A comprehensive method for modeling and simulating ion exchange chromatography of complex mixtures.

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.

ClearColi as a platform for untagged pneumococcal surface protein A production: cultivation strategy, bioreactor culture, and purification.

Several studies have searched for new antigens to produce pneumococcal vaccines that are more effective and could provide broader coverage, given the great number of serotypes causing pneumococcal diseases. One of the promising subunit vaccine candidates is untagged recombinant pneumococcal surface protein A (PspA4Pro), obtainable in high quantities using recombinant Escherichia coli as a microbial factory. However, lipopolysaccharides (LPS) present in E. coli cell extracts must be removed, in order to obtain the target protein at the required purity, which makes the downstream process more complex and expensive. Endotoxin-free E. coli strains, which synthesize a nontoxic mutant LPS, may offer a cost-effective alternative way to produce recombinant proteins for application as therapeutics. This paper presents an investigation of PspA4Pro production employing the endotoxin-free recombinant strain ClearColi® BL21(DE3) with different media (defined, auto-induction, and other complex media), temperatures (27, 32, and 37 °C), and inducers. In comparison to conventional E. coli cells in a defined medium, ClearColi presented similar PspA4Pro yields, with lower productivities. Complex medium formulations supplemented with salts favored PspA4Pro yields, titers, and ClearColi growth rates. Induction with isopropyl-ß-D-thiogalactopyranoside (0.5 mM) and lactose (2.5 g/L) together in a defined medium at 32 °C, which appeared to be a promising cultivation strategy, was reproduced in 5 L bioreactor culture, leading to a yield of 146.0 mg PspA4Pro/g dry cell weight. After purification, the cell extract generated from ClearColi led to 98% purity PspA4Pro, which maintained secondary structure and biological function. ClearColi is a potential host for industrial recombinant protein production. KEY POINTS: • ClearColi can produce as much PspA4Pro as conventional E. coli BL21(DE3) cells. • 10.5 g PspA4Pro produced in ClearColi bioreactor culture using a defined medium. • Functional PspA4Pro (98% of purity) was obtained in ClearColi bioreactor culture. Graphical abstract.

Machine learning applied for metabolic flux-based control of micro-aerated fermentations in bioreactors.

Various bio-based processes depend on controlled micro-aerobic conditions to achieve a satisfactory product yield. However, the limiting oxygen concentration varies according to the micro-organism employed, while for industrial applications, there is no cost-effective way of measuring it at low levels. This study proposes a machine learning procedure within a metabolic flux-based control strategy (SUPERSYS_MCU) to address this issue. The control strategy used simulations of a genome-scale metabolic model to generate a surrogate model in the form of an artificial neural network, to be used in a micro-aerobic fermentation strategy (MF-ANN). The meta-model provided setpoints to the controller, allowing adjustment of the inlet air flow to control the oxygen uptake rate. The strategy was evaluated in micro-aerobic batch cultures employing industrial Saccharomyces cerevisiae yeast, with defined medium and glucose as the carbon source, as a case study. The performance of the proposed control scheme was compared with a conventional fermentation and with three previously reported micro-aeration strategies, including respiratory quotient-based control and constant air flow rate. Due to maintenance of the oxidative balance at the anaerobiosis threshold, the MF-ANN provided volumetric ethanol productivity of 4.16 g·L-1 ·h-1 and a yield of 0.48 gethanol .gsubstrate-1 , which were higher than the values achieved for the other conditions studied (maximum of 3.4 g·L-1 ·h-1 and 0.35-0.40 gethanol ·gsubstrate-1 , respectively). Due to its modular character, the MF-ANN strategy could be adapted to other micro-aerated bioprocesses.

A hierarchical state estimation and control framework for monitoring and dissolved oxygen regulation in bioprocesses.

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.

Production and purification of an untagged recombinant pneumococcal surface protein A (PspA4Pro) with high-purity and low endotoxin content.

