No description available.

No description available.

No description available.

No description available.

No description available.

The bio-manufacturing of recombinant proteins from mammalian cell cultures requires robust processes that can maximize protein yield while ensuring the efficacy of these proteins as human therapeutics. Recognizing that the challenge of improving protein yield and quality can be met through various approaches, this paper presents three strategies currently being developed in our group. A method for rapidly selecting subpopulations of cells with high production characteristics is proposed. This method combines the efficiency of green fluorescent protein/fluorescence-activated cell sorting (GFP/FACS)–based screening with homologous recombination to generate and select high-producing subclones. Next, the development of chemically defined, protein-free media for enhancing monoclonal antibody production is described. Analysis of culture media effects on the genome-wide transcriptional program of the cell is presented as a means to optimize the culture media and identify potential targets for genetic manipulation. Finally, we propose a method for increasing the extent of intracellular sialylation by improving the transport of CMP-sialic acid into the trans-Golgi. This is hypothesized to increase the sialic acid availability, and may enhance the degree of sialylation in the glycoprotein product. / Singapore-MIT Alliance (SMA)

Cell culture media used in the manufacture of biopharmaceuticals conventionally contain many animal derived components. These components can harbor adventitious agents which can be transmitted through biotherapeutics, employed in the medical treatment of immunocompromised patients. An ADCF (animal derived component free) medium formulation obviates this concern. A rational method for the rapid and efficient screening and optimization of ADCF media while preserving, if not enhancing, cellular growth and protein productivity is needed. CHO (Chinese Hamster Ovary) cells, widely used as a production platform in industry, expressing a recombinant protein, were employed as a model system. Design of Experiment (DOE) and statistical analysis were employed to assess the impact of media formulation on cellular physiology. Metabolic flux, cellular growth, and protein productivity were evaluated as the measures of ADCF media formulation success. Measurements of extracellular metabolites were determined by HPLC and enzymatic methods. Recombinant protein production was measured by HPLC. This research demonstrates the successful screening and optimization of four plant hydrolysate mixtures (2 soy and 2 wheat) as a replacement for animal derived components. / NSERC, ABIN, MABNET

The requirement of DNA synthesis for the release of Ɣ-radiation-induced mitotic inhibition in mammalian cells has been studied. Mammalian cells in which DNA synthesis had been inhibited by treatment with fluorodeoxyuridine (FUdR) were not released from radiation-induced mitotic inhibition until the FUdR block was removed. After removal of the block, mitotic figures reappeared, but only after a time equivalent to the usual mitotic delay caused by the particular radiation dose employed. This suggests that repair of the mitotic inhibition lesion can not proceed unless the pathway for DNA synthesis is intact. Further evidence for the requirement of DNA synthesis in the release of mitotic inhibition came from the observation of radiation-induced synthesis of DNA during G₂, a stage in the cell cycle normally not associated with such synthesis. / Thesis / Master of Science (MSc)

Chloramphenicol has been used to study mitochondrial biogenesis in mammalian cells by examining its effect on: the incorporation of radioactive amino acids into protein by isolated mitochondria, the growth of L cells, the level of representative enzymes and cytochromes in the mitochondria and cytoplasm and the structure of mitochondria and L cells. A reversible inhibition of synthesis of cytochrome c oxidase was obtained by treating cells with D-threo-chloramphenicol for 90 hr. Recovery of cytochrome c oxidase activity was inhibited by cycloheximide, an inhibitor of cytoplasmic protein synthesis. Cycloheximide also reversibly inhibited cytochrome c oxidase formation in cells which were not treated with D-chloramphenicol. It is suggested that the mitochondria and the nucleus have a joint control in the formation of a functionally active cytochrome c oxidase enzyme. / Thesis / Doctor of Philosophy (PhD)

No description available.