Author(s): Pereira, João Nuno dos Santos
Date: 2011
Persistent ID: http://hdl.handle.net/10362/6149
Origin: Repositório Institucional da UNL
Subject(s): Transient gene expression; Recombinant protein production; CHO cells; Conditioned medium; Scale-up
Dissertação para obtenção do Grau de Mestre em Biotecnologia
Transient gene expression (TGE) allows for fast protein production in mammalian cells and has become a very important technology in the product development pipeline of biopharmaceuticals. Polyethylenimine (PEI) mediated, high-density transfections have allowed for transient processes exceeding ~300mg/L in CHO-DG44 cells. As such, the bottleneck of TGE is no more in the titers, but in the scale-up to volumes higher than 1L, because of the need for a medium exchange before transfection. It is known that if the transfection is done in a running culture, without a medium exchange (i.e in conditioned medium), the yields obtained are very low (~5 mg/L). In CHO-DG44 cells, this problem was explored from the point of view of transfection efficiency, gene delivery and transcription. A new insight is presented in this work: The low productivities are not due to a deficient gene delivery, but instead, to lower mRNA levels that we hypothesize to be related to a lower gene accessibility of the transfected plasmid. Further, the yields were improved from ~5mg/L to ~90mg/L (18-fold) by optimizing the conditions for transfecting in conditioned medium and utilizing sodium butyrate as a transcription enhancer. These results are expected to open paths for the successful scale-up of TGE.
