产顺,顺-粘康酸细胞工厂的构建与优化

顺,顺-粘康酸是重要的平台化学品。目前,生物合成顺,顺-粘康酸还缺乏高性能菌株,已报道的主要工程菌株不仅需要诱导表达,遗传不稳定,而且发酵培养基组分复杂,不利于大规模工业化生产。构建能利用简单无机盐培养基、遗传稳定且不需要诱导表达的新型工程菌受到人们的关注。本研究在实验室前期构建的产三脱氢莽草酸工程菌株WJ060中,整合合成顺,顺-粘康酸的3个外源基因(aro Z、aro Y、cat A),并且利用3个不同强度的组成型启动子进行组合调控,成功构建了27株顺,顺-粘康酸工程菌,得到的最优工程菌MA30的产量达到1.7 g/L。为了进一步提高顺,顺-粘康酸工程菌的生产能力,利用基因组复制工程构建突变体库,结合高通量筛选方法,经过两轮筛选,成功筛选到了顺,顺-粘康酸产量提高超过8%的大肠杆菌MA30-G2。利用5 L发酵罐进行分批补料发酵,MA30-G2的顺,顺-粘康酸产量达到了11.5 g/L。本研究采用组合调控和高通量筛选相结合的策略不仅促进了顺,顺-粘康酸的生物合成,同时也为其他生物基化学品的生物制造提供了重要参考。     

Cell culture medium improvement by rigorous shuffling of components using media blending

A novel high-throughput methodology for the simultaneous optimization of many cell culture media components is presented. The method is based on the media blending approach which has several advantages as it works with ready-to-use media. In particular it allows precise pH and osmolarity adjustments and eliminates the need of concentrated stock solutions, a frequent source of serious solubility issues. In addition, media blending easily generates a large number of new compositions providing a remarkable screening tool. However, media blending designs usually do not provide information on distinct factors or components that are causing the desired improvements. This paper addresses this last point by considering the concentration of individual medium components to fix the experimental design and for the interpretation of the results. The extended blending strategy was used to reshuffle the 20 amino acids in one round of experiments. A small set of 10 media was specifically designed to generate a large number of mixtures. 192 mixtures were then prepared by media blending and tested on a recombinant CHO cell line expressing a monoclonal antibody. A wide range of performances (titers and viable cell density) was achieved from the different mixtures with top titers significantly above our previous results seen with this cell line. In addition, information about major effects of key amino acids on cell densities and titers could be extracted from the experimental results. This demonstrates that the extended blending approach is a powerful experimental tool which allows systematic and simultaneous reshuffling of multiple medium components.     

Rational development of a serum-free medium and fed-batch process for a GS-CHO cell line expressing recombinant antibody

A serum-free medium (CHO-SFM) together with a fed-batch process was developed for the cultivation of a recombinant GS-CHO cell line producing TNFR-Fc. According to the metabolic characteristics of GS-CHO cell, a basal medium was prepared by supplementing DMEM:F12:RPMI1640 (2:1:1) with amino acids, insulin, transferrin, Pluronic F68 and some other ingredients. Statistical optimization approaches based on Plackett–Burman and central composite designs were then adopted to identify additional positive determinants and determine their optimal concentrations, which resulted in the final CHO-SFM medium formulations. The maximum antibody titer reached was 90.95 mg/l in the developed CHO-SFM, which was a 18 % and 10 fold higher than that observed in the commercial EX-CELL™ 302 medium (76.95 mg/l) and basal medium (8.28 mg/l), respectively. Subsequently, a reliable, reproducible and robust fed-batch strategy was designed according to the offline measurement of glucose, giving a final antibody yield of 378 mg/l, which was a threefold improvement over that in conventional batch culture (122 mg/l) using CHO-SFM. In conclusion, the use of design of experiment (DoE) method facilitated the development of CHO-SFM medium and fed-batch process for the production of recombinant antibody using GS-CHO cells.     