Streptococcus pneumoniae is the main cause of pneumonia, meningitis, and other conditions that kill thousands of children every year worldwide. The replacement of pneumococcal serotypes among the vaccinated population has evidenced the need for new vaccines with broader coverage and driven the research for protein-based vaccines. Pneumococcal surface protein A (PspA) protects S. pneumoniae from the bactericidal effect of human apolactoferrin and prevents complement deposition. Several studies indicate that PspA is a very promising target for novel vaccine formulations. Here we describe a production and purification process for an untagged recombinant fragment of PspA from clade 4 (PspA4Pro), which has been shown to be cross-reactive with several PspA variants. PspA4Pro was obtained using lactose as inducer in Phytone auto-induction batch or glycerol limited fed-batch in 5-L bioreactor. The purification process includes two novel steps: (i) clarification using a cationic detergent to precipitate contaminant proteins, nucleic acids, and other negatively charged molecules as the lipopolysaccharide, which is the major endotoxin; and (ii) cryoprecipitation that eliminates aggregates and contaminants, which precipitate at -20 °C and pH 4.0, leaving PspA4Pro in the supernatant. The final process consisted of cell rupture in a continuous high-pressure homogenizer, clarification, anion exchange chromatography, cryoprecipitation, and cation exchange chromatography. This process avoided costly tag removal steps and recovered 35.3 ± 2.5% of PspA4Pro with 97.8 ± 0.36% purity and reduced endotoxin concentration by >99.9%. Circular dichroism and lactoferrin binding assay showed that PspA4Pro secondary structure and biological activity were preserved after purification and remained stable in a wide range of temperatures and pH values.

Recombinant Escherichia coli cultivation in a pressurized airlift bioreactor: assessment of the influence of temperature on oxygen transfer and uptake rates.

Temperature influences the rates of oxygen transfer (OTR) and uptake (q O2) in aerobic bioprocesses. Hence, joint analysis of q O2 and OTR at variable temperature is essential for bioprocess optimization and control. However, no such analyses have yet been reported for cultures of engineered E. coli producing recombinant proteins. E. coli cultivations at different temperatures (27-37 °C) were performed using a 5-L stirred tank bioreactor (STB), and a 5-L airlift bioreactor (ALB) was used to measure k L a and validate models of q O2 and OTR. The equations were then employed to evaluate the cultivation process in the ALB at different pressures (0.1-0.4 MPa) and temperatures (27-37 °C). The results showed that the positive effect of temperature on k L a was more pronounced than the negative influence on oxygen solubility, increasing the OTR in the ALB. The specific growth rate and temperature influenced q O2. In contrast to previous reports, the results showed that q O2 was not explicitly affected by recombinant protein synthesis. In addition, model predictions revealed that biomass concentration and productivity were greatly improved by pressurization of the system and use of a lower temperature.

Oxygen transfer in a pressurized airlift bioreactor.

Airlift bioreactors (ALBs) offer advantages over conventional systems, such as simplicity of construction, reduced risk of contamination, and efficient gas-liquid dispersion with low power consumption. ALBs are usually operated under atmospheric pressure. However, in bioprocesses with high oxygen demand, such as high cell density cultures, oxygen limitation may occur even when operating with high superficial gas velocity and air enriched with oxygen. One way of overcoming this drawback is to pressurize the reactor. In this configuration, it is important to assess the influence of bioreactor internal pressure on the gas hold-up, volumetric oxygen transfer coefficient (k(L)a), and volumetric oxygen transfer rate (OTR). Experiments were carried out in a concentric-tube airlift bioreactor with a 5 dm(3) working volume, equipped with a system for automatic monitoring and control of the pressure, temperature, and inlet gas flow rate. The results showed that, in disagreement with previous published results for bubble column and external loop airlift reactors, overpressure did not significantly affect k(L)a within the studied ranges of pressure (0.1-0.4 MPa) and superficial gas velocity in the riser (0.032-0.065 m s(-1)). Nevertheless, a positive effect on OTR was observed: it increased up to 5.4 times, surpassing by 2.3 times the oxygen transfer in a 4 dm(3) stirred tank reactor operated under standard cultivation conditions. These results contribute to the development of non-conventional reactors, especially pneumatic bioreactors operated using novel strategies for oxygen control.

High-throughput strategies for penicillin G acylase production in rE. coli fed-batch cultivations.

