Oxygen-limited fed-batch process: an alternative control for Pichia pastoris recombinant protein processes

Abstract

An oxygen-limited fed-batch technique (OLFB) was compared to traditional methanol-limited fed-batch technique (MLFB) for the production of recombinant Thai Rosewood beta-glucosidase with Pichia pastoris. The degree of energy limitation, expressed as the relative rate of respiration (q(O)/q(O,max)), was kept similar in both the types of processes. Due to the higher driving force for oxygen transfer in the OLFB, the oxygen and methanol consumption rates were about 40% higher in the OLFB. The obligate aerobe P. pastoris responded to the severe oxygen limitation mainly by increased maintenance demand, measured as increased carbon dioxide production per methanol, but still somewhat higher cell density (5%) and higher product concentrations (16%) were obtained. The viability was similar, about 90-95%, in both process types, but the amount of total proteins released in the medium was much less in the OLFB processes resulting in substantially higher (64%) specific enzyme purity for input to the downstream processing.

Combined Use of Fluorescent Dyes and Flow Cytometry To Quantify the Physiological State ofPichia pastorisduring the Production of Heterologous Proteins in High-Cell-Density Fed-Batch Cultures

“…The optimum pH value for Pichia growth is approximately 5 (6), but the pH value is commonly reduced to counteract proteolysis (9,32) or to increase both the stability and productivity of heterologous proteins (5,29,56). However, a pH value of 4 or lower can compromise cells during the production process (27).…”

Two-phase fed-batch modification for 48 hour peak expression of hepatitis B surface antigen in Pichia pastoris shake flask system

“…In the past, in order to avoid oxygen limiting conditions in Pichia cultures, it was common practice for most protocols either to dilute and maintain cell biomass at low levels or to reduce methanol supply [13]. However, two studies have shown that reduced oxygen conditions may be advantageous for the production of recombinant proteins by using P. pastoris AOX1 expression system [17,27]. A model explaining the relationship between specific growth rate and methanol concentration in P. pastoris suggested that the production of hydrogen peroxidase and formaldehyde metabolites is more dependent on methanol concentration than on oxygen intake [21].…”

“…However, previous study of the Mut + phenotype for HBsAg expression was regarded as unfavourable, mainly due to its high demand for oxygen in high density cell cultures that creates oxygen limitation [16]. On the other hand, more recent studies have shown that oxygen limited fed-batch expression may prove advantageous in producing higher cell growth density and obtaining higher product concentration [8,17].…”

“…Under these conditions, the concentration of methanol used as carbon source is usually low and the growth rate of the cultures is limited to ensure a smooth adaptation of cells from glycerol to methanol as the sole carbon source [17]. Glycerol and methanol mix-feeding fed-batch has also been introduced, which could lead to an increase in production time and cost [19].…”

ALGINATE PRODUCTION BY Azotobacter vinelandii AS A VIRTUAL SENSOR TO ESTIMATE EFFECTIVE SHEAR RATE ON STIRRED TANK FERMENTERS

“…Under these conditions, the relative respiration rate (RRR) of A. vinelandii diminished drastically; this parameter has been defined as the current respiration rate divided by the maximum respiration rate (Charoenrat et al, 2005). (Díaz-Barrera et al, 2007;Díaz-Barrera et al, 2009) .…”