Microbubble fermentation of recombinant Pichia pastoris for human serum albumin production

Abstract

The high cell density fermentation of recombinant Pichia pastoris for human serum albumin (HSA) production is a high oxygen demand process. The oxygen demand is usually met by increased agitation rate and use of oxygen-enriched air. Microbubble fermentation however can supply adequate oxygen to the microorganisms at relatively low agitation rates because of improved mass transfer of the microbubbles used for the sparging. Conventionally sparged fermentations were conducted for the production of HSA using P. pastoris at agitation rates of 350, 500, and 750 rpm, and were compared to MBD sparged fermentation at 150, 350, and 500 rpm agitation rates. The MBD improved the volumetric oxygen transfer coefficient (k L a) and subsequently increased the cell mass and protein production compared to conventional fermentation.Cell production in MBD fermentation at 350 rpm was 4.6 times higher than that in the conventional fermentation at 350 rpm, but similar to that in the conventional 750 rpm. The volumetric oxygen transfer coefficient k L a was 1011.9 h -1 in MBD 350 rpm, which was 6.1 times higher than that in the conventional 350 rpm (164.9 h -1 ) but was similar to the conventional 750 rpm (1098 h -1 ). Therefore, MBD fermentation results at low agitation of 350 rpm were similar to those in the conventional fermentation at high agitation of 750 rpm. There was considerable improvement in oxygen transfer to the microorganism using MBD sparging relative to the conventional sparging.Conventional fermentations were conducted both in a Biostat Q fermenter (small) at 500 rpm, 750 rpm, and 1000 rpm, and in a Bioflo III fermenter (large) at 350 rpm, 500 rpm, and 750 rpm. At the same agitation rate of 500 rpm, cell production in the large reactor was 3.8 times higher than that in the small one, and no detectable protein was produced in the small reactor at 500 rpm. At the same agitation rate of 750 rpm, both cell production and protein production in the large reactor were 4.6 times higher than the small reactor. Thus, the Bioflo III fermenter showed higher oxygen transfer efficiency than the Biostat Q fermenter, because of the more efficient aeration design of the Bioflo III fermenter. ACKNOWLEDGMENTS

Fed‐batch methanol feeding strategy for recombinant protein production by Pichia pastoris in the presence of co‐substrate sorbitol

“…The K L a values were generally highest at t = 12 h of the bioprocess, and had values of K L a = 0.067/s, 0.017/s, 0.043/s and 0.050/s for the M‐0.03, MS‐0.02, MS‐0.03 and MS‐0.04 conditions, respectively. The K L a values obtained were in the lower range of previously reported values for P. pastoris fermentations (0.01–0.3/s; Cunha et al , 2004; Zhang et al , 2005; Isett et al , 2007), probably due to the dimensions of the bioreactor used.…”

Effects of different glycerol feeding strategies on S-adenosyl-l-methionine biosynthesis by PGAP-driven Pichia pastoris overexpressing methionine adenosyltransferase

“…Another efficient means was employing the microbubble sparging dispersion to elevate k L a (Zhang et al, 2005). In the current paper, different glycerol feeding strategies were developed to increase OTR.…”

Oxygen transfer model in recombinant Pichia pastoris and its application in biomass estimation

“…The solubility of O 2 in pure water is 0.244 mmol l -1 at 25°C. In culture medium, it can be lower due to the presence of salts and other nutrients (Zhang et al 2005). O 2 requirements are usually met by regulating agitation and aeration rates.…”