Improved production of monoclonal antibodies through oxygen-limited cultivation of glycoengineered yeast

Glycoengineering technology can elucidate and exploit glycan related structure-function relationships for therapeutic proteins. Glycoengineered yeast has been established as a safe, robust, scalable, and economically viable expression platform. It has been found that specific productivity of antibodies in glycoengineered Pichia pastoris is a non-linear function of specific growth rate that is dictated by a limited methanol feed rate. The optimal carbon-limited cultivation requires an exponential methanol feed rate with an increasing biomass concentration and more significantly an increase in heat and mass transfer requirements that often become the limiting factor in scale-up. Both heat and mass transfer are stoichiometrically linked to the oxygen uptake rate. Consequently an oxygen-limited cultivation approach was evaluated to limit the oxygen uptake rate and ensure robust and reliable scale-up. The oxygen-limited process not only limited the maximum oxygen uptake rate (and consequently the required heat removal rate) in mut+ P. pastoris strains but also enabled extension of the induction phase leading to an increased antibody concentration (1.9 g L⁻¹ vs. 1.2 g L⁻¹), improved N-glycan composition and galactosylation, and reduced antibody fragmentation. Furthermore, the oxygen-limited process was successfully scaled to manufacturing pilot scale and thus presents a promising process option for the glycoengineered yeast protein expression platform.     

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

The induction using substrate mixtures is an operational strategy for improving the productivity of heterologous protein production with Pichia pastoris. Glycerol as a cosubstrate allows for growth at a higher specific growth rate, but also has been reported to be repressor of the expression from the AOX1 promoter. Thus, further insights about the effects of glycerol are required for designing the induction stage with mixed substrates. The production of Rhizopus oryzae lipase (ROL) was used as a model system to investigate the application of methanol‐glycerol feeding mixtures in fast metabolizing methanol phenotype. Cultures were performed in a simple chemostat system and the response surface methodology was used for the evaluation of both dilution rate and methanol‐glycerol feeding composition as experimental factors. Our results indicate that productivity and yield of ROL are strongly affected by dilution rate, with no interaction effect between the involved factors. Productivity showed the highest value around 0.04–0.06 h^?1, while ROL yield decreased along the whole dilution rate range evaluated (0.03–0.1 h^?1). Compared to production level achieved with methanol‐only feeding, the highest specific productivity was similar in mixed feeding (0.9 UA g‐biomass^?1 h^?1), but volumetric productivity was 70% higher. Kinetic analysis showed that these results are explained by the effects of dilution rate on specific methanol uptake rate, instead of a repressor effect caused by glycerol feeding. It is concluded that despite the effect of dilution rate on ROL yield, mixed feeding strategy is a proper process option to be applied to P. pastoris Mut⁺ phenotype for heterologous protein production. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:707–714, 2015     

GAP promoter‐based fed‐batch production of highly bioactive core streptavidin by Pichia pastoris

Streptavidin is a homotetrameric protein binding the vitamin biotin and peptide analogues with an extremely high affinity, which leads to a large variety of applications. The biotin‐auxotrophic yeast Pichia pastoris has recently been identified as a suitable host for the expression of the streptavidin gene, allowing both high product concentrations and productivities. However, so far only methanol‐based expression systems have been applied, bringing about increased oxygen demand, strong heat evolution and high requirements for process safety, causing increased cost. Moreover, common methanol‐based processes lead to large proportions of biotin‐blocked binding sites of streptavidin due to biotin‐supplemented media. Targeting these problems, this paper provides strategies for the methanol‐free production of highly bioactive core streptavidin by P. pastoris under control of the constitutive GAP promoter. Complex were superior to synthetic production media regarding the proportion of biotin‐blocked streptavidin. The optimized, easily scalable fed‐batch process led to a tetrameric product concentration of up to 4.16 ± 0.11 µM of biotin‐free streptavidin and a productivity of 57.8 nM h^?1 based on constant glucose feeding and a successive shift of temperature and pH throughout the cultivation, surpassing the concentration in un‐optimized conditions by a factor of 3.4. Parameter estimation indicates that the optimized conditions caused a strongly increased accumulation of product at diminishing specific growth rates (μ ≈ D < 0.01 h^?1), supporting the strategy of feeding. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:855–864, 2016     

Pichia surface display: display of proteins on the surface of glycoengineered Pichia pastoris strains

Expression of proteins on the surface of yeasts has a wide range of applications in biotechnology, such as directed evolution of proteins for increased affinity and thermal stability, screening of antibody libraries, epitope mapping, and use as whole-cell biocatalysts. However, hyperglycosylation can interfere with overall protein accessibility on the surface. Therefore, the less elaborate hyperglycosylation in wild type Pichia pastoris and the availability of glycoengineered strains make this yeast an excellent alternative for surface display of glycoproteins. Here, we report the implementation of the well-established a-agglutinin-based yeast surface display technology in P. pastoris. Four heterologous proteins were expressed on the surface of a wild type and a glycoengineered strain. Surface display levels were monitored by Western blot, immunofluorescence microscopy, and FACS analysis. The availability of glycoengineered strains makes P. pastoris an excellent alternative for surface display of glycoproteins and paves the way for new applications. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Two‐compartment processing as a tool to boost recombinant protein production

