RESUMO
In recent years, many biological-based products have been developed, representing a significant fraction of income in the pharmaceutical market. Ion exchange chromatography is an important downstream step for the purification of target recombinant proteins present in clarified cell extracts, together with many other unknown impurities. This work develops a robust approach to model and simulate the purification of untagged heterologous proteins, so that the improved conditions to carry out an ion exchange chromatography are identified in a rational basis prior to the real purification run itself. Purification of the pneumococcal surface protein A (PspA4Pro) was used as a case study. This protein is produced by recombinant Escherichia coli and is a candidate for the manufacture of improved pneumococcal vaccines. The developed method combined experimental and computational procedures. Different anion exchange operating conditions were mapped in order to gather a broad range of representative experimental data. The equilibrium dispersive and the steric mass action equations were used to model and simulate the process. A training strategy to fit the model and separately describe the elution profiles of PspA4Pro and other proteins of the cell extract was applied. Based on the simulation results, a reduced ionic strength was applied for PspA4Pro elution, leading to increases of 14.9% and 11.5% for PspA4Pro recovery and purity, respectively, compared to the original elution profile. These results showed the potential of this method, which could be further applied to improve the performance of ion exchange chromatography in the purification of other target proteins under real process conditions.
Assuntos
Produtos Biológicos , Misturas Complexas , Cromatografia por Troca Iônica/métodos , Proteínas Recombinantes/química , Misturas Complexas/metabolismo , Produtos Biológicos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismoRESUMO
In the last years, Salmonella has been extensively studied not only due to its importance as a pathogen, but also as a host to produce pharmaceutical compounds. However, the full exploitation of Salmonella as a platform for bioproduct delivery has been hampered by the lack of information about its metabolism. Genome-scale metabolic models can be valuable tools to delineate metabolic engineering strategies as long as they closely represent the actual metabolism of the target organism. In the present study, a 13C-MFA approach was applied to map the fluxes at the central carbon pathways of S. typhimurium LT2 growing at glucose-limited chemostat cultures. The experiments were carried out in a 2L bioreactor, using defined medium enriched with 20% 13C-labeled glucose. Metabolic flux distributions in central carbon pathways of S. typhimurium LT2 were estimated using OpenFLUX2 based on the labeling pattern of biomass protein hydrolysates together with biomass composition. The results suggested that pentose phosphate is used to catabolize glucose, with minor fluxes through glycolysis. In silico simulations, using Optflux and pFBA as simulation method, allowed to study the performance of the genome-scale metabolic model. In general, the accuracy of in silico simulations was improved by the superimposition of estimated intracellular fluxes to the existing genome-scale metabolic model, showing a better fitting to the experimental extracellular fluxes, whereas the intracellular fluxes of pentose phosphate and anaplerotic reactions were poorly described.
Assuntos
Mapeamento Cromossômico/métodos , Análise do Fluxo Metabólico/métodos , Redes e Vias Metabólicas/genética , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Biomassa , Reatores Biológicos , Isótopos de Carbono , Simulação por Computador , Cromatografia Gasosa-Espectrometria de Massas , Glucose/metabolismo , Glicólise , Engenharia Metabólica/métodosRESUMO
The integration of state estimation and control is a promising approach to overcome challenges related to unavailable or noisy online measurements and plant-model mismatch. Extended Kalman filter (EKF) and moving horizon estimator (MHE) are widely used methods that have complementary features. EKF provides fast estimation and MHE optimal performance. In this paper, a novel hierarchical EKF/MHE approach combined with a dynamic matrix controller (DMC), denoted as EKF/MHE-DMC, is proposed for process monitoring and dissolved oxygen control in airlift bioreactors. The approach is successfully tested in simulated cultivations of Escherichia coli for recombinant protein production, considering specific scenarios of step set point tracking, step disturbance rejection, plant-model mismatch, and measurement noise. Results also show that, given a model that describes the measured dissolved oxygen precisely, as assumed in this study for the in silico experiments, the EKF/MHE-DMC approach is able to estimate the cell, protein, substrate, and dissolved oxygen concentrations based only on the measurement of the latter, reducing the estimation error by 93.8% when compared to a benchmark case employing EKF and DMC. The general structure of the proposed EKF/MHE-DMC framework could be adapted for implementation on other relevant bioprocess systems employing their derived process models.