Blend uniformity analysis using stream sampling and near infrared spectroscopy

A near infrared spectroscopic method was developed to determine drug content in a 20% (wt/wt) ibuprofen and spray-dried hydous lactose blend. A blending profile was obtained after blending for 0.5, 1, 3, 5, 10, and 20 minutes. Stream sampling was used to collect about 20 blend samples at each of the blending times from a laboratory scale V-blender. The samples collected were used to develop a near infrared calibration model. The calibration model was then used to determine the drug content of unknown samples from 2 validation blends. The validation blends were not included in the calibration model; they were used to evaluate the effectiveness of the calibration model. A total of 45 samples from the 2 validation blends were predicted by the near infrared calibration model and then analyzed by a validated UV spectrophotometric method. The root mean square error of prediction for the first validation blend was 5.69 mg/g and 3.30 mg/g for the samples from the second blend. A paired t test at the 95% confidence level did not indicate any differences between the drug content predicted by the near infrared spectroscopy (NIRS) method and the validated UV method for the 2 blends. The results show that the NIRS method could be developed while the blending profile is generated and used to thoroughly characterize a new formulation during development by analyzing a large number of samples. The new formulation could be transferred to a manufacturing plant with an NIRS method to facilitate blend uniformity analysis.     

Production of excreted human epidermal growth factor (hEGF) by an efficient recombinant Escherichia coli system

Recombinant Escherichia coli JM101 strains harbouring plasmids pWKW2 or lacUV5par8EGF, both encoding human epidermal growth factor (hEGF), were used in fermentations to optimize levels of excreted hEGF. Medium composition, inducer level, growth stage at induction and culture conditions, were optimized with respect to volumetric production of the recombinant protein. MMBL medium, with glucose at 5 g/l and tryptone as nitrogen source, was chosen. Isopropyl-β- -thiogalactopyranoside(IPTG) concentrations of 0.1 mM for E. coli JM101[pWKW2] and 0.2 mM for E. coli K-12 JM101[lacUV5par8EGF], were found to give the best hEGF production levels. The volumetric yields of hEGF were maximal when the cultures were induced in the mid-logarithmic phase. Growth temperature had a significant effect on hEGF yield. A simple continuous fed-batch process for cultivation of E. coli JM101[pWKW2] was developed. The maximum concentration of excreted hEGF attained in continuous fed-batch cultivation was 325 mg/l, as compared to 175 mg/l, in batch cultivation. The hEGF produced from the continuous fed-batch cultivation was substantiated by SDS-PAGE and immunoblotting.     

Modular development enables rapid design of media for alternative hosts

Developing media to sustain cell growth and production is an essential and ongoing activity in bioprocess development. Modifications to media can often address host or product-specific challenges, such as low productivity or poor product quality. For other applications, systematic design of new media can facilitate the adoption of new industrially relevant alternative hosts. Despite manifold existing methods, common approaches for optimization often remain time and labor-intensive. We present here a novel approach to conventional media blending that leverages stable, simple, concentrated stock solutions to enable rapid improvement of measurable phenotypes of interest. We applied this modular methodology to generate high-performing media for two phenotypes of interest: biomass accumulation and heterologous protein production, using high-throughput, milliliter-scale batch fermentations of Pichia pastoris as a model system. In addition to these examples, we also created a flexible open-source package for modular blending automation on a low-cost liquid handling system to facilitate wide use of this method. Our modular blending method enables rapid, flexible media development, requiring minimal labor investment and prior knowledge of the host organism, and should enable developing improved media for other hosts and phenotypes of interest.     

High-end pH-controlled delivery of glucose effectively suppresses lactate accumulation in CHO fed-batch cultures

A simple method for control of lactate accumulation in suspension cultures of Chinese hamster ovary (CHO) cells based on the culture's pH was developed. When glucose levels in culture reach a low level (generally below 1 mM) cells begin to take up lactic acid from the culture medium resulting in a rise in pH. A nutrient feeding method has been optimized which delivers a concentrated glucose solution triggered by rising pH. We have shown that this high-end pH-controlled delivery of glucose can dramatically reduce or eliminate the accumulation of lactate during the growth phase of a fed-batch CHO cell culture at both bench scale and large scale (2,500 L). This method has proven applicable to the majority of CHO cell lines producing monoclonal antibodies and other therapeutic proteins. Using this technology to enhance a 12-day fed-batch process that already incorporated very high initial cell densities and highly concentrated medium and feeds resulted in an approximate doubling of the final titers for eight cell lines. The increase in titer was due to additional cell growth and higher cell specific productivity.     