Pichia pastoris is used extensively as a production platform for many recombinant proteins. The dissolved oxygen (DO) is one of the most important factors influencing protein production. The influence of the DO on productivity has not been studied independent from the feed rate. In this work, various DO levels were investigated independent from the feed rate. The model system was recombinant P. pastoris under the control of methanol‐induced alcohol oxidase promoter, which expressed HRP as the target protein. No significant effect was observed in terms of titer and specific productivity, which is a confirmation of the fact that the DO in a one‐compartment system cannot boost productivity for the model system under study. Hence, a two‐compartment system (a single reactor coupled with a plug flow reactor) was designed and implemented in order to apply oxygen‐related stress in the plug flow reactor and allow the cells to be recovered in the main reactor. Doing so, more than two‐fold increase in the titer and productivity and three‐fold increase in protein‐specific activity were achieved. Hence, partial application of oxygen‐related stress in the two‐compartment system was proposed as a process technology to enhance protein production.     

Oxygen-limited control of methanol uptake for improved production of a single-chain antibody fragment with recombinant Pichia pastoris

The yeast Pichia pastoris is a suitable production system for recombinant proteins due to its strong methanol-inducible AOX1 promoter. A key parameter of the production process is the specific methanol uptake rate. To control the methanol uptake and simultaneously maintain a constant methanol concentration during the production phase, two strategies were developed to generate purposeful oxygen limitation and to feed-forward control the specific methanol uptake rate into the optimum range. First, the cell density at induction was adjusted by prolonged preinduction glycerol feeding. Alternatively, the airflow rate was restricted and increased in parallel with the biomass. While the product accumulation started 20 h earlier with the first approach, the specific production rate of a single-chain antibody fragment was three times higher in the latter case. After 70 h of production, both schemes yielded product concentrations in the gram-per-liter range. Moreover, they release the requirement for dosage of pure oxygen and thereby can facilitate the scale-up of the production process. The different production profiles indicate that the impact of specific methanol uptake rate on protein production by recombinant P. pastoris depends on the control mode.     

Dextranase production by recombinant Pichia pastoris under operational volumetric mass transfer coefficient (kLa) and volumetric gassed power input (Pg/V) attainable at commercial large scale

Abstract

Most of the reported bioprocesses carried out by the methylotrophic yeast Pichia pastoris have been performed at laboratory scale using high power inputs and pure oxygen, such conditions are not feasible for industrial large-scale processes. In this study, volumetric mass transfer (k_La) and volumetric gassed power input (Pg/V) were evaluated within values attainable in large-scale production as scale-up criteria for recombinant dextranase production by Mut^S P. pastoris strain. Cultures were oxygen limited when the volumetric gassed power supply was limited to 2?kW m^?3. Specific growth rate, and then dextranase production, increased as k_La and Pg/V did. Meanwhile, specific production and methanol consumption rates were constant, due to the limited methanol condition also achieved at 2?L bioprocesses. The specific dextranase production rate was two times higher than the values previously reported for a Mut⁺ strain. After a scale-up process, at constant k_La, the specific growth rate was kept at 30?L bioprocess, whereas dextranase production decreased, due to the effect of methanol accumulation. Results obtained at 30?L bioprocesses suggest that even under oxygen-limited conditions, methanol saturated conditions are not adequate to express dextranase with the promoter alcohol oxidase. Bioprocesses developed within feasible and scalable operational conditions are of high interest for the commercial production of recombinant proteins from Pichia pastoris.     

