Hybrid fed‐batch bioreactor operation design: control of substrate uptake enhances recombinant protein production in high‐cell‐density fermentations

Abstract

BACKGROUND The hybrid fed‐batch bioreactor (HFBB) operation is a novel design method for high‐cell‐density fermentations. It employs fed‐batch and batch operations, starting with fed‐batch that in turn shifts to the next, and proceeds in successive cycles to enhance production and productivity by controlling the substrate uptake rate. RESULTS The method was tested for the production of recombinant human growth hormone (rhGH) in Pichia pastoris under GAP promoter with carbon sources of (i) glucose and (ii) pre‐treated hydrolyzed sugar‐beet molasses containing equimolar glucose and fructose. The superior strategies were: (i) GH2, a HFBB with glucose feeding composed of ΔtFB = 1.5 h fed‐batch periods with continuous‐feed stream designed with the pre‐determined growth rate µ = 0.10 h−1, which shift in turn to ΔtB = 0.5 h batch periods; and (ii) MH2, a HFBB with molasses feeding designed in a similar way to GH2. The highest rhGH production attained in GH2 was 611 mg dm−3 at 8 h, whereas it was 625 mg dm−3 at 13.5 h in MH2. In GH2, rhGH production increased 1.37‐fold in half the cultivation time of GH2. However, in MH2 with dual carbon sources enriched with other substrates, higher rhGH production was obtained at the cost of lower productivity. CONCLUSION The HFBB is a powerful design method with a single carbon source which improves the productivity of high‐cell‐density fermentations. However, with molasses upon shifting to batch operations, the uptake of accumulated fructose is continued. Thus, the glamour of the HFBB is hindered for complex media fermentations to an extent, because multi‐carbon sources contribute to the growth during the batch periods by lengthening the cultivation time. © 2018 Society of Chemical Industry

Innovative Bioprocess Strategies Combining Physiological Control and Strain Engineering of Pichia pastoris to Improve Recombinant Protein Production

“…Additionally, the application of different types of cell stress has been demonstrated to have a significant positive effect on RPP, so the established process strategies can be remarkably improved (García-Ortega et al, 2019;Chen et al, 2021). Some examples are the application of substrate starving periods (Garcia-Ortega et al, 2016;Çalık et al, 2018) and the implementation of oxygen limiting conditions (Baumann et al, 2008), which have shown a positive effect on RPP under P GAP regulation.…”

Comparative study of μ‐stat methanol feeding control in fed‐batch fermentation of Pichia pastoris producing HBsAg: an open‐loop control versus recurrent artificial neural network‐based feedback control

“…By applying the proposed feedback feeding control system based on RNN, the efficiency of already established large‐scale production plants producing HBsAg in methylotrophic yeasts could be enhanced without considerable time and money investment. In general, this tool can be combined with other feeding strategies and other fed‐batch designs, especially novel and more complicated ones, to reduce any unexpected deviations in key parameters . The combination of mathematical models with computational models, based on machine learning, in the future, could significantly reduce the costs and time related to bioprocess development and scale‐up by decreasing the number of experimental investigations.…”

“…In general, this tool can be combined with other feeding strategies and other fed-batch designs, especially novel and more complicated ones, to reduce any unexpected deviations in key parameters. 11,21,36 The combination of mathematical models with computational models, based on machine learning, in the future, could significantly reduce the costs and time related to bioprocess development and scale-up by decreasing the number of experimental investigations.…”

“…In many previous studies, the classic AOX1 promoter (P AOX1 ), including P AOX1 ‐derived variants, was mostly used for recombinant protein expression in P. pastoris owing to its strong and tightly regulated features . From the industrial perspective, however, the main drawbacks of this type of strain are the high amount of heat production and oxygen requirement as well as the presence of methanol as a toxic and flammable substance in the upstream and sometimes downstream stages . Glyceraldehyde 3‐phosphate dehydrogenase (GAP) promoter is one of the alternative natural promoters for methanol‐free protein expression without any requirement for induction by shifting the culture from one carbon source to another .…”

“…2,3 From the industrial perspective, however, the main drawbacks of this type of strain are the high amount of heat production and oxygen requirement as well as the presence of methanol as a toxic and flammable substance in the upstream and sometimes downstream stages. [11][12][13][14] Glyceraldehyde 3-phosphate dehydrogenase (GAP) promoter is one of the alternative natural promoters for methanol-free protein expression without any requirement for induction by shifting the culture from one carbon source to another. 14 Apart from natural promoters, since the last decade, there has also been growing interest in using other synthetic-engineered promoters to increase the production rate without the major challenges related to P AOX1.…”