High cell density culture of the diatom Nitzschia laevis for eicosapentaenoic acid production: fed-batch development

A fed-batch process was developed for high cell density culture of the diatom Nitzschia laevis for enhanced production of eicosapentaenoic acid (EPA). Firstly, among the various medium components, glucose (Glu) was identified as the limiting substrate while nitrate (NO₃⁻), tryptone (Tr) and yeast extract (Ye) were found to promote cell growth by enhancing specific growth rate. Therefore, these components were considered essential and were included in the feed medium for subsequent fed-batch cultivation. With the optimized ratio of NO₃⁻:Tr:Ye being 1:2.6:1.3 (by weight), the relative proportions of glucose to the nitrogen sources in the feed were investigated. The optimal ratios of Glu:NO₃⁻ for specific growth rate and EPA productivity were both determined to be 32:1 (by weight). Finally, based on the residual glucose concentration in the culture, a continuous medium feeding strategy for fed-batch fermenter cultivation was developed, with which, the maximal cell dry weight and EPA yield obtained were 22.1 g l⁻¹ and 695 mg l⁻¹, respectively, which were great improvements over those of batch cultures.     

Application of factorial design to accelerate identification of CHO growth factor requirements

To accelerate recombinant CHO media and process development, we describe a simple approach to integrating multiple tasks associated with these processes including initial media design, serum-free adaptation, stability analysis and first generation scale-up. Factorial design techniques and normal probability chart representation of the results were first applied to identify potent parental CHO cell growth factors in a lean basal medium. These results were then applied to identify a suitable manufacturing medium from a panel of commercial and proprietary media formulations. When this approach was applied to recombinant CHO cell line, rapid adaptation of the cell line to an appropriate production medium occurred during culture expansion in the presence of the identified growth factor(s). This approach allows media component screening to be naturally integrated into the adaptation and scale-up processes since components that have little or no relative effect on cell proliferation are selected against as the "best" cultures are moved forward. The rapidity of the adaptation process allowed cell line stability studies to be initiated relatively early in the development process, thus providing preliminary stability information by the time the "outgrowing" culture could be scaled to 100-L reactors some 30 days after adaptation commenced. The application of full factorial design techniques allowed us to calculate the maximum number of interaction effects, the interpretation of which we believe can provide insights into growth factor biology.     

Fed-batch microbioreactor platform for scale down and analysis of a plasmid DNA production process

The rising costs of bioprocess research and development emphasize the need for high-throughput, low-cost alternatives to bench-scale bioreactors for process development. In particular, there is a need for platforms that can go beyond simple batch growth of the organism of interest to include more advanced monitoring, control, and operation schemes such as fed-batch or continuous. We have developed a 1-mL microbioreactor capable of monitoring and control of dissolved oxygen, pH, and temperature. Optical density can also be measured online for continuous monitoring of cell growth. To test our microbioreactor platform, we used production of a plasmid DNA vaccine vector (pVAX1-GFP) in Escherichia coli via a fed-batch temperature-inducible process as a model system. We demonstrated that our platform can accurately predict growth, glycerol and acetate concentrations, as well as plasmid copy number and quality obtained in a bench-scale bioreactor. The predictive abilities of the micro-scale system were robust over a range of feed rates as long as key process parameters, such as dissolved oxygen, were kept constant across scales. We have highlighted plasmid DNA production as a potential application for our microbioreactor, but the device has broad utility for microbial process development in other industries as well.     

In Vitro Growth of Curcuma longa L. in Response to Five Mineral Elements and Plant Density in Fed-Batch Culture Systems

Plant density was varied with P, Ca, Mg, and KNO₃ in a multifactor experiment to improve Curcuma longa L. micropropagation, biomass and microrhizome development in fed-batch liquid culture. The experiment had two paired D-optimal designs, testing sucrose fed-batch and nutrient sucrose fed-batch techniques. When sucrose became depleted, volume was restored to 5% m/v sucrose in 200 ml of modified liquid MS medium by adding sucrose solutions. Similarly, nutrient sucrose fed-batch was restored to set points with double concentration of treatments’ macronutrient and MS micronutrient solutions, along with sucrose solutions. Changes in the amounts of water and sucrose supplementations were driven by the interaction of P and KNO₃ concentrations. Increasing P from 1.25 to 6.25 mM increased both multiplication and biomass. The multiplication ratio was greatest in the nutrient sucrose fed-batch technique with the highest level of P, 6 buds/vessel, and the lowest level of Ca and KNO₃. The highest density (18 buds/vessel) produced the highest fresh biomass at the highest concentrations of KNO₃ and P with nutrient sucrose fed-batch, and moderate Ca and Mg concentrations. However, maximal rhizome dry biomass required highest P, sucrose fed-batch, and a moderate plant density. Different media formulations and fed-batch techniques were identified to maximize the propagation and storage organ responses. A single experimental design was used to optimize these dual purposes.     