Increasing Secretion of a Bivalent Anti-T-Cell Immunotoxin by Pichia pastoris

The bivalent anti-T-cell immunotoxin A-dmDT390-bisFv(G₄S) was developed for treatment of T-cell leukemia and autoimmune diseases and for tolerance induction for transplantation. This immunotoxin was produced extracellularly in toxin-sensitive Pichia pastoris JW102 (Mut⁺) under control of the AOX1 promoter. There were two major barriers to efficient immunotoxin production, the toxicity of the immunotoxin for P. pastoris and the limited capacity of P. pastoris to secrete the immunotoxin. The immunotoxin toxicity resulted in a decrease in the methanol consumption rate, cessation of cell growth, and low immunotoxin productivity after the first 22 h of methanol induction. Continuous cell growth and continuous immunotoxin secretion after the first 22 h of methanol induction were obtained by adding glycerol to the methanol feed by using a 4:1 methanol-glycerol mixed feed as an energy source and by continuously adding a yeast extract solution during methanol induction. The secretory capacity was increased from 22.5 to 37 mg/liter by lowering the induction temperature. A low temperature reduced the methanol consumption rate and protease activity in the supernatant but not cell growth. The effects of adding glycerol and yeast extract to the methanol feed were synergistic. Adding yeast extract primarily enhanced methanol utilization and cell growth, while adding glycerol primarily enhanced immunotoxin production. The synergy was further enhanced by decreasing the induction temperature from 23 to 15°C, which resulted in a robust process with a yield of 37 mg/liter, which was sevenfold greater than the yield previously reported for a toxin-resistant CHO cell expression system. This methodology should be applicable to other toxin-related recombinant proteins in toxin-sensitive P. pastoris.     

Recombinant Protein Production with Pichia pastoris in Continuous Fermentation – Kinetic Analysis of Growth and Product Formation

Continuous fermentation was applied to the production of recombinant human chymotrypsinogen B (hCTRB) by the methylotrophic yeast Pichia pastoris as a tool for the kinetic analysis of growth and product formation. Using methanol as the sole source of carbon, energy, and induction, cell growth could be described by a non‐competitive Monod approach. Maximum growth rate μ_max was determined to 0.084 h^‐‐1 and the K_M‐value for methanol to 0.22 g·L^‐‐1, respectively. With respect to product formation, a similar model was established exhibiting a methanol concentration of 0.13 g·L^‐‐1 as the K_M‐value and a maximum biomass‐specific product‐formation rate of π_max = 0.23 mg·g^‐‐1·h^‐‐1. The production of hCTRB was strictly growth‐coupled. The data provided covers the range of methanol concentrations between 0 and 4 g·L^‐‐1. Substrate concentrations exceeding this upper value led to a complete collapse of product formation. This change in phenotype turned out to be irreversible indicating a genetic instability of transformed Pichia pastoris caused by excess methanol.     

Mixed-feed exponential feeding for fed-batch culture of recombinant methylotrophic yeast

A simple, accurate model capable of predicting cell growth and methanol utilization during the mixed substrate fed-batch fermentation of Mut^S recombinant Pichia pastoris was developed and was used to design an exponential feeding strategy for mixed substrate fed-batch fermentation at a constant specific growth rate. Mixed substrate feeding has been shown to boost productivity in recombinant fed-batch culture of P. pastoris, while fixed growth rate exponential feeding during fed-batch culture is a useful tool in process optimization and control.     

Metabolic flux analysis for recombinant protein production by Pichia pastoris using dual carbon sources: Effects of methanol feeding rate

The intracellular metabolic fluxes through the central carbon pathways in the bioprocess for recombinant human erythropoietin (rHuEPO) production by Pichia pastoris (Mut⁺) were calculated to investigate the metabolic effects of dual carbon sources (methanol/sorbitol) and the methanol feed rate, and to obtain a deeper understanding of the regulatory circuitry of P. pastoris, using the established stoichiometry‐based model containing 102 metabolites and 141 reaction fluxes. Four fed‐batch operations with (MS‐) and without (M‐) sorbitol were performed at three different constant specific growth rates (h^?1), and denoted as M‐0.03, MS‐0.02, MS‐0.03, and MS‐0.04. Considering the methanol consumption pathway, the M‐0.03 and MS‐0.02 conditions produced similar effects and had >85% of formaldehyde flux towards the assimilatory pathway. In contrast, the use of the dual carbon source condition generated a shift in metabolism towards the dissimilatory pathway that corresponded to the shift in dilution rate from MS‐0.03 to MS‐0.04, indicating that the methanol feed exceeded the metabolic requirements at the higher µ₀. Comparing M‐0.03 and MS‐0.03 conditions, which had the same methanol feeding rates, sorbitol addition increased the rHuEPO synthetic flux 4.4‐fold. The glycolysis, gluconeogenesis, and PPP pathways worked uninterruptedly only at MS‐0.02 condition. PPP and TCA cycles worked with the highest disturbances at MS‐0.04 condition, which shows the stress of increased feeding rates of methanol on cell metabolism. Biotechnol. Bioeng. 2010; 105: 317–329. © 2009 Wiley Periodicals, Inc.     

Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway

Βackground  

The methylotrophic yeast Pichia pastoris has become an important host organism for recombinant protein production and is able to use methanol as a sole carbon source. The methanol utilization pathway describes all the catalytic reactions, which happen during methanol metabolism. Despite the importance of certain key enzymes in this pathway, so far very little is known about possible effects of overexpressing either of these key enzymes on the overall energetic behavior, the productivity and the substrate uptake rate in P. pastoris strains.     

