Maximizing recombinant human serum albumin production in a Mut^sPichia pastoris strain

Abstract

Human serum albumin (HSA) is a cysteine rich molecule that is most abundant in human blood plasma. To remain viable in the market due to lower marketing costs for HSA, it is important to produce a large quantity in an economical manner by recombinant technology. The objective of this study was to maximize recombinant HSA (rHSA) production using a Mut(s) Pichia pastoris strain by fermentation process optimization. We evaluated the impact of process parameters on the production of rHSA, including induction cell density (wet cell weight, g/L) and the control of specific growth rate at induction. In this study, we demonstrated that induction cell density is a critical factor for high level production of rHSA under controlled specific growth rate. We observed higher specific productivities at higher induction cell densities (285 g/L) and at lower specific growth rates (0.0022-0.0024/h) during methanol induction phase, and achieved the broth titer of rHSA up to 10 g/L. The temperature shift from 24 to 28(o) C was effective to control the specific growth rate at low level (≤0.0024/h) during methanol induction phase while maintaining high specific productivity [0.0908 mgrHSA /(gwcw h)].

Characterization of methanol utilization negative Pichia pastoris for secreted protein production: New cultivation strategies for current and future applications

“…HSA production with Mut + or Mut S strains has already been attempted by some researchers, and by selecting for multicopy clones and optimizing the process, they obtained end titers of up to 10 g/L (Mallem et al, 2014; Zhu et al, 2018). As our work is still at a proof of concept stage, we used single copy clones and did not further optimize the process.…”

Cultivation strategies to enhance productivity of Pichia pastoris: A review

“…At the beginning of the methanol induction-/production-phase, CDW concentrations between 20 g l -1 to 100 g l -1 were reported (Table 4). The biomass concentration at the beginning of the production phase is a critical factor that should be considered for optimising high-level production (Mallem et al, 2014, Zhang et al, 2007. By Invitrogen protocols (2002), an initial biomass concentration of 180 g l -1 to 220 g l -1 WCW, which corresponds to approximately 50 g l -1 CDW, is recommended.…”

“…Typically production kinetics were investigated for µ < 0.08 h -1 (Curvers et al, 2001b, Zhang et al, 2005 or even below 0.03 h -1 (Hang et al, 2008, Kobayashi et al, 2000, Min et al, 2010, Potgieter et al, 2010. Possibly the relatively low specific growth rates were chosen since they are favourable for the design of a production process with a high titre and productivity (Mallem et al, 2014). For fully growth-associated GAP-regulated production, kinetics was typically investigated between 0.02 h -1 and 0.19 h -1 (Buchetics et al, 2011, Maurer et al, 2006.…”

“…However, the application was for GAPcontrolled recombinant protein production with a single substrate for growth and production. For AOX1-controlled processes, biomass concentration at the start of the methanol induction-/production-phase is variable and has to be optimised, together with the time course for µ during the production phase (Mallem et al, 2014, Zhang et al, 2007. A maximisation model for AOX1-controlled processes was established by Kobayashi et al (2000) and Zhang et al (2005).…”

Perspectives and Prospects for Whole Cell Catalysis

“…The methanol also serves as a carbon and energy source that can support product synthesis. A recent example of using P. pastoris is the production of recombinant human serum albumin at a titer of 10 g/liter [24]. The product can be used to treat hypoalbuminemia and because it is free of blood-borne pathogens (e.g.…”