Influence of specific growth rate on specific productivity and glycosylation of a recombinant avidin produced by a Pichia pastoris Mut⁺ strain

Abstract

A recombinant avidin-producing Mut+ Pichia pastoris strain was used as a model organism to study the influence of the methanol feeding strategy on the specific product productivity (q(p)) and protein glycosylation. Fed-batch cultivations performed at various specific growth rates (micro) and residual methanol concentrations showed that the specific avidin productivity is growth-dependent. The specific productivity increases strongly with the specific growth rate for micro ranging from 0 to 0.02 h(-1), and increases only slightly with the specific growth rate above this limit. N-terminal glycosylation was also found to be influenced by the specific growth rate, since 9-mannose glycans were the most abundant form at low growth rates, whereas 10-mannose carbohydrate chains were favored at higher micro. These results show that culture parameters, such as the specific growth rate, may significantly affect the activity of glycoproteins produced in Pichia pastoris. In terms of process optimization, this suggests that a compromise on the specific growth rate may have to be found, in certain cases, to work with an acceptable productivity while avoiding the addition of many mannoses.

Antibody expression kinetics in glycoengineered Pichia pastoris

“…Similar to the findings of various other investigators, the relationship between specific growth rate and specific productivity is non-linear and not predictable a priori (Cunha et al, 2004;Kobayashi et al, 2000;Schenk et al, 2008;Zhang et al, 2000b). The imbalances between energy supply for growth and production is usually postulated as the mechanism to explain the impact of growth rate on specific productivity (Zhang et al, 2005).…”

“…Based on comparison with relationships between specific growth rate and specific productivity reported in the literature (Cunha et al, 2004;Kobayashi et al, 2000;Schenk et al, 2008;Zhang et al, 2000b), this correlation seems empirical in nature and cannot be established a priori.…”

“…It has been postulated that correlations may exists between secretion kinetics, protein folding, degradation and N-linked glycosylation homogeneity (Damasceno et al, 2007;Jahic et al, 2002;Schenk et al, 2008;Sinha et al, 2004). Figure 4 shows that although the reduced secretion rate at increased growth rates may be attributed to either misfolding, aggregation or degradation, the fraction of IgG1 secreted by glycoengineered yeast that binds to protein A remained essentially unaffected over the range of growth rates tested.…”

Effect of dilution rate and methanol‐glycerol mixed feeding on heterologous Rhizopus oryzae lipase production with Pichia pastorisMut+ phenotype in continuous culture

“…Thus, the process requires maintaining the methanol concentration above a level able to allow full induction from the promoter, but below the cell growth inhibition level. 6,7 These facts affect the kinetics and productivity of the system and they are the basis developing fed-batch culture strategies at high cell density, such as those with constant specific growth rate (m), 8 or constant methanol concentration using an automatic control system. 9,10 The induction stage can also be operated using feeding mixtures of methanol-glycerol substrates.…”

Reaction rate reconstruction from biomass concentration measurement in bioreactors using modified second-order sliding mode algorithms

“…In this paper we focus on the estimation of reaction rates and, particularly, of specific growth rates. The motivation is that control specifications are often related with the growth rate of microorganisms, whether the objective is to maximize biomass production or to maintain a metabolic steady state [19,34,36,40]. In addition, growth rate estimators provide essential information to monitor the development of microorganisms.…”