Human Chymotrypsinogen B Production with <i>Pichia</i><i>pastoris</i> by Integrated Development of Fermentation and Downstream Processing. Part 1. Fermentation

Curvers, Simon; Brixius, Peter; Klauser, Thomas; Thömmes, Jörg; Weuster‐Botz, Dirk; Takors, Ralf; Wandrey, Christian

doi:10.1021/bp000164j

Cited by 78 publications

(64 citation statements)

References 31 publications

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“…Thus, the common concept of a toxicity threshold for extracellular methanol concentration may be questioned. However, production strategies under AOX1 promoter control of Mut ϩ strains usually require control of methanol addition to achieve residual (extracellular) concentrations ranging from 1 to 5 g liter Ϫ1 (10,11,19,21,57,60). Concentrations of more than 3.5 g liter Ϫ1 are reported to inhibit biomass growth (60), while concentrations of more than 8 g liter Ϫ1 cause a decrease in productivity (57).…”

Section: Discussionmentioning

confidence: 99%

Combined Use of Fluorescent Dyes and Flow Cytometry To Quantify the Physiological State ofPichia pastorisduring the Production of Heterologous Proteins in High-Cell-Density Fed-Batch Cultures

Hyka

Züllig

Ruth

et al. 2010

Appl Environ Microbiol

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Matching both the construction of a recombinant strain and the process design with the characteristics of the target protein has the potential to significantly enhance bioprocess performance, robustness, and reproducibility. The factors affecting the physiological state of recombinant Pichia pastoris Mut ؉ (methanol utilization-positive) strains and their cell membranes were quantified at the individual cell level using a combination of staining with fluorescent dyes and flow cytometric enumeration. Cell vitalities were found to range from 5 to 95% under various process conditions in high-cell-density fed-batch cultures, with strains producing either porcine trypsinogen or horseradish peroxidase extracellularly. Impaired cell vitality was observed to be the combined effect of production of recombinant protein, low pH, and high cell density. Vitality improved when any one of these stress factors was excluded. At a pH value of 4, which is commonly applied to counter proteolysis, recombinant strains exhibited severe physiological stress, whereas strains without heterologous genes were not affected. Physiologically compromised cells were also found to be increasingly sensitive to methanol when it accumulated in the culture broth. The magnitude of the response varied when different reporters were combined with either the native AOX1 promoter or its d6* variant, which differ in both strength and regulation. Finally, the quantitative assessment of the physiology of individual cells enables the implementation of innovative concepts in bioprocess development. Such concepts are in contrast to the frequently used paradigm, which always assumes a uniform cell population, because differentiation between the individual cells is not possible with methods commonly used.Changes to the product quantity and quality as well as the robustness of bioprocesses can be triggered by a number of factors which affect the physiological state of the microorganisms. The expression of a foreign gene, the processing of the recombinant protein, and the exposure of the cell to metabolites, inductors, substrates, unfavorable environmental conditions, high cell density (HCD), and/or "aging" can all result in markedly different physiological responses (17).The interpretation of bioprocess data often fails to reflect the actual state of individual cells within a population. It is based typically on performance characterization using concentration measurements and the simplifying assumption of uniform ("averaged") performance of each cell. This practice (39) provides an example of the failure to distinguish between a homogenous population of equally compromised microorganisms and a heterogeneous population of cells each performing differently. An understanding of the state of the individual cells is critical in achieving high efficiency in recombinant protein production. Only by knowing the number of (vital) cells that express the target molecule at the highest possible rate can the number of such cells within the population be maximized and the propo...

