Design of methanol Feed control in Pichia pastoris fermentations based upon a growth model

The methylotrophic yeast Pichia pastoris is an effective system for recombinant protein productions that utilizes methanol as an inducer, and also as carbon and energy source for a Mut(+) (methanol utilization plus) strain. Pichia fermentation is conducted in a fed-batch mode to obtain a high cell density for a high productivity. An accurate methanol control is required in the methanol fed-batch phase (induction phase) in the fermentation. A simple "on-off" control strategy is inadequate for precise control of methanol concentrations in the fermentor. In this paper we employed a PID (proportional, integral and derivative) control system for the methanol concentration control and designed the PID controller settings on the basis of a Pichia growth model. The closed-loop system was built with four components: PID controller, methanol feed pump, fermentation process, and methanol sensor. First, modeling and transfer functions for all components were derived, followed by frequency response analysis, a powerful method for calculating the optimal PID parameters K(c) (controller gain), tau(I) (controller integral time constant), and tau(D) (controller derivative time constant). Bode stability criteria were used to develop the stability diagram for evaluating the designed settings during the entire methanol fed-batch phase. Fermentations were conducted using four Pichia strains, each expressing a different protein, to verify the control performance with optimal PID settings. The results showed that the methanol concentration matched the set point very well with only small overshoot when the set point was switched, which indicated that a very good control performance was achieved. The method developed in this paper is robust and can serve as a framework for the design of other PID feedback control systems in biological processes.     

Adaptive dissolved oxygen control through the glycerol feeding in a recombinant Pichia pastoris cultivation in conditions of oxygen transfer limitation

In high cell density cultivation processes the productivity is frequently constrained by the bioreactor maximum oxygen transfer capacity. The productivity can often be increased by operating the process at low dissolved oxygen concentrations close to the limitation level. This may be accomplished with a closed-loop controller that regulates the dissolved oxygen concentration by manipulating the dominant carbon source feeding rate. In this work we study this control problem in a pilot 50l bioreactor with a high cell density recombinant P. pastoris cultivation in complex media. The study focuses on the design of accurate stable adaptive controllers, with guaranteed exponential convergence and its relation with the calibration of controller parameters. Two adaptive control strategies were tested in the pilot bioreactor: a model reference adaptive controller with a linear reference model and an integral feedback controller with adaptive gain. The latter alternative proved to be more robust to errors in the measurements of the off-gas composition. Concerning the instrumentation, algorithms were derived assuming that both the dissolved oxygen tension and off-gas composition are measured on-line, but also the case of only dissolved oxygen being measured is addressed. It was verified that the measurement of off-gas composition might not improve the controller performance due to measurement and process time delays.     

Design of a novel automated methanol feed system for pilot-scale fermentation of Pichia pastoris

Large-scale fermentation of Pichia pastoris requires a large volume of methanol feed during the induction phase. However, a large volume of methanol feed is difficult to use in the processing suite because of the inconvenience of constant monitoring, manual manipulation steps, and fire and explosion hazards. To optimize and improve safety of the methanol feed process, a novel automated methanol feed system has been designed and implemented for industrial fermentation of P. pastoris. Details of the design of the methanol feed system are described. The main goals of the design were to automate the methanol feed process and to minimize the hazardous risks associated with storing and handling large quantities of methanol in the processing area. The methanol feed system is composed of two main components: a bulk feed (BF) system and up to three portable process feed (PF) systems. The BF system automatically delivers methanol from a central location to the portable PF system. The PF system provides precise flow control of linear, step, or exponential feed of methanol to the fermenter. Pilot-scale fermentations with linear and exponential methanol feeds were conducted using two Mut(+) (methanol utilization plus) strains, one expressing a recombinant therapeutic protein and the other a monoclonal antibody. Results show that the methanol feed system is accurate, safe, and efficient. The feed rates for both linear and exponential feed methods were within ± 5% of the set points, and the total amount of methanol fed was within 1% of the targeted volume.     

Fermentation strategies for recombinant protein expression in the methylotrophic yeastPichia pastoris

Fermentation strategies for recombinant protein production inPichia pastoris have been investigated and are reviewed here. Characteristics of the expression system, such as phenotypes and carbon utilization, are summarized. Recently reported results such as growth model establishment, application of a methanol sensor, optimization of substrate feeding strategy, DOstat controller design, mixed feed technology, and perfusion and continuous culture are discussed in detail.     