Determination of metabolic flux changes during fed-batch cultivation from measurements of intracellular amino acids by LC-MS/MS

Metabolic flux analysis using (13)C-labeled substrates is a well-developed method for investigating cellular behavior in steady-state culture condition. To extend its application, in particular to typical industrial conditions, such as batch and fed-batch cultivations, a novel method of (13)C metabolic flux analysis is proposed. An isotopomer balancing model was developed to elucidate flux distributions in the central metabolism and all amino acids synthetic pathways. A lysine-producing strain of Escherichia coli was cultivated by fed-batch mode in a growth medium containing yeast extract. Mass distribution data was derived from both intracellular free amino acids and proteinogenic amino acids measured by LC-MS/MS, and a correction parameter for the protein turnover effect on the mass distributions of intracellular amino acids was introduced. Metabolic flux distributions were determined in both exponential and stationary phases. Using this new approach, a culture phase-dependent metabolic shift was detected in the fed-batch culture. The approach presented here has great potential for investigating cellular behavior in industrial processes, independent of cultivation modes, metabolic phase and growth medium.     

Biosynthesis of lovastatin and (+)-geodin by Aspergillus terreus in batch and fed-batch culture in the stirred tank bioreactor

The simultaneous formation of mevinolinic acid (lovastatin; antihypercholesterolemia drug) and (+)-geodin (by-product) by Aspergillus terreus ATCC 20542 in the batch and fed-batch cultivation in the stirred tank bioreactor was investigated and described in this paper. The main factors influencing the formation of these two secondary metabolites were the initial nitrogen concentration and the aeration rate of the medium. The experiments aimed at achieving as high as possible lovastatin titre accompanied by as low as possible (+)-geodin concentration. The application of lactose-fed discontinuous fed-batch process allowed increasing lovastatin formation, in comparison with the batch process. Nevertheless (+)-geodin titre increased too. But the control of pH at the levels of 7.6 and 7.8 was successfully applied to repress the formation of the by-product both in batch and fed-batch experiments. Additionally, apart from pH control, the supplementation of the medium with nicotinamide and calcium pantothenate was used to facilitate the formation of lovastatin. The simultaneous pH control and B-group vitamin supplementation allowed achieving the best results in the batch cultivation.     

Natural wool/cellulose acetate blends regenerated from the ionic liquid 1-butyl-3-methylimidazolium chloride

Dissolution and blending one of the most commonly used natural polymers, i.e., wool using a green solvent ionic liquid is described. The cleaned natural wool from merino sheep was directly dissolved and regenerated from 1-butyl-3-methylimidazolium chloride (BMIMCl) without any modifications. BMIMCl was subsequently used to develop wool/cellulose acetate (CA) blends. Blending modification of wool in this IL green solvent led to significant increase in glass transition temperature (T_g) and thermal stability compared to the pure components. It was found that there exist strong intermolecular hydrogen bonding interactions between regenerated wool and CA. Moreover homogeneous surface morphology was observed in the blends with higher CA concentrations. At the final stage of the blending process, the IL solvent was recycled completely. This work presents a green processing route for development of novel natural wool blended materials.     

Improving lactate metabolism in an intensified CHO culture process: productivity and product quality considerations

In this study, we discussed the development and optimization of an intensified CHO culture process, highlighting medium and control strategies to improve lactate metabolism. A few strategies, including supplementing glucose with other sugars (fructose, maltose, and galactose), controlling glucose level at <0.2 mM, and supplementing medium with copper sulfate, were found to be effective in reducing lactate accumulation. Among them, copper sulfate supplementation was found to be critical for process optimization when glucose was in excess. When copper sulfate was supplemented in the new process, two-fold increase in cell density (66.5 ± 8.4 × 10⁶ cells/mL) and titer (11.9 ± 0.6 g/L) was achieved. Productivity and product quality attributes differences between batch, fed-batch, and concentrated fed-batch cultures were discussed. The importance of process and cell metabolism understanding when adapting the existing process to a new operational mode was demonstrated in the study.     

