Development Of Fed-batch Process Research Articles

Microaerobic growth-decoupled production of α-ketoglutarate and succinate from xylose in a one-pot process using Corynebacteriumglutamicum.

Lignocellulosic biomass is the most abundant raw material on earth. Its efficient use for novel bio-based materials is essential for an emerging bioeconomy. Possible building blocks for such materials are the key TCA-cycle intermediates α-ketoglutarate and succinate. These organic acids have a wide range of potential applications, particularly in use as monomers for established or novel biopolymers. Recently, Corynebacteriumglutamicum was successfully engineered and evolved towards an improved utilization of d-xylose via the Weimberg pathway, yielding the strain WMB2evo . The Weimberg pathway enables a carbon-efficient C5-to-C5 conversion of d-xylose to α-ketoglutarate and a shortcut route to succinate as co-product in a one-pot process. C.glutamicumWMB2evo was grown under dynamic microaerobic conditions on d-xylose, leading to the formation of comparably high amounts of succinate and only small amounts of α-ketoglutarate. Subsequent carbon isotope labeling experiments verified the targeted production route for both products in C.glutamicumWMB2evo . Fed-batch process development was initiated and the effect of oxygen supply and feeding strategy for a growth-decoupled co-production of α-ketoglutarate and succinate were studied in detail. The finally established fed-batch production process resulted in the formation of 78.4mmolL-1 (11.45gL-1 ) α-ketoglutarate and 96.2mmolL-1 (11.36gL-1 ) succinate. The developed one-pot process represents a promising approach for the combined supply of bio-based α-ketoglutarate and succinate. Future work will focus on tailor-made down-stream processing of both organic acids from the fermentation broth to enable their application as building blocks in chemical syntheses. Alternatively, direct conversion of one or both acids via whole-cell or cell-free enzymatic approaches can be envisioned; thus, extending the network of value chains starting from cheap and renewable d-xylose.

Enhancing the functionality of a microscale bioreactor system as an industrial process development tool for mammalian perfusion culture.

Without a scale‐down model for perfusion, high resource demand makes cell line screening or process development challenging, therefore, potentially successful cell lines or perfusion processes are unrealized and their ability untapped. We present here the refunctioning of a high‐capacity microscale system that is typically used in fed‐batch process development to allow perfusion operation utilizing in situ gravity settling and automated sampling. In this low resource setting, which involved routine perturbations in mixing, pH and dissolved oxygen concentrations, the specific productivity and the maximum cell concentration were higher than 3.0 × 106 mg/cell/day and 7 × 10 7 cells/ml, respectively, across replicate microscale perfusion runs conducted at one vessel volume exchange per day. A comparative analysis was conducted at bench scale with vessels operated in perfusion mode utilizing a cell retention device. Neither specific productivity nor product quality indicated by product aggregation (6%) was significantly different across scales 19 days after inoculation, thus demonstrating this setup to be a suitable and reliable platform for evaluating the performance of cell lines and the effect of process parameters, relevant to perfusion mode of culturing.

Development of fed-batch process for high-yielding β-glucosidase displayed on cell surface of industrial yeast Saccharomyces cerevisiae

Here, we developed a fed-batch process for the cultivation of multistress tolerant Saccharomyces cerevisiae engineered to display β-glucosidase (BGL) on the cell surface. Although cell growth was promoted by increasing the amount of oxygen supplied, increasing the overall volumetric oxygen transfer coefficient rather decreased the specific BGL activity of yeast cells probably because of the high agitation speed used. To suppress the formation of ethanol, the method of feeding glucose was investigated. Namely, the timely addition of glucose and glucose feed rate were found crucial for increasing both cell concentration and specific BGL activity. When the lowest concentration of dissolved oxygen at 9h was used as an indicator, the lowest glucose feed rate of 1g/(L·h) provided the maximum BGL activity of 673.6U/g-dry cell, which is 1.6-fold higher than that in batch cultivation. By employing the fed-batch technique together with the improved gene cassette containing the SED1 promoter and anchoring domain, we found that the cell concentration and specific BGL activity continued to increase, reaching 15.4 g-dry cell/L and 1866.6U/g-dry cell after 48h, respectively. The results show the advantage of the fed-batch process for realizing the potential of cell-surface display systems in S. cerevisiae.

Study on Interactions between Fed-Batch and Batch Operating Parameters for the Development of Monoclonal Antibody Fed-Batch using Design of Experiments

In conventional fed batch process development approaches, batch operating parameters (such as pH, temperature, seeding density, dissolved oxygen concentration) are kept constant and only feeding parameters such as feeding time, post-feed concentration are manipulated. The batch and fed batch operating parameters are assumed to be independent of each other. This approach to process development ignores any interactions that might exist between the batch and fed-batch operating parameters are therefore not evaluated. However in a complex bioprocess, none of the factors affecting the process can be assumed to be independent of each other and mutually exclusive. Therefore in this study an attempt was made to study the interaction between fed-batch operating parameter-post feed glucose concentration (A) and batch operating parameters- seeding density (B), temperature (C), and dissolved oxygen concentration (D) by their simultaneous manipulation, as well as the effect of these interactions on cell growth and monoclonal antibody (mAb) production. NS0 cell line producing the mAb’s against carcino-embryogenic antigen (Anti-CEA) was used. The final mAb concentration, viable cell density and integral of viable cell concentration (IVCC) were the responses evaluated. Statistical analysis experimental data showed that parameter (A) and its interaction with parameter (B) were the main factors affecting both the response variables. In comparison to the batch run which yielded 5.21 mg/L mAb, the developed fed batch process increased the mAb titer by 10 fold (59.40 mg/mL), and the IVCC was increased by 7 fold. The maximum VCD value (3.46×106 cells/mL) of the developed fed batch process was 1.25 over fold the value for batch.