表达重组咖啡豆α-半乳糖苷酶的酵母工程菌的高密度发酵

用5 L发酵罐优化了重组咖啡豆α-半乳糖苷酶酵母工程菌pPIC9K-Gal/GS115(本室构建)的高密度发酵工艺.通过对发酵条件的优化,包括甘油补充量及补充时机、甲醇诱导量及诱导时机、溶氧控制、诱导时间等,重组咖啡豆α-半乳糖苷酶在毕赤酵母中得到了高效表达.利用所确定的最适条件进行发酵,菌体密度最终达到368 g/L以上,每批发酵液离心后可获得3.5 L的发酵上清,上清中的蛋白含量达到3 g/L以上,目的蛋白占上清总蛋白的50%以上,含量约为1.5 g/L,上清中α-半乳糖苷酶的活性维持在80 U/ml左右.确立工艺后又进行了3次发酵试验,证明了工艺的可行性和稳定性.为重组咖啡豆α-半乳糖苷酶在B→O血型改造和酶解大豆低聚糖方面的应用奠定了基础.     

Pathway analysis of Pichia pastoris to elucidate methanol metabolism and its regulation for production of recombinant proteins

This research rationally analyzes metabolic pathways of Pichia pastoris to study the metabolic flux responses of this yeast under methanol metabolism. A metabolic model of P. pastoris was constructed and analyzed by elementary mode analysis (EMA). EMA was used to comprehensively identify the cell's metabolic flux profiles and its underlying regulation mechanisms for the production of recombinant proteins from methanol. Change in phenotypes and flux profiles during methanol adaptation with varying feed mixture of glycerol and methanol was examined. EMA identified increasing and decreasing fluxes during the glycerol–methanol metabolic shift, which well agreed with experimental observations supporting the validity of the metabolic network model. Analysis of all the identified pathways also led to the determination of the metabolic capacities as well as the optimum metabolic pathways for recombinant protein synthesis during methanol induction. The network sensitivity analysis revealed that the production of proteins can be improved by manipulating the flux ratios at the pyruvate branch point. In addition, EMA suggested that protein synthesis is optimum under hypoxic culture conditions. The metabolic modeling and analysis presented in this study could potentially form a valuable knowledge base for future research on rational design and optimization of P. pastoris by determining target genes, pathways, and culture conditions for enhanced recombinant protein synthesis. The metabolic pathway analysis is also of considerable value for production of therapeutic proteins by P. pastoris in biopharmaceutical applications. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:28–37, 2014     

Analysis of single-chain antibody production in Pichia pastoris using on-line methanol control in fed-batch and mixed-feed fermentations.

In the last few years the Pichia pastoris expression system has been gaining more and more interest for the expression of recombinant proteins. Many groups have employed fermentation technology in their investigations because the system is fairly easy to scale up and suitable for the production in the milligram to gram range. A large number of heterologous proteins from different sources has been expressed, but the fermentation process technology has been investigated to a lesser extent. A large number of fermentations are carried out in standard bioreactors that may be insufficiently equipped to meet the demands of high-cell-density fermentations of methylotrophic yeasts. In particular, the lack of on-line methanol analysis leads to fermentation protocols that may impair the optimal expression of the desired products. We have used a commercially available methanol sensor to investigate in detail the effects of supplementary glycerol feeding while maintaining a constant methanol concentration during the induction of a Mut(+) strain of Pichia pastoris. Specific glycerol feed rates in the range of 38-4.2 mg. g(-1). h(-1) (mg glycerol per gram fresh weight per hour) were investigated. Expression of the recombinant scFv antibody fragment was only observed at specific feed rates below 6 mg. g(-1). h(-1). At low specific feed rates, growth was even lower than with methanol as the sole carbon source and the harvest expression level of the scFv was only half of that found in the control fermentation. These results show that glycerol inhibits expression driven by the AOX1 promoter even at extremely limited availability and demonstrate the benefits of on-line methanol control in Pichia fermentation research.     