BACKGROUND: Penicillin G acylase (PGA) is used industrially to catalyze the hydrolysis of penicillin G to obtain 6-aminopenicillanic acid. In Escherichia coli, the most-studied microorganism for PGA production, this enzyme accumulates in the periplasmic cell space, and temperature plays an important role in the correct synthesis of its subunits. RESULTS: This work investigates the influence of medium composition, cultivation strategy, and temperature on PGA production by recombinant E. coli cells. Shake flask cultures carried out using induction temperatures ranging from 18 to 28°C revealed that the specific enzyme activity achieved at 20°C (3000 IU gDCW-1) was 6-fold higher than the value obtained at 28°C. Auto-induction and high cell density fed-batch bioreactor cultures were performed using the selected induction temperature, with both defined and complex media, and IPTG and lactose as inducers. Final biomass concentrations of 100 and 120 gDCW L-1, and maximum enzyme productivities of 7800 and 5556 IU L-1 h-1, were achieved for high cell density cultures using complex and defined media, respectively. CONCLUSIONS: To the best of our knowledge, the volumetric enzyme activity and productivity values achieved using the complex medium are the highest ever reported for PGA production using E. coli. Overall PGA recovery yields of 64 and 72% after purification were achieved for crude extracts obtained from cells cultivated in defined and complex media, respectively. The complex medium was the most cost-effective for PGA production, and could be used in both high cell density and straightforward auto-induction protocols.

Modeling and simulation of anion exchange chromatography for purification of proteins in complex mixtures.

Ion exchange chromatography is extensively used in the purification of biological compounds. Reliable mathematical models describing this chromatographic technique are available and can be used to improve the performance of this separation step. However, the use of synthetic mixtures for model development hampers the application of this approach with real cell extracts processed in downstream operations. This work presents an original approach for handling non-synthetic genuine mixtures of proteins, which was applied in the purification of an untagged recombinant pneumococcal surface protein A (PspA4Pro). First, evaluation was made of the efficiency of steric mass action (SMA) and modified Langmuir isotherms, which were separately used together with the equilibrium dispersive model (EDM). The data used for parameter estimation and model validation were obtained from anion exchange chromatography runs (employing Q-Sepharose FF), applied to real cell extracts produced by different cultivation strategies. Simulations showed that the models were able to describe the complex mixtures of unknown proteins. Next, the EDM and SMA approaches were used to separately describe the profile of PspA4Pro and the pool of protein impurities eluted together. The simulations showed that PspA4Pro tended to elute at the beginning of the peak, enabling the establishment of an alternative elution schedule that provided a 34% increase in the purity achieved using the anion exchange chromatography.

Cost analysis based on bioreactor cultivation conditions: Production of a soluble recombinant protein using Escherichia coli BL21(DE3).

The impact of cultivation strategy on the cost of recombinant protein production is crucial for defining cost-effective bioreactor operation conditions. This paper presents a methodology to estimate and compare cost impacts related to utilities as well as medium composition, using simple design equations and accessible data. Data from batch bioreactor cultures were used as case study involving the production of pneumococcal surface protein A, a soluble recombinant protein, employing E. coli BL21(DE3). Cultivation strategies and corresponding process costs covered a wide range of operational conditions, including different media, inducers, and temperatures. The core expenses were related to the medium and cooling. When the price of peptone was above the threshold value of US$ 30/kg, defined medium became the best choice. IPTG and temperatures around 32 °C led to shorter cultures and lower PspA4Pro production costs. The procedure offers a simple, accessible theoretical tool to identify cost-effective production strategies using bioreactors.

ClearColi as a platform for untagged pneumococcal surface protein a production: cultivation strategy, bioreactor culture, and purification