Analyzing the dynamics of cell growth and protein production in mammalian cell fed-batch systems using logistic equations

The logistic modeling approach was used to describe experimental viable cell density (X) and product concentration (P) data from two industrial fed-batch mammalian cell culture processes with maximum product concentrations in the 3.0-9.4 g/l range. In both cases, experimental data were well described by the logistic equations and the resulting specific growth rate and protein productivity profiles provided useful insights into the process kinetics. Subsequently, sensitivity equations for both the X and P models were analyzed which helped characterize the influence of model parameters on X and P time courses. This was augmented by conventional sensitivity analyses where five values of each model parameter, 25% apart, were used to generate X and P time courses. Finally, results from sensitivity analysis were used to simulate X and P time courses that were reflective of typical early- and late-stage fed-batch cell culture processes. Different combinations of the logistic model parameters were used to arrive at the same final product concentration demonstrating the ability of the logistic approach to describe the multitude of process paths that result in the same final product concentration. Overall, the capability of the logistic equations to well describe X and P data from fed-batch cultures, coupled with their ability to simulate the multitude of paths leading up to the desired cell density and product concentration profiles, make them a useful tool during mammalian cell fed-batch process development.

On-line monitoring of responses to nutrient feed additions by multi-frequency permittivity measurements in fed-batch cultivations of CHO cells

Changes in the nutrient availability of mammalian cell cultures are reflected in the beta-dispersion parameter characteristic frequency (f ( C )) and the on-line dual frequency permittivity signal. Multi-frequency permittivity measurements were therefore evaluated in fed-batch cultivations of two different CHO cell lines. Similar responses to nutrient depletions and discontinuous feed additions were monitored in different cultivation phases and experimental setups. Sudden increases in permittivity and f ( C ) occurred when feed additions were conducted. A constant or declining permittivity value in combination with a decrease in f ( C ) indicated nutrient limitations. f ( C ) correlated well with changes in oxygen uptake rate when cell diameter remained constant, indicating that metabolic activity is reflected in the value of f ( C ). When significant cell size changes occurred during the cultivations, the analysis of the beta-dispersion parameters was rendered complex. For the application of our findings in other systems it will be hence required to conduct additional off-line measurements. Based on these results, it is hypothesized that multi-frequency permittivity measurements can give information on the intracellular or physiological state in fed-batch mode. Similar observations were made when using different cell lines and feeding strategies, indicating that the findings are transferable to other cell lines and systems. The results should lead to an improved understanding of routine fed-batch processes. Additional studies are, however, required to explore how these observations can be used for fed-batch process development and optimization.

Industrial production of heterologous proteins by fed-batch cultures of the yeast Saccharomyces cerevisiae.

This review concerns the issues involved in the industrial development of fed-batch culture processes with Saccharomyces cerevisiae strains producing heterologous proteins. Most of process development considerations with fed-batch recombinant cultures are linked to the reliability and reproducibility of the process for manufacturing environments where quality assurance and quality control aspects are paramount. In this respect, the quality, safety and efficacy of complex biologically active molecules produced by recombinant techniques are strongly influenced by the genetic background of the host strain, genetic stability of the transformed strain and production process factors. An overview of the recent literature of these culture-related factors is coupled with our experience in yeast fed-batch process development for producing various therapeutic grade proteins. The discussion is based around three principal topics: genetics, microbial physiology and fed-batch process design. It includes the fundamental aspects of yeast strain physiology, the nature of the recombinant product, quality control aspects of the biological product, features of yeast expression vectors, expression and localization of recombinant products in transformed cells and fed-batch process considerations for the industrial production of Saccharomyces cerevisiae recombinant proteins. It is our purpose that this review will provide a comprehensive understanding of the fed-batch recombinant production processes and challenges commonly encountered during process development.

Industrial production of heterologous proteins by fed-batch cultures of the yeast Saccharomyces cereuisiae

This review concerns the issues involved in the industrial development of fed-batch culture processes with Saccharomyces cereriviae strains producing heterologous proteins. Most of process development considerations with fed-batch recombinant cultures are linked to the reliability and reproducibility of the process for manufacturing environments where quality assurance and quality control aspects are paramount. In this respect, the quality, safety and efficacy of complex biologically active molecules produced by recombinant techniques are strongly influenced by the genetic background of the host strain, genetic stability of the transformed strain and production process factors. An overview of the recent literature of these culture-related factors is coupled with our experience in yeast fed-batch process development for producing various therapeutic grade proteins. The discussion is based around three principal topics: genetics, microbial physiology and fed-batch process design. It includes the fundamental aspects of yeast strain physiology, the nature of the recombinant product, quality control aspects of the biological product, features of yeast expression vectors, expression and localization of recombinant products in transformed cells and fed-batch process considerations for the industrial production of Saccharomyces cerevisiae recombinant proteins. It is our purpose that this review will provide a comprehensive understanding of the fed-batch recombinant production processes and challenges commonly encountered during process development.