Intensification of high cell-density cultivations of rE. coli for production of S. pneumoniae antigenic surface protein, PspA3, using model-based adaptive control

Horta, Antonio Carlos Luperni; Sargo, Cíntia Regina; Silva, A. J. da; Gonzaga, Marina de Carvalho; Santos, M. P.; Gonçalves, Viviane Maimoni; Zangirolami, Teresa Cristina; Giordano, Roberto de Campos

doi:10.1007/s00449-012-0714-4

Cited by 26 publications

(33 citation statements)

References 28 publications

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“…The protein was produced and purified using previously published methods with slight modifications Horta et al, 2012;Horta et al, 2014). The production of recombinant PspA4Pro was performed in 5 L bioreactors using; fed-batch cultivation with defined medium containing glycerol as carbon source and lactose as inducer (Horta et al, 2012) or batch cultivation with complex medium containing glucose, glycerol and lactose for auto-induction (Horta et al, 2014). The purification method consisted of cell disruption in a continuous high pressure homogenizer, precipitation of the homogenate with cetyltrimethylammonium bromide, pellet removal by centrifugation, anion exchange chromatography in Q-Sepharose, cryoprecipitation and cation exchange chromatography in SP-Sepharose .…”

Section: Production and Purification Of Pspa4promentioning

confidence: 99%

Pulmonary dry powder vaccine of pneumococcal antigen loaded nanoparticles

Kunda

Alfagih

Miyaji

et al. 2015

International Journal of Pharmaceutics

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Pneumonia, caused by Streptococcus pneumoniae, mainly affects the immune compromised, the very young and the old, and remains one of the leading causes of death.A steady rise in disease numbers from non-vaccine serotypes necessitates a new vaccine formulation that ideally has better antigen stability and integrity, does not require cold-chain and can be delivered non-invasively. In this study, a dry powder vaccine containing an important antigen of S. pneumoniae, pneumococcal surface protein A (PspA) that has shown cross-reactivity amongst serotypes to be delivered via the pulmonary route has been formulated. The formulation contains the antigen PspA adsorbed onto the surface of polymeric nanoparticles encapsulated in L-leucine microparticles that can be loaded into capsules and delivered via an inhaler. We have successfully synthesized particles of ~150 nm and achieved ~ 20 µg of PspA adsorption per mg of NPs. In addition, the spray-dried powders displayed a FPF of 74.31±1.32% and MMAD of 1.70±0.03 μm suggesting a broncho-alveolar lung deposition facilitating the uptake of the nanoparticles by dendritic cells. Also, the PspA released from the dry powders maintained antigen stability (SDS-PAGE), integrity (Circular dichroism) and activity (lactoferrin binding assay). Moreover, the released antigen also maintained its antigenicity as determined by ELISA.

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Section: Production and Purification Of Pspa4promentioning

confidence: 99%

Pulmonary dry powder vaccine of pneumococcal antigen loaded nanoparticles

Kunda

Alfagih

Miyaji

et al. 2015

International Journal of Pharmaceutics

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“…Cx_perm was further used for inference of the growth rate (r x ) and specific growth rate (μ), as described in Horta et al (2012). Bioreactor volume (Vn, in L) was also continuously updated as a function of sample withdrawal (V sample ) and feeding medium supplied as described by Equation (5).…”

Section: Supervisory and Control Toolmentioning

confidence: 99%

A supervision and control tool based on artificial intelligence for high cell density cultivations

Horta

Silva

Sargo

et al. 2014

Braz. J. Chem. Eng.

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-High cell density cultivations of recombinant E. coli have been increasingly used for the production of heterologous proteins. However, it is a challenge to maintain these cultivations within the desired conditions, given that some variables such as dissolved oxygen concentration (DOC) and feed flow rate are difficult to control. This paper describes the software SUPERSYS_HCDC, a tool developed to supervise fed-batch cultures of rE. coli with biomass concentrations up to 150 g DCW /L and cell productivities up to 9 g DCW .L -1 .h -1 . The tool includes automatic control of the DOC by integrated action of the stirrer speed as well as of the air and oxygen flow rates; automatic start-up of the feed flow of fresh medium (system based on a neural network committee); and automatic slowdown of feeding when oxygen consumption exceeds the maximum capacity of the oxygen supply.

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“…Fed-batch cultivation is considered to be the most efficient process for the production of inducible heterologous proteins, including PGA, because the nutrient supply can be modulated to firstly achieve high biomass formation and then proceed with the induction [25]. However, performing a high cell density culture (HCDC) is a challenge, mainly because control of both the dissolved oxygen concentration and the flow rate of the feed medium is problematic at high biomass concentrations [24,26]. …”

Section: Introductionmentioning

confidence: 99%

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

et al. 2014

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BackgroundPenicillin 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.ResultsThis 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.ConclusionsTo 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.

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Intensification of high cell-density cultivations of rE. coli for production of S. pneumoniae antigenic surface protein, PspA3, using model-based adaptive control

Cited by 26 publications

References 28 publications

Pulmonary dry powder vaccine of pneumococcal antigen loaded nanoparticles

Pulmonary dry powder vaccine of pneumococcal antigen loaded nanoparticles

A supervision and control tool based on artificial intelligence for high cell density cultivations

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

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