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Section: Discussionmentioning

confidence: 99%

Combined Use of Fluorescent Dyes and Flow Cytometry To Quantify the Physiological State ofPichia pastorisduring the Production of Heterologous Proteins in High-Cell-Density Fed-Batch Cultures

Hyka

Züllig

Ruth

et al. 2010

Appl Environ Microbiol

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“…Chung (2000) estimated that 0.64 mol of oxygen is required per mol of methanol consumed based on methanol metabolism stoichiometry and a correlation between specific oxygen and methanol uptake rates obtained from steady state fed-batch cultivation conditions. Curvers et al (2001) used the measured yield of biomass from methanol (0.32 g DCW/g CH 3 OH) combined with an elemental analysis of biomass (CH 1.68 O 0.61 N 0.12 ) grown on methanol to calculate that 1.08 mol of O 2 is required per mole of methanol consumed. Cunha et al (2004) as well as Jahic et al (2003) measured the oxygen consumption rate as a function of the methanol uptake rate in fed-batch cultivations to be 0.8-1.1 mol O 2 /mol methanol consumed.…”

Section: Introductionmentioning

confidence: 99%

Antibody expression kinetics in glycoengineered Pichia pastoris

Potgieter

Kersey²,

Mallem³

et al. 2010

Biotech & Bioengineering

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Growth of the antibody market has fueled the development of alternative expression systems such as glycoengineered yeast. Although intact antibody expression levels in excess of 1 g L(-1) have been demonstrated in glycoengineered yeast, this is still significantly below the titers reported for antibody fragments in fungal expression systems. This study presents a simplified approach to estimate antibody secretion kinetics and oxygen uptake rate requirements as a function of growth-rate controlled by a limiting methanol feed rate in glycoengineered Pichia pastoris. The yield of biomass from methanol and the specific oxygen requirements predicted in this study compare well with values reported in the literature for wild-type P. pastoris, indicating the intrinsic nature of these yields independent of glycoengineering or the heterologous protein expressed. Specific productivity was found to be a non-linear function of specific growth rate. Based on comparison with relationships between specific growth rate and specific productivity reported in the literature this correlation seems empirical in nature and cannot be established a priori. These correlations were then used in a simple mass balance based model to predict the cultivation performance of carbon limited cultivations under oxygen transfer limited conditions to indicate the usefulness of this approach to predict large scale performance and aid in process development.

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“…Continuous bioprocess using methanol was performed via online methanol measurement and control using a minimal-variance-controller and a semi-continuous Kalman-Filter (Curvers et al, 2001).…”

Section: Introductionmentioning

confidence: 99%

A simple model‐based control for Pichia pastoris allows a more efficient heterologous protein production bioprocess

Cós

Ramon

Montesinos

et al. 2006

Biotech & Bioengineering

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A predictive control algorithm coupled with a PI feedback controller has been satisfactorily implemented in the heterologous Rhizopus oryzae lipase production by Pichia pastoris methanol utilization slow (Mut(s)) phenotype. This control algorithm has allowed the study of the effect of methanol concentration, ranging from 0.5 to 1.75 g/L, on heterologous protein production. The maximal lipolytic activity (490 UA/mL), specific yield (11,236 UA/g(biomass)), productivity (4,901 UA/L . h), and specific productivity (112 UA/g(biomass)h were reached for a methanol concentration of 1 g/L. These parameters are almost double than those obtained with a manual control at a similar methanol set-point. The study of the specific growth, consumption, and production rates showed different patterns for these rates depending on the methanol concentration set-point. Results obtained have shown the need of implementing a robust control scheme when reproducible quality and productivity are sought. It has been demonstrated that the model-based control proposed here is a very efficient, robust, and easy-to-implement strategy from an industrial application point of view.

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Human Chymotrypsinogen B Production with Pichiapastoris by Integrated Development of Fermentation and Downstream Processing. Part 1. Fermentation

Cited by 78 publications

References 31 publications

Combined Use of Fluorescent Dyes and Flow Cytometry To Quantify the Physiological State ofPichia pastorisduring the Production of Heterologous Proteins in High-Cell-Density Fed-Batch Cultures

Combined Use of Fluorescent Dyes and Flow Cytometry To Quantify the Physiological State ofPichia pastorisduring the Production of Heterologous Proteins in High-Cell-Density Fed-Batch Cultures

Antibody expression kinetics in glycoengineered Pichia pastoris

A simple model‐based control for Pichia pastoris allows a more efficient heterologous protein production bioprocess

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