Rosario, Juan C.

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    Effects of apoptotic inhibitor addition to cell viability prolongation of suspended bench-scale CHO culture and analysis of raman spectra in off-line bioreactor samples
    (2010) Rosario, Juan C.; Saliceti-Piazza, Lorenzo; College of Engineering; Carlos Sáez, Juan; Silva, Walter; Romañach, Rodolfo; Department of Chemical Engineering; Ramírez-Vick, Jaime E.
    The productivity of suspended mammalian cell cultures plays a major role in the overall manufacturing costs of biotechnology products. Upstream cell culture product variability can lead to problems in downstream purification and even in the quality of the final product. Amino acids and carbohydrates are the major raw substrates of mammalian cell metabolism. Knowing the ways in which the cells use these nutrients, researchers can gain a better understanding of what factors contribute to productivity, variability and apoptosis in cell culture processes. To achieve this knowledge, Raman spectroscopy can be used to develop a non-invasive, in-line tool to monitor and better understand the mammalian cell growth process in real-time. Our work is focused in implementing this analytical method as a way to monitor off-line cell metabolism and death under different culture conditions, such as two levels of temperature set points and presence or absence in the culture medium of an apoptosis inhibitor (Cytochrome C inhibitor). Techniques such as HPLC and biochemical analyzers were used as reference methods to validate the Raman spectra as a routine measurement in suspended mammalian cell culture processes and to monitor metabolic pathway changes. It is expected that the implementation of this methodology in-line will result in a better comprehension of mammalian cell culture in real-time and an improvement of productivity in such systems, as well as a tool towards implementation of process analytical technologies (PAT). Eight batches of suspended cultures of Chinese hamster ovary (CHO) cells were performed under four different conditions. The best parameters were a temperature shift of 35-33°C in 2.5 days with addition in the media of the apoptotic inhibitor (Cytochrome C inhibitor). In this case, twelve run days were achieved with viability between ninety and eighty percent with a substantial increment in L-lactate of approximately 35% as a secondary metabolite and a constant cell density on the entire runs. In addition to this condition a cell viability decay rate was observed. For conditions with temperature shift of 37-33°C in 2.5 days and overexpression or addition of the anti-apoptotic molecule, data obtained show that even though a higher viability was achieved, the run days could not be extended, an increment of approximately 35% on L-lactate production was also observed and the cell density decay rate was faster. This increment shows the possibility of double inhibition by the over-expression of the apoptotic inhibitor. As a consequence, these simultaneous inhibitions affect the cell metabolism and avoid the normal process of gluconeogenesis as published by Centocor with the use of another apoptotic inhibitor. These present increments on L-lactate provoke the use of more base in the process to stabilize and maintain pH measurements among the runs showing increments in osmolality values too. The proposed use of Raman to monitor cell metabolism and apoptosis was assayed in an offline mode providing promising results for cell metabolism monitoring (secondary and primary metabolites). The case of cell viability identification is still a matter of study. Peaks of some intracellular components were identified and they would constitute a subject of study to model any increase that could be associated with cellular survival or death.