Protease production by Bacillus subtilis in oxygen-controlled,glucose fed-batch fermentations

Summary An open-loop, on-off control system using the dissolved oxygen level to control a glucose feed was used in a study of growth and production of protease by Bacillus subtilis CNIB 8054. With this system, both glucose and oxygen were controlled at low concentrations. In batch fermentations, protease activity in the fermentation broth was maximum when growth had stopped. During oxygen-controlled, glucose fed-batch fermentations, growth and the production of protease activity continued during glucose feeding. Oxygen-controlled, glucose fed-batch fermentations produced more protease activity than batch fermentations, depending upon the set point for dissolved oxygen. These results indicate that control of glucose and oxygen concentrations can result in improvements in protease production.     

The design of controllers for batch bioreactors

The implementation of control algorithms to batch bioreactors is often complicated by variations in process dynamics that occur during the course of fermentation. Such a wide operating range often renders the performance of fixed gain proportional-integral-differential (PID) controllers unsatisfactory. In this work, detailed studies on the control of batch fermentations are per formed. Two simple controller designs are presented with the intent to compensate for changing process dynamics. One design incorporates the concepts of static feedforward-feedback control. While this technique produces tighter control than feedback alone, it is not as successful as a controller based on gain scheduling. The gain-scheduling controller, a subclass of adaptive controllers, uses the oxygen uptake rate as an auxiliary variable to fine-tune the PID controller parameters. The control of oxygen tension in the bioreactor is used as a vehicle to convey the proposed ideas, analyses, and results. Simulation experiments indicate significant improvement in controller performance can be achieved by both of the proposed approaches even in the presence of measurement noise.     

Twenty-four-well plate miniature bioreactor high-throughput system: assessment for microbial cultivations

High-throughput (HT) miniature bioreactor (MBR) systems are becoming increasingly important to rapidly perform clonal selection, strain improvement screening, and culture media and process optimization. This study documents the initial assessment of a 24-well plate MBR system, Micro (micro)-24, for Saccharomyces cerevisiae, Escherichia coli, and Pichia pastoris cultivations. MBR batch cultivations for S. cerevisiae demonstrated comparable growth to a 20-L stirred tank bioreactor fermentation by off-line metabolite and biomass analyses. High inter-well reproducibility was observed for process parameters such as on-line temperature, pH and dissolved oxygen. E. coli and P. pastoris strains were also tested in this MBR system under conditions of rapidly increasing oxygen uptake rates (OUR) and at high cell densities, thus requiring the utilization of gas blending for dissolved oxygen and pH control. The E. coli batch fermentations challenged the dissolved oxygen and pH control loop as demonstrated by process excursions below the control set-point during the exponential growth phase on dextrose. For P. pastoris fermentations, the micro-24 was capable of controlling dissolved oxygen, pH, and temperature under batch and fed-batch conditions with subsequent substrate shot feeds and supported biomass levels of 278 g/L wet cell weight (wcw). The average oxygen mass transfer coefficient per non-sparged well were measured at 32.6 +/- 2.4, 46.5 +/- 4.6, 51.6 +/- 3.7, and 56.1 +/- 1.6 h(-1) at the operating conditions of 500, 600, 700, and 800 rpm shaking speed, respectively. The mixing times measured for the agitation settings 500 and 800 rpm were below 5 and 1 s, respectively.     

重组毕赤酵母表达工程植酸酶发酵过渡相参数相关分析

微生物发酵是一个涉及不同尺度的互相关联的复杂生物系统的过程 ,将重组毕赤酵母表达工程植酸酶过渡相的在线和离线参数进行了相关分析研究。通过对发酵过程的在线细胞代谢生理参数 (OUR)和环境参数 (DO)的变化进行相关分析表明 :甘油和葡萄糖碳源对AOX合成的阻遏强度不同 ,葡萄糖的阻遏性明显强于甘油 ,相对于醇氧化酶启动子 ,葡萄糖为强阻遏性底物。根据甲醇代谢途径关键酶酶活性变化 ,推测出各代谢途径流量分布的变化 ,即甲醇诱导后糖酵解途径和三羧酸循环途径代谢流比例下降 ,而磷酸戊糖途径中代谢流通量上升 ,甲醇完全氧化代谢流成为主要代谢流 ,与过渡相在线参数pH、OUR(CER)和RQ等相关分析的甲醇代谢途径的变化结果一致。此外 ,建立了生产过程在线控制与分析的标准 :当OUR和CER逐渐增大 ,则可判断甲醇已被利用和启动子已被甲醇成功诱导 ,即工程植酸酶开始启动表达.     

