Stabilizing Porcine Interferon-α Fermentation Performance By On-line Controlling Ethanol Concentration In Glycerol Feeding Phase

In this dissertation, porcine interferon-(pIFN-α) production by recombinant Pichiapastoris was conducted in a5L fermentor under high cell density environment. However,pIFN-α fermentation performance is unstable. pIFN-α antiviral activities or titers largely varyeven though the cell concentrations at methanol induction initiating instant are the same andthe induction conditions (methanol concentration, dissolved oxygen concentration DO, etc.)are similar in each fermentation batch. To solve this problem, different glycerol feedingstrategies during cell cultivation phase were investigated and the relevant fermentationperformance was compared, using the process control methodolges, aiming toincrease/stabilize pIFN-α titers. The major results were summarized as follow:1) It was found that ethanol accumulation occurred at the late cultivation phase, whencell concentration reached a certain higher level (80-100g-DCW·L–1), if traditional DO-Statmethod was adopted for glycerol feeding. The initiating instant of ethanol accumulation andthe highest ethanol concentration were affected by the cultivation conditions and difficult tocontrol. Methanol induction could not be initiated if the cells were subject to high ethanolconcentration environment (>6g·L–1) for longer time (>4h). In this case, DO and oxygenuptake rate (OUR) stayed at levels of of100%and0without any recovery trends during avery long period (>16h). However, the accumulated ethanol could be completely consumed.2) Analzing the transcription levels of the key enzymes in methanol metabolism routesrevealed that, the long-term and high level accumulation of ethanol during cultivation phaseirreversibly inhibited the activity and transcriptional level of alcohol oxidase (AOX), whichdeteriorated methanol induction and pIFN-α fermentation stability in turn.3) A commercialized methanol electrode was used to on-line measure ethanolconcentration during cultivation phase and methanol concentration during induction phase. Animproved DO-Stat glycerol feeding strategy based on on-line ethanol measurement was thusproposed, to alternatively utilize both glycerol and the accumulated ethanol, and to controlethanol concentrations at any desired levels. pIFN-α concentration could be stabilized athigher levels of2.70-3.65g·L–1if ethanol was controlled at lower level (2g·L–1), regardlesswhat kind of induction conditions (methanol/sorbitol co-feeding rate ratio of1:1or4:1) wereadopted. The pIFN-α concentration obtained in these cases was more than29.0%higher thanthe highest value obtained in the batches without ethanol concentration control. On the otherhand, pIFN-α concentrations stayed at very low levels of0.22-0.32g·L–1if ethanol wascontrolled at higher levels (12g·L–1). Stabilizing pIFN-α fermentation performance bycontrolling ethanol concentration during cultivation phase was experimentally verified.4) Parameters such as methanol concentration, DO, etc. in induction phase, are also thekey factors affecting heterologous protein fermentation performance. For the strain used inthis studay, the low DO environment, which supplied the maximum oxygen transfer force,was beneficial to pIFN-α production under moderate methanol induction intensity (5g·L–1).This could relieve oxygen supply load and improve fermentation performance furthermore.