Assuntos
Modelos Químicos , Oxigênio/química , Escherichia coli/crescimento & desenvolvimento , Oxigênio/metabolismo , Proteínas Recombinantes/biossínteseRESUMO
One of the main challenges of second generation (2G) ethanol production is the high quantities of phenolic compounds and furan derivatives generated in the pretreatment of the lignocellulosic biomass, which inhibit the enzymatic hydrolysis and fermentation steps. Fast monitoring of these inhibitory compounds could provide better control of the pretreatment, hydrolysis, and fermentation processes by enabling the implementation of strategic process control actions. We investigated the feasibility of monitoring these inhibitory compounds by ultraviolet-visible (UV-Vis) spectroscopy associated with partial least squares (PLS) regression. Hydroxymethylfurfural, furfural, vanillin, and ferulic and p-coumaric acids generated during different severities of liquid hot water pretreatment of sugarcane bagasse were quantified with highly accuracy. In cross-validation (leave-one-out), the PLS-UV-Vis method presented root mean square error of prediction (RMSECV) of around only 5.0%. The results demonstrated that the monitoring performance achieved with PLS-UV-Vis could support future studies of optimization and control protocols for application in industrial processes.
Assuntos
Etanol , Fermentação , Biomassa , Hidrólise , Análise dos Mínimos Quadrados , SaccharumRESUMO
In recent years, many biological-based products have been developed, representing a significant fraction of income in the pharmaceutical market. Ion exchange chromatography is an important downstream step for the purification of target recombinant proteins present in clarified cell extracts, together with many other unknown impurities. This work develops a robust approach to model and simulate the purification of untagged heterologous proteins, so that the improved conditions to carry out an ion exchange chromatography are identified in a rational basis prior to the real purification run itself. Purification of the pneumococcal surface protein A (PspA4Pro) was used as a case study. This protein is produced by recombinant Escherichia coli and is a candidate for the manufacture of improved pneumococcal vaccines. The developed method combined experimental and computational procedures. Different anion exchange operating conditions were mapped in order to gather a broad range of representative experimental data. The equilibrium dispersive and the steric mass action equations were used to model and simulate the process. A training strategy to fit the model and separately describe the elution profiles of PspA4Pro and other proteins of the cell extract was applied. Based on the simulation results, a reduced ionic strength was applied for PspA4Pro elution, leading to increases of 14.9% and 11.5% for PspA4Pro recovery and purity, respectively, compared to the original elution profile. These results showed the potential of this method, which could be further applied to improve the performance of ion exchange chromatography in the purification of other target proteins under real process conditions.
RESUMO
Competition with well-established, fine-tuned chemical processes is a major challenge for the industrial implementation of the enzymatic synthesis of beta-lactam antibiotics. Enzyme-based routes are acknowledged as an environmental-friendly approach, avoiding organochloride solvents and working at room temperatures. Among different alternatives, the kinetically controlled synthesis, using immobilized penicillin G acylase (PGA) in aqueous environment, with the simultaneous crystallization of the product, is the most promising one. However, PGA may act either as a transferase or as a hydrolase, catalyzing two undesired side reactions: the hydrolysis of the acyl side-chain precursor (an ester or amide, a parallel reaction) and the hydrolysis of the antibiotic itself (a consecutive reaction). This review focuses specially on aspects of the reactions' kinetics that may affect the performance of the enzymatic reactor.
Assuntos
Antibacterianos/síntese química , Reatores Biológicos , Penicilina Amidase/química , beta-Lactamas/síntese química , Antibacterianos/química , Catálise , Enzimas Imobilizadas/química , Escherichia coli/enzimologia , Cinética , Modelos Químicos , beta-Lactamas/químicaRESUMO
Rapid, efficient, and low-cost technologies for monitoring the fermentation process during second generation (2G) or cellulosic ethanol production are essential for the successful implementation of this process at the commercial scale. Here, the use of near-infrared (NIR) spectroscopy associated with partial least squares (PLS) regression was investigated as a tool for monitoring the production of 2G ethanol from lignocellulosic sugarcane residues including bagasse, straw, and tops. The spectral data was based on a set of 103 alcoholic fermentation samples. Models based on different pre-processing techniques were evaluated. The best root mean square error of prediction (RMSEP) values obtained in the external validation were around 3.02 g/L for ethanol and 6.60 g/L for glucose. The findings showed that the PLS-NIR methodology was efficient in accurately predicting the glucose and ethanol concentrations during the production of 2G ethanol, demonstrating potential for use in monitoring and control of large-scale industrial processes.