Production of human serum albumin by immobilized yeast
Date of Award
Doctor of Philosophy (PhD)
Biomedical and Chemical Engineering
Albumin, Calcium alginate
Biomedical Engineering and Bioengineering | Chemical Engineering
Production of human serum albumin (HSA) in fermentations using the yeast P. pastoris , immobilized in a calcium alginate matrix was explored. HSA, produced by the P. pastoris GS115 Mut s strain, was able to diffuse through the calcium alginate gel into the supernatant. The immobilized cell fermentations were conducted in both shake flask and bioreactor scales. The effects of several parameters (bead size, initial cell loading and ratio of immobilized cell beads to liquid medium) were investigated. An automatic data acquisition system and methanol control scheme were developed using a low-cost, automobile exhaust gas analyzer. Mathematical models were also constructed to describe cell growth using glycerol and HSA production during the methanol induction phase.
Glutamate medium was more effective than several other media (BMMY, MMY and MM medium) in stabilizing the calcium alginate immobilized cells. Production of HSA using the glutamate medium during suspended cell fermentation, however, suggested the presence of nutrient limitation. The production of HSA in calcium alginate immobilized cell fermentation was affected by the pH of the liquid medium, and it is hypothesized that electrostatic interactions between HSA and the uronic acids comprising the alginate could inhibit diffusion of HSA out of the immobilized cell beads. Immobilized cells beads were metabolically active in producing HSA, and remained intact for at least 38 days in shake flask fermentation and 17 days in the bioreactor.
Although HSA concentrations in immobilized cell fermentation were less than that in suspended cell fermentation, the maximum specific total protein production rates of immobilized cell and suspended cell fermentations in glutamate medium were comparable, 0.00109 and 0.00148 mg total protein/(mg cell dry weight day), respectively. The maximum specific total protein production rates in both suspended and immobilized cells were not sustained throughout glutamate fermentation presumably due to nutrient limitation. This immobilized fermentation using calcium alginate entrapment seems promising as an alternative means to produce recombinant protein. Further studies to optimize glutamate medium, investigate the pH effect, explore an extended period of fermentation, and examine alternative immobilized reactor types merit consideration.
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Thipayarat, Aluck, "Production of human serum albumin by immobilized yeast" (2002). Biomedical and Chemical Engineering - Dissertations. 25.