Several studies have searched for new antigens to produce pneumococcal vaccines that are more effective and could provide broader coverage, given the great number of serotypes causing pneumococcal diseases. One of the promising subunit vaccine candidates is untagged recombinant pneumococcal surface protein A (PspA4Pro), obtainable in high quantities using recombinant Escherichia coli as a microbial factory. However, lipopolysaccharides (LPS) present in E. coli cell extracts must be removed, in order to obtain the target protein at the required purity, which makes the downstream process more complex and expensive. Endotoxin-free E. coli strains, which synthesize a nontoxic mutant LPS, may offer a cost-effective alternative way to produce recombinant proteins for application as therapeutics. This paper presents an investigation of PspA4Pro production employing the endotoxin-free recombinant strain ClearColi® BL21(DE3) with different media (defined, auto-induction, and other complex media), temperatures (27, 32, and 37 °C), and inducers. In comparison to conventional E. coli cells in a defined medium, ClearColi presented similar PspA4Pro yields, with lower productivities. Complex medium formulations supplemented with salts favored PspA4Pro yields, titers, and ClearColi growth rates. Induction with isopropyl-β-D-thiogalactopyranoside (0.5 mM) and lactose (2.5 g/L) together in a defined medium at 32 °C, which appeared to be a promising cultivation strategy, was reproduced in 5 L bioreactor culture, leading to a yield of 146.0 mg PspA4Pro/g dry cell weight. After purification, the cell extract generated from ClearColi led to 98% purity PspA4Pro, which maintained secondary structure and biological function. ClearColi is a potential host for industrial recombinant protein production.

A comprehensive method for modeling and simulating ion exchange chromatography of complex mixtures

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.

Modeling and simulation of anion exchange chromatography for purification of proteins in complex mixtures

Ion exchange chromatography is extensively used in the purification of biological compounds. Reliable mathematical models describing this chromatographic technique are available and can be used to improve the performance of this separation step. However, the use of synthetic mixtures for model development hampers the application of this approach with real cell extracts processed in downstream operations. This work presents an original approach for handling non-synthetic genuine mixtures of proteins, which was applied in the purification of an untagged recombinant pneumococcal surface protein A (PspA4Pro). First, evaluation was made of the efficiency of steric mass action (SMA) and modified Langmuir isotherms, which were separately used together with the equilibrium dispersive model (EDM). The data used for parameter estimation and model validation were obtained from anion exchange chromatography runs (employing Q-Sepharose FF), applied to real cell extracts produced by different cultivation strategies. Simulations showed that the models were able to describe the complex mixtures of unknown proteins. Next, the EDM and SMA approaches were used to separately describe the profile of PspA4Pro and the pool of protein impurities eluted together. The simulations showed that PspA4Pro tended to elute at the beginning of the peak, enabling the establishment of an alternative elution schedule that provided a 34% increase in the purity achieved using the anion exchange chromatography.

Cost analysis based on bioreactor cultivation conditions: production of a soluble recombinant protein using Escherichia coli BL21(DE3)

The impact of cultivation strategy on the cost of recombinant protein production is crucial for defining cost-effective bioreactor operation conditions. This paper presents a methodology to estimate and compare cost impacts related to utilities as well as medium composition, using simple design equations and accessible data. Data from batch bioreactor cultures were used as case study involving the production of pneumococcal surface protein A, a soluble recombinant protein, employing E. coli BL21(DE3). Cultivation strategies and corresponding process costs covered a wide range of operational conditions, including different media, inducers, and temperatures. The core expenses were related to the medium and cooling. When the price of peptone was above the threshold value of US$ 30/kg, defined medium became the best choice. IPTG and temperatures around 32°C led to shorter cultures and lower PspA4Pro production costs. The procedure offers a simple, accessible theoretical tool to identify cost-effective production strategies using bioreactors.

Salmonella typhimurium and Escherichia coli dissimilarity: Closely related bacteria with distinct metabolic profiles.

Live attenuated strains of Salmonella typhimurium have been extensively investigated as vaccines for a number of infectious diseases. However, there is still little information available concerning aspects of their metabolism. S. typhimurium and Escherichia coli show a high degree of similarity in terms of their genome contents and metabolic networks. However, this work presents experimental evidence showing that significant differences exist in their abilities to direct carbon fluxes to biomass and energy production. It is important to study the metabolism of Salmonella to elucidate the formation of acetate and other metabolites involved in optimizing the production of biomass, essential for the development of recombinant vaccines. The metabolism of Salmonella under aerobic conditions was assessed using continuous cultures performed at dilution rates ranging from 0.1 to 0.67 h(-1), with glucose as main substrate. Acetate assimilation and glucose metabolism under anaerobic conditions were also investigated using batch cultures. Chemostat cultivations showed deviation of carbon towards acetate formation, starting at dilution rates above 0.1 h(-1). This differed from previous findings for E. coli, where acetate accumulation was only detected at dilution rates exceeding 0.4 h(-1), and was due to the lower rate of acetate assimilation by S. typhimurium under aerobic conditions. Under anaerobic conditions, both microorganisms mainly produced ethanol, acetate, and formate. A genome-scale metabolic model, reconstructed for Salmonella based on an E. coli model, provided a poor description of the mixed fermentation pattern observed during Salmonella cultures, reinforcing the different patterns of carbon utilization exhibited by these closely related bacteria.