High cell density induces spontaneous bifurcations of dissolved oxygen controllers during CHO cell fermentations

High cell density cultures of CHO cells growing in a bioreactor under dissolved oxygen control were found to undergo spontaneous bifurcations and a subsequent loss of stability some time into the fermentation. This loss of stability was manifested by sustained and amplified oscillations in the bioreactor dissolved oxygen concentration and in the oxygen gas flow rate to the reactor. To identify potential biological and operational causes for the phenomenon, linear stability analysis was applied in a neighborhood of the experimentally observed bifurcation point. The analysis revealed that two steady state process gains, K(P1) and K(P2), regulated k(l)a and gas phase oxygen concentration inputs, respectively, and the magnitude of K(P1) was found to determine system stability about the bifurcation point. The magnitude of K(P1), and hence the corresponding open-loop steady state gain K(OL1), scaled linearly with the bioreactor cell density, increasing with increasing cell density. These results allowed the generation of a fermentation stability diagram, which partitioned K(C)-N operating space into stable and unstable regions separated by the loci of predicted critically stable controller constants, K(C,critical), as a function of bioreactor cell density. This consistency of this operating diagram with experimentally observed changes in system stability was demonstrated. We conclude that time-dependent increases in cell density are the cause of the observed instabilities and that cell density is the critical bifurcation parameter. The results of this study should be readily applicable to the design of a more robust controller.     

重组毕赤酵母产青霉素G酰化酶的分批发酵动力学研究

构建了重组毕赤酵母产青霉素G酰化酶的分批发酵动力学模型。实验考察了分批发酵过程中甘油消耗、甲醇浓度、菌体浓度、溶氧、补料时间对青霉素G酰化酶活力的影响。应用Matlab软件,对菌体生长、基质消耗和产物生成方程进行最优参数估算和非线性拟合,得到相应的动力学模型。模型的计算值与实验值能较好地拟合,表明所建模型能较好反映重组毕赤酵母产青霉素G酰化酶的分批发酵过程。     

电子嗅在线反馈控制毕赤酵母糖化酶发酵过程中甲醇浓度新方法的应用

探索了电子嗅传感仪直接通过发酵尾气进行发酵液中甲醇浓度在线检测的方法,建立了毕赤酵母表达糖化酶过程中甲醇浓度的自动化反馈补料控制模型,可准确实现发酵过程中甲醇浓度的精确控制;研究表明,当利用电子嗅将培养液中甲醇浓度稳定控制在(890±35)ppm水平下,发酵诱导培养到128h时目的蛋白糖化酶酶活达到了8 153U/ml,与甲醇浓度控制在(350±26)ppm时的发酵水平相比提升了48.8%。该方法具有无需前处理、与发酵液非接触、快速和准确性的优点,为提升工程酵母在工业发酵培养过程工艺的优化控制具有重要的指导作用。     

Production of recombinant human bile salt-stimulated lipase in Pichia pastoris.

Recombinant human bile salt-stimulated lipase (rhBSSL) was efficiently expressed under the control of the AOX1 gene promoter in Pichia pastoris. Human BSSL has 16 successively repeated sequences in the carboxy terminal region. The sequence consists of 11 amino acid residues. The coding sequence for the middle 11 of the 16 repeats was removed from hBSSL cDNA to facilitate efficient secretory expression. The clone used for fermentation was a transformant of GS115 (his4) integrated with four copies of the expression cassette containing the modified hBSSL cDNA. Unique fermentation conditions were required for efficient expressions of rhBSSL in the high cell-density fermentation. A sufficient glycerol feed at 30 degrees C and pH 4 under an adequate concentration of dissolved oxygen in the growth phase make the cells active over a long induction period of approximately 15 days. On methanol induction, the concentration of dissolved oxygen should be maintained very low in the presence of sorbitol and skimmed milk at 20 degrees C and pH 5.7. Under these conditions, 0.8-1 g of rhBSSL was secreted in 1 liter of the medium. By immunoelectron microscopy, rhBSSL-tagged gold particles were located in secretion microbodies after the beginning of methanol induction. The secreted rhBSSL was efficiently captured and purified by expanded bed adsorption chromatography.     

