Description of Evandromyia piperiformis sp. nov. (Diptera: Psychodidae).

Brazil presents great sand fly species diversity, with new species constantly being described, as new areas are surveyed to monitor sand flies. In captures undertaken in Porto Velho, Rondônia state-Brazil, a new species, Evandromyia (Aldamyia) piperiformissp. nov. (Godoy, Cunha & Galati 2017), was found and is here described. Both sexes of this new species may be distinguished from those of the Aldamyia subgenus through morphometric and morphological characters. Further, we present an identification key for the subgenus Aldamyia.

Fast development of Pichia pastoris GS115 Mut(+) cultures employing batch-to-batch control and hybrid semi-parametric modeling.

In this paper, we implemented a model-based optimization platform for fast development of Pichia pastoris cultures employing batch-to-batch control and hybrid semi-parametric modeling. We illustrate the methodology with a P. pastoris GS115 strain expressing a single-chain antibody fragment (scFv) by determining the optimal time profiles of temperature, pH, glycerol feeding and methanol feeding that maximize the endpoint scFv titer. The first hybrid model was identified from data of six exploratory experiments carried out in a pilot 50-L reactor. This model was subsequently used to maximize the final scFv titer of the proceeding batch employing a dynamic optimization program. Thereupon, the optimized time profiles of control variables were implemented in the pilot reactor and the resulting new data set was used to re-identify the hybrid model and to re-optimize the next batch. The iterative batch-to-batch optimization was stopped after 4 complete optimized batches with the final scFv titer stabilizing at 49.5 mg/L. In relation to the baseline batch (executed according to the Pichia fermentation guidelines by Invitrogen) a more than fourfold increase in scFv titer was achieved. The biomass concentration at induction and the methanol feeding rate profile were found to be the most critical control degrees of freedom to maximize scFv titer.

Application of adaptive DO-stat feeding control to Pichia pastoris X33 cultures expressing a single chain antibody fragment (scFv).

In this study, fed-batch cultures of a Pichia pastoris strain constitutively expressing a single chain antibody fragment (scFv) under the control of the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter were performed in a pilot 50 L bioreactor. Due to the very high cell density achieved within the first 75 h, typically between 140 and 160 g-DCW/L of dry cell weight (DCW), most of the scFv is produced under hard oxygen transfer limitation. To improve scFv productivity, a direct adaptive dissolved oxygen (DO)-stat feeding controller that maximizes glycerol feeding under the constraint of available oxygen transfer capacity was developed and applied to this process. The developed adaptive controller enabled to maximize glycerol feeding through the regulation of DO concentration between 3 and 5 % of saturation, thereby improving process productivity. Set-point convergence dynamics are characterized by a fast response upon large perturbations to DO, followed by a slower but very robust convergence in the vicinity of the boundary with almost imperceptible overshoot. Such control performance enabled operating closer to the 0 % boundary for longer periods of time when compared to a traditional proportional-integral-derivative algorithm, which tends to destabilize with increasing cell density.

Predicting sporulation events in a bioreactor using an electronic nose.

An electronic nose (EN) based on a non- specific multi-sensor array was used to accurately estimate sporulation events and the spore concentration of Bacillus subtilis cultures. The array included 6 metal oxide sensors (MOS), 10 metal oxide semiconductor field effect transistors (MOSFET), one CO(2) infrared sensor and one humidity sensor. The EN was used to monitor the gas emissions from B. subtilis bioreactions during both batch and fed-batch operation. The signal pattern produced by the sensors was evaluated by principal component analysis (PCA) and training cultivations were used to build a model. The arc length of the PCA trajectories was successfully correlated to the off-line spore count; a strong linear correlation (R(2) = 0.992) between the numerical integration of the curves and the measured spore concentration was established. The fast responses of the sensors in combination with the robust correlation with the off-line determination of spore concentration establish this EN device as a convenient tool for monitoring sporulation events in bioprocesses.

Modelling and optimization of a recombinant BHK-21 cultivation process using hybrid grey-box systems.