Modeling and simulation of anion exchange chromatography for purification of proteins in complex mixtures

Ion exchange chromatography is extensively used in the purification of biological compounds. Reliable mathematical models describing this chromatographic technique are available and can be used to improve the performance of this separation step. However, the use of synthetic mixtures for model development hampers the application of this approach with real cell extracts processed in downstream operations. This work presents an original approach for handling non-synthetic genuine mixtures of proteins, which was applied in the purification of an untagged recombinant pneumococcal surface protein A (PspA4Pro). First, evaluation was made of the efficiency of steric mass action (SMA) and modified Langmuir isotherms, which were separately used together with the equilibrium dispersive model (EDM). The data used for parameter estimation and model validation were obtained from anion exchange chromatography runs (employing Q-Sepharose FF), applied to real cell extracts produced by different cultivation strategies. Simulations showed that the models were able to describe the complex mixtures of unknown proteins. Next, the EDM and SMA approaches were used to separately describe the profile of PspA4Pro and the pool of protein impurities eluted together. The simulations showed that PspA4Pro tended to elute at the beginning of the peak, enabling the establishment of an alternative elution schedule that provided a 34% increase in the purity achieved using the anion exchange chromatography.

Cost analysis based on bioreactor cultivation conditions: production of a soluble recombinant protein using Escherichia coli BL21(DE3)

The impact of cultivation strategy on the cost of recombinant protein production is crucial for defining cost-effective bioreactor operation conditions. This paper presents a methodology to estimate and compare cost impacts related to utilities as well as medium composition, using simple design equations and accessible data. Data from batch bioreactor cultures were used as case study involving the production of pneumococcal surface protein A, a soluble recombinant protein, employing E. coli BL21(DE3). Cultivation strategies and corresponding process costs covered a wide range of operational conditions, including different media, inducers, and temperatures. The core expenses were related to the medium and cooling. When the price of peptone was above the threshold value of US$ 30/kg, defined medium became the best choice. IPTG and temperatures around 32°C led to shorter cultures and lower PspA4Pro production costs. The procedure offers a simple, accessible theoretical tool to identify cost-effective production strategies using bioreactors.

Production and purification of an untagged recombinant pneumococcal surface protein A (PspA4Pro) with high-purity and low endotoxin content

Streptococcus pneumoniae is the main cause of pneumonia, meningitis, and other conditions that kill thousands of children every year worldwide. The replacement of pneumococcal serotypes among the vaccinated population has evidenced the need for new vaccines with broader coverage and driven the research for protein-based vaccines. Pneumococcal surface protein A (PspA) protects S. pneumoniae from the bactericidal effect of human apolactoferrin and prevents complement deposition. Several studies indicate that PspA is a very promising target for novel vaccine formulations. Here we describe a production and purification process for an untagged recombinant fragment of PspA from clade 4 (PspA4Pro), which has been shown to be cross-reactive with several PspA variants. PspA4Pro was obtained using lactose as inducer in Phytone auto-induction batch or glycerol limited fed-batch in 5-L bioreactor. The purification process includes two novel steps: (i) clarification using a cationic detergent to precipitate contaminant proteins, nucleic acids, and other negatively charged molecules as the lipopolysaccharide, which is the major endotoxin; and (ii) cryoprecipitation that eliminates aggregates and contaminants, which precipitate at -20 A degrees C and pH 4.0, leaving PspA4Pro in the supernatant. The final process consisted of cell rupture in a continuous high-pressure homogenizer, clarification, anion exchange chromatography, cryoprecipitation, and cation exchange chromatography. This process avoided costly tag removal steps and recovered 35.3 +/- 2.5% of PspA4Pro with 97.8 +/- 0.36% purity and reduced endotoxin concentration by > 99.9%. Circular dichroism and lactoferrin binding assay showed that PspA4Pro secondary structure and biological activity were preserved after purification and remained stable in a wide range of temperatures and pH values.