Integrated single‐cell analysis shows Pichia pastoris secretes protein stochastically

The production of heterologous proteins by secretion from cellular hosts is an important determinant for the cost of biotherapeutics. A single‐cell analytical method called microengraving was used to examine the heterogeneity in secretion by the methylotrophic yeast Pichia pastoris. We show that constitutive secretion of a human Fc fragment by P. pastoris is not cell‐cycle dependent, but rather fluctuates between states of high and low productivity in a stochastic manner. Biotechnol. Bioeng. 2010;106: 319–325. © 2010 Wiley Periodicals, Inc.     

Operational strategies, monitoring and control of heterologous protein production in the methylotrophic yeast Pichia pastoris under different promoters: A review

The methylotrophic yeast Pichia pastoris has been widely reported as a suitable expression system for heterologous protein production. The use of different phenotypes under PAOX promoter, other alternative promoters, culture medium, and operational strategies with the objective to maximize either yield or productivity of the heterologous protein, but also to obtain a repetitive product batch to batch to get a robust process for the final industrial application have been reported. Medium composition, kinetics growth, fermentation operational strategies from fed-batch to continuous cultures using different phenotypes with the most common PAOX promoter and other novel promoters (GAP, FLD, ICL), the use of mixed substrates, on-line monitoring of the key fermentation parameters (methanol) and control algorithms applied to the bioprocess are reviewed and discussed in detail.     

Enhanced truncated-t-PA (CT-b) expression in high-cell-density fed-batch cultures of <Emphasis Type="Italic">Pichia pastoris</Emphasis> through optimization of a mixed feeding strategy by response surface methodology

Recently, Pichia pastoris has been the focal point of interest as an expression system for production of many recombinant proteins. The study and optimization of feeding strategy are of major importance to achieve maximum volumetric productivity in fed-batch cultivations. Among different feeding strategies used in P. pastoris fed-batch cultures, those trying to maintain a constant specific growth rate have usually resulted in superior productivities. The objective of the present study was to investigate and optimize the co-feeding of glycerol and methanol to attain maximum expression of t-PA in P. pastoris fed-batch cultures with constant specific growth rate. The experiments were designed by response surface methodology, considering the specific feeding rates of methanol and glycerol as independent variables. In each experiment, glycerol and methanol were fed according to a predetermined equation to maintain a constant specific growth rate. It was found that with glycerol feeding for higher specific growth rates, the inhibitory properties of glycerol are more pronounced, while the best expression level was achieved when the ratio of µ _set glycerol to that of methanol was around 1.67. In all specific growth rates tested, almost a similar ratio of the specific glycerol feeding rate to that of methanol led to the maximum protein production and activity. The statistical model predicted the optimal operating conditions for µ _set glycerol and that of methanol to be 0.05 and 0.03 h^?1, respectively. Applying the optimum strategy, maximum of 52 g/L biomass, 300 mg/L t-PA and 340,000 IU/mL enzyme activity were obtained.     

Optimization of a glycoengineered Pichia pastoris cultivation process for commercial antibody production

Glycoengineering enabled the production of proteins with human N-linked glycans by Pichia pastoris. This study used a glycoengineered P. pastoris strain which is capable of producing humanized glycoprotein with terminal galactose for monoclonal antibody production. A design of experiments approach was used to optimize the process parameters. Followed by further optimization of the specific methanol feed rate, induction duration, and the initial induction biomass, the resulting process yielded up to 1.6 g/L of monoclonal antibody. This process was also scaled-up to 1,200-L scale, and the process profiles, productivity, and product quality were comparable with 30-L scale. The successful scale-up demonstrated that this glycoengineered P. pastoris fermentation process is a robust and commercially viable process.     

Enhanced heterologous protein production in Pichia pastoris under increased air pressure

Pichia pastoris is a widely used host for the production of heterologous proteins. In this case, high cell densities are needed and oxygen is a major limiting factor. The increased air pressure could be used to improve the oxygen solubility in the medium and to reach the high oxygen demand of methanol metabolism. In this study, two P. pastoris strains producing two different recombinant proteins, one intracellular (β‐galactosidase) and other extracellular (frutalin), were used to investigate the effect of increased air pressure on yeast growth in glycerol and heterologous protein production, using the methanol AOX1‐inducible system. Experiments were carried out in a stainless steel bioreactor under total air pressure of 1 bar and 5 bar. The use of an air pressure raise of up to 5 bar proved to be applicable for P. pastoris cultivation. Moreover, no effects on the kinetic growth parameters and methanol utilization (Mut) phenotype of strains were found, while an increase in recombinant β‐galactosidase‐specific activity (ninefold) and recombinant frutalin production was observed. Furthermore, the air pressure raise led to a reduction in the secreted protease specific activity. This work shows for the first time that the application of an air pressure of 5 bar may be used as a strategy to decrease protease secretion and improve recombinant protein production in P. pastoris. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1040–1047, 2014