Conversion of a CHO cell culture process from perfusion to fed-batch technology without altering product quality

During the development of a new drug product, it is a common strategy to develop a first-generation process with the aim to rapidly produce material for pre-clinical and early stage clinical trials. At a later stage of the development, a second-generation process is then introduced with the aim to supply late-stage clinical trials as well as market needs. This work was aimed at comparing the performance of two different CHO cell culture processes (perfusion and fed-batch) used for the production of a therapeutically active recombinant glycoprotein at industrial pilot-scale. The first-generation process was based on the Fibra-Cel packed-bed perfusion technology. It appeared during the development of the candidate drug that high therapeutic doses were required (>100mg per dose), and that future market demand would exceed 100 kg per year. This exceeded by far the production capacity of the first-generation process, and triggered a change of technology from a packed-bed perfusion process with limited scale-up capabilities to a fed-batch process with scale-up potential to typical bioreactor sizes of 15m(3) or more. The productivity per bioreactor unit volume (in product m(-3)year(-1)) of the fed-batch process was about 70% of the level reached with the first-generation perfusion process. However, since the packed-bed perfusion system was limited in scale (0.6m(3) maximum) compared to the volumes reached in suspension cultures (15m(3)), the fed-batch was selected as second-generation process. In fact, the overall process performance (in product year(-1)) was about 18-fold higher for the fed-batch compared to the perfusion mode. Data from perfusion and fed-batch harvests samples indicated that comparable product quality (relative abundance of monomers dimers and aggregates; N-glycan sialylation level; isoforms distribution) was obtained in both processes. To further confirm this observation, purification to homogeneity of the harvest material from both processes, followed by a complementary set of studies (e.g. full physico-chemical characterization, assessment of in vitro and in vivo bioactivity, comparative pharmacokinetics and pharmacodynamics studies in relevant species, etc.) would be required. Finally, this illustrates the need to fix the production process early during the development of a new drug product in order to minimize process conversion efforts and to shorten product development time lines.     

Electroantennographic bioassay as a screening tool for host plant volatiles

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host plant. When a host plant is an important agronomic commodity feeding damage by insect pests can inflict serious economic losses to growers. Accordingly, kairomones can be used as attractants to lure or confuse these insects and, thus, offer an environmentally friendly alternative to pesticides for insect control. Unfortunately, plants can emit a vast number volatiles with varying compositions and ratios of emissions dependent upon the phenology of the commodity or the time of day. This makes identification of biologically active components or blends of volatile components an arduous process. To help identify the bioactive components of host plant volatile emissions we employ the laboratory-based screening bioassay electroantennography (EAG). EAG is an effective tool to evaluate and record electrophysiologically the olfactory responses of an insect via their antennal receptors. The EAG screening process can help reduce the number of volatiles tested to identify promising bioactive components. However, EAG bioassays only provide information about activation of receptors. It does not provide information about the type of insect behavior the compound elicits; which could be as an attractant, repellent or other type of behavioral response. Volatiles eliciting a significant response by EAG, relative to an appropriate positive control, are typically taken on to further testing of behavioral responses of the insect pest. The experimental design presented will detail the methodology employed to screen almond-based host plant volatiles by measurement of the electrophysiological antennal responses of an adult insect pest navel orangeworm (Amyelois transitella) to single components and simple blends of components via EAG bioassay. The method utilizes two excised antennae placed across a "fork" electrode holder. The protocol demonstrated here presents a rapid, high-throughput standardized method for screening volatiles. Each volatile is at a set, constant amount as to standardize the stimulus level and thus allow antennal responses to be indicative of the relative chemoreceptivity. The negative control helps eliminate the electrophysiological response to both residual solvent and mechanical force of the puff. The positive control (in this instance acetophenone) is a single compound that has elicited a consistent response from male and female navel orangeworm (NOW) moth. An additional semiochemical standard that provides consistent response and is used for bioassay studies with the male NOW moth is (Z,Z)-11,13-hexdecadienal, an aldehyde component from the female-produced sex pheromone.     

A new approach to define optimized range of medium composition for enhancement of hairy root production in fed-batch process

This work proposes a new methodology to identify the best medium concentrations for fed-batch production of hairy root using Datura innoxia as a model. Firstly, the role of each component on the growth rate is investigated separately. Then, an experimental design allows refining the optimization studying the interactions between the major species. The result analysis let to define concentration range optimized for fed-batch process. The work novelties lie in two aspects. Firstly, concentrations have been kept constant during each run. Thus, biomass uptakes do not affect the optimization and the growth rate is maintained constant during the exponential phase. Secondly, the effects of salts are generally studied. In this work, the influences of each ion are investigated in order to avoid bias due to the counter-ion effects. Compared to the classical B₅ medium, the optimized medium shows a significant improvement leading to more than 80% increase of final biomass production.