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Determination of optimal bioreactor operational strategy and unstructured macrokinetic model for recombinant human growth hormone production on ethanol by a novel pichia pastoris PADH2-Cat8-L2 based system

Wehbe Al Masri, Omar
In recombinant protein (r-protein) production, the host Pichia pastoris (Komagataella phaffii) is used with the methanol-inducible alcohol oxidase 1 promoter (PAOX1), one of the strongest naturally occurring promoters, due to its tight regulation and exceptional strength. For the pharmaceutical and food sectors, since the use of methanol as a substrate in r-protein productions may complicate separation processes, and required documentations, methanol-free production by green bio-processes is encouraged but challenging. In this context, using the non-toxic carbon and energy source ethanol under our newly developed alcohol dehydrogenase 2 (ADH2) hybrid promotor PADH2-Cat8-L2, ethanol feeding strategies were developed for rhGH production by P. pastoris. Prior to pilot-scale experiments, the effects of ethanol on the growth of P. pastoris were investigated in laboratory-scale batch-bioreactors at T=30°C and N=200 min-1 (rpm). An increase in ethanol concentration lead to an increase in the final cell concentration, however, at CEtOH > 3 g L-1, the substrate inhibited the growth marked by a decrease in specific cell growth rates and the longer cultivation time required to reach the same cell densities. This inhibition behavior was also mathematically modelled where the Haldane model was able to clearly depict the inhibitory effect of ethanol at high concentrations with a critical substrate concentration of 1.97 g L-1 and a maximum specific growth rate of 0.194 h-1. The effects of two different continuous feed stream (CFS) operational strategies on the specific growth rate, specific ethanol uptake rate and specific rhGH production rate were investigated as follows: CFS designed with three pre-determined specific growth rate (μ) values of 0.020, 0.035 and 0.050 h-1, and ethanol-stat CFS designed with three constant CEtOH values of 0.5, 1.0, and 1.5 g L-1 in the cultivation medium. In the ethanol-stat fed-batch bioreactors, approximately 1.6-fold higher maximum cell concentration and 2.1-fold higher maximum rhGH concentration (91 mg L-1) were obtained compared to those obtained by the fed-batch bioreactors designed with pre-determined μ values. In addition, 2-fold higher maximum volumetric productivity was obtained by the ethanol-stat fed-batch bioreactors. The ethanol-stat strategy of CEtOH,set=0.5 g L-1 was the most favorable operational strategy due to it having the highest rhGH titer, along with high volumetric productivity and protein on biomass yield (Yp/x). In addition, high ethanol concentrations inhibited the specific growth rate, the specific ethanol uptake rate and the protein production rate, where the inhibition pattern was mathematically described according to the Haldane model. The relationship between the specific ethanol uptake rate and the specific growth rate was described by the linear Pirt model, whereas the relationship between the specific protein production rate and the specific growth rate was not linear, and thus could not be described by the Luedeking-Piret model. Lastly, cell generation and rhGH production were simulated based on the Haldane models relating the specific growth rate and specific rhGH production rate to the residual ethanol concentration.