate synthesis.

OBJECTIVE: Over-express galactokinase (Galk1) in tissue plasminogen activator (tPA) producing CHO cells as a potential strategy to improve cell growth and product synthesis. RESULTS: tPA producing CHO cells were transfected with the galactokinase (Galk1) gene. CHO-Galk1 cells showed a 39% increase of the specific growth rate in galactose. Moreover, clones were able to use this hexose as their main carbon source to sustain growth contrary to their parental cell line. Metabolic Flux Analysis revealed that the CHO-Galk1 selected clone shows an active metabolism towards biomass and product synthesis, characterized by higher fluxes in the TCA cycle, which is consistent with increased cellular densities and final product concentration. CONCLUSION: This cellular engineering strategy, where modifications of key points of alternative carbon sources metabolism lead to an improved metabolism of these sugars, is a starting point towards the generation of new cell lines with reduced lactate synthesis and increased cell growth and productivity.

Plasmid DNA production for therapeutic applications.

Plasmid DNA (pDNA) is the base for promising DNA vaccines and gene therapies against many infectious, acquired, and genetic diseases, including HIV-AIDS, Ebola, Malaria, and different types of cancer, enteric pathogens, and influenza. Compared to conventional vaccines, DNA vaccines have many advantages such as high stability, not being infectious, focusing the immune response to only those antigens desired for immunization and long-term persistence of the vaccine protection. Especially in developing countries, where conventional effective vaccines are often unavailable or too expensive, there is a need for both new and improved vaccines. Therefore the demand of pDNA is expected to rise significantly in the near future. Since the injection of pDNA usually only leads to a weak immune response, several milligrams of DNA vaccine are necessary for immunization protection. Hence, there is a special interest to raise the product yield in order to reduce manufacturing costs. In this chapter, the different stages of plasmid DNA production are reviewed, from the vector design to downstream operation options. In particular, recent advances on cell engineering for improving plasmid DNA production are discussed.

Adaptation of the "Dynamic Method" for measuring the specific respiration rate in oxygen transfer systems through diffusion membrane.

Monitoring the specific respiration rate (Q(O2)) is a valuable tool to evaluate cell growth and physiology. However, for low Q(O2) values the accuracy may depend on the measurement methodology, as it is the case in animal cell culture. The widely used "Dynamic Method" imposes serious difficulties concerning oxygen transfer cancellation, especially through membrane oxygenation. This paper presents an improved procedure to this method, through an automated control of the gas inlet composition that can minimize the residual oxygen transfer driving force during the Q(O2) measurement phase. The improved technique was applied to animal cell cultivation, particularly three recombinant S2 (Drosophila melanogaster) insect cell lines grown in a membrane aeration bioreactor. The average measurements of the proposed method reached 98% of stationary liquid phase balance method, taken as a reference, compared to 21% when the traditional method was used. Furthermore, this methodology does not require knowledge of the volumetric transfer coefficient k(L)a, which may vary during growth.