Evaluation of a chemically defined medium for Pichia pastoris high cell density fermentation process

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Dokumentart: Bachelor Thesis
Institut: Department Biotechnologie
Sprache: Englisch
Erstellungsjahr: 2013
Publikationsdatum:
SWD-Schlagwörter: Pichia pastoris , Fermentation
DDC-Sachgruppe: Biowissenschaften, Biologie

Kurzfassung auf Englisch:

Microbial processes are important in modern biotechnology, due to the low requirements of the organisms in comparison to cell culture. Special relevance has Pichia pastoris in the production of biopharmaceuticals. It is a eukaryotic protein expression system and able to perform many protein modifications common in mammalian cells. For a successful protein expression an optimised process is important. Therefore, a fed batch process was evaluated in an established stainless steel system for the comparison of the medium to other media. The process was optimised for maximal cell density. Afterwards, a process transfer to a single use rocking motion type bioreactor was accomplished. Single use systems have the advantages of high flexibility and a decreased effort for cleaning and preparation. The system was originally designed for cell culture applications. For microbial processes the oxygen transfer rate was the limiting parameter in this system. The development of processes for microbial high cell density cultivations are challenging in this system. An important point was the optimisation of the feeding strategy in reference to the oxygen uptake rate. In batch processes the limit of the oxygen transfer rate was reviewed to calculated values. Fed batch processes conducted to increase the maximal cell density. Finally protein expression of human serum albumin with a methanol feed phase was performed. In the reusable system a maximal cell density of 387 gWCW/L (113 gDCW/L) was reached in a glycerol fed batch process. The results were comparable to industrial high cell density cultivations. In the single use rocking motion type bioreactor the cell density increased up to 292 gWCW/L (82 gDCW/L) in a glycerol fed batch process and up to 315 gWCW/L (92 gDCW/L) in a fed batch process with methanol induction phase. Limitations in the oxygen transfer rate avoid a further increase of the cell density. The present work shows the possibility of high cell density cultivations of Pichia pastoris in single use systems. Furthermore, the expression of the human serum albumin was possible in the single use system.

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