毕赤酵母高密度发酵工艺的研究

高密度发酵是毕赤酵母提高蛋白表达量的一种重要策略,发酵工艺是高密度发酵的一个重要因素。采用下列措施均可以有效地提高表达水平：调节基础培养基,采用变pH和变温发酵,提高DO,选择最适的诱导前菌体密度和比生长速率并降低甘油初始浓度和采用分段式指数流加进行调控。选择合适的甲醇补料策略：甲醇限制补料（MLFB）、氧气限制补料（OLFB）、甲醇不限制补料（MNLFB）和温度限制补料（TLFB）。采用两种方式调控补料：诱导阶段菌体生长时,甲醇比消耗速率(qMeOH)为0.02-0.03gg-1h-1,而菌体不生长时,qMeOH采用较高值。     

Recombinant protein expression in Pichia pastoris

The methylotrophic yeast Pichia pastoris is now one of the standard tools used in molecular biology for the generation of recombinant protein. P. pastoris has demonstrated its most powerful success as a large-scale (fermentation) recombinant protein production tool. What began more than 20 years ago as a program to convert abundant methanol to a protein source for animal feed has been developed into what is today two important biological tools: a model eukaryote used in cell biology research and a recombinant protein production system. To date well over 200 heterologous proteins have been expressed in P. pastoris. Significant advances in the development of new strains and vectors, improved techniques, and the commercial availability of these tools coupled with a better understanding of the biology of Pichia species have led to this microbe's value and power in commercial and research labs alike.     

基于途径分析的L-异亮氨酸发酵溶氧控制研究

利用途径分析方法对黄色短杆菌（Brevibacterium flavum）TC-21 生产L-异亮氨酸的途径进行了分析,确定了黄色短杆菌TC-21生产L-异亮氨酸的最佳途径的通量分布,根据途径分析的结果,TCA循环的代谢流量对L-异亮氨酸产量有明显影响,而TCA循环与发酵过程中的溶氧密切相关,因此可以通过控制溶氧来提高L-异亮氨酸产量。在发酵过程的不同阶段,根据菌体生长和产酸的需求,改变TCA代谢流量,可以有效提高产酸率。实验证明,通过溶氧分阶段控制发酵生产L-异亮氨酸,比溶氧恒定控制方式发酵产率提高了15.77％。实验结果说明,用途径分析的结果指导发酵过程中的溶氧可以大幅度提高L-异亮氨酸的产量。     

Feed rate control in fed‐batch fermentations based on frequency content analysis

A new strategy for controlling substrate feed in the exponential growth phase of aerated fed‐batch fermentations is presented. The challenge in this phase is typically to maximize specific growth rate while avoiding the accumulation of overflow metabolites which can occur at high substrate feed rates. In the new strategy, regular perturbations to the feed rate are applied and the proximity to overflow metabolism is continuously assessed from the frequency spectrum of the dissolved oxygen signal. The power spectral density for the frequency of the external perturbations is used as a control variable in a controller to regulate the substrate feed. The strategy was implemented in an industrial pilot scale fermentation set up and calibrated and verified using an amylase producing Bacillus licheniformis strain. It was shown that a higher biomass yield could be obtained without excessive accumulation of harmful overflow metabolites. The general applicability of the strategy was further demonstrated by implementing the controller in another process using a Bacillus licheniformis strain currently used in industrial production processes. In addition, in this case a higher growth rate and decreased accumulation of overflow metabolites in the exponential growth phase was achieved in comparison to the reference controller. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:817–824, 2013     

Control of continuous fed-batch fermentation process using neural network based model predictive controller

Cell growth and metabolite production greatly depend on the feeding of the nutrients in fed-batch fermentations. A strategy for controlling the glucose feed rate in fed-batch baker’s yeast fermentation and a novel controller was studied. The difference between the specific carbon dioxide evolution rate and oxygen uptake rate (Q _c − Q _o) was used as controller variable. The controller evaluated was neural network based model predictive controller and optimizer. The performance of the controller was evaluated by the set point tracking. Results showed good performance of the controller.