In this work a model-based optimization study of fed-batch BHK-21 cultures expressing the human fusion glycoprotein IgG1-IL2 was performed. It was concluded that due to the complexity of the BHK metabolism it is rather difficult to develop a kinetic model with sufficient accuracy for optimization studies. Many kinetic expressions and a large number of parameters are involved resulting in a complex identification problem. For this reason, an alternative more cost-effective methodology based on hybrid grey-box models was adopted. Several model structures combining the a priori reliable first principles knowledge with black-box models were investigated using data from batch and fed-batch experiments. It has been reported in previous studies that the BHK metabolism exhibits modulation particularities when compared to other mammalian cell lines. It was concluded that these mechanisms were effectively captured by the hybrid model, this being of crucial importance for the successful optimization of the process operation. A method was proposed to monitor the risk of hybrid model unreliability and to constraint the optimization results to acceptable risk levels. From the optimization study it was concluded that the process productivity may be considerably increased if the glutamine and glucose concentrations are maintained at low levels during the growth phase and then glutamine feeding is increased.

Adaptive dissolved oxygen control through the glycerol feeding in a recombinant Pichia pastoris cultivation in conditions of oxygen transfer limitation.

In high cell density cultivation processes the productivity is frequently constrained by the bioreactor maximum oxygen transfer capacity. The productivity can often be increased by operating the process at low dissolved oxygen concentrations close to the limitation level. This may be accomplished with a closed-loop controller that regulates the dissolved oxygen concentration by manipulating the dominant carbon source feeding rate. In this work we study this control problem in a pilot 50l bioreactor with a high cell density recombinant P. pastoris cultivation in complex media. The study focuses on the design of accurate stable adaptive controllers, with guaranteed exponential convergence and its relation with the calibration of controller parameters. Two adaptive control strategies were tested in the pilot bioreactor: a model reference adaptive controller with a linear reference model and an integral feedback controller with adaptive gain. The latter alternative proved to be more robust to errors in the measurements of the off-gas composition. Concerning the instrumentation, algorithms were derived assuming that both the dissolved oxygen tension and off-gas composition are measured on-line, but also the case of only dissolved oxygen being measured is addressed. It was verified that the measurement of off-gas composition might not improve the controller performance due to measurement and process time delays.

Methanol induction optimization for scFv antibody fragment production in Pichia pastoris.

Fibronectin splice variant ED B (extracellular domain B) is a promising marker for angiogenesis in growing solid tumors. Currently, recombinant antibodies against ED B are being investigated concerning their potential use, for either therapeutic or diagnostic purposes. Single-chain antibody fragments directed against the ED B can be efficiently expressed in Pichia pastoris; thus, a recombinant strain of the methylotropic yeast P. pastoris was used for this work. Three different forms of scFv antibody fragment are found in the supernatant from this fermentation: covalent homodimer, associative homodimer, and monomer. Both homodimeric forms can be converted to the monomeric form (under reducing conditions) and be efficiently radiolabeled, whereas the monomeric form of scFv already present in the supernatant cannot. It was also found that the fraction of protein in the monomeric form is highly dependent on the mode of induction rather than scFv concentration. This suggests that the monomeric form of the scFv present in the supernatant might be a result of events occurring at the expression, secretion, or folding level. A high cell density fermentation protocol was developed by optimizing methanol induction, yielding the highest scFv antibody fragment production rate and product quality; cell concentration at the induction point and specific methanol uptake rate were found to be the most important control variables. A decrease in specific methanol uptake rate led to a higher specific production rate for the scFv antibody fragment (5.4 microg g(cell) h(-1)). Product quality, i.e., percentage of product in a homodimeric form, also increased with the decrease in methanol uptake rate. Furthermore, the volumetric productivity depended on cell concentration at the induction point, increasing with the increase of cell concentration up to 320 g L(-1) wet cell weight (WCW). The reduction of the methanol feeding rate for induction, and consequently of the oxygen uptake rate, have important consequences for optimizing product titers and quality and thus on the scale-up of this production process; hence one of the major limitations upon high cell density cultivation in bioreactors is keeping the high oxygen transfer rate required. From the results obtained, a scale-up strategy was developed based on the available oxygen transfer rates at larger scales, allowing the definition of the optimum biomass concentration for induction and methanol feeding strategy for maximization of product titer and quality.

Process development of a recombinant antibody/interleukin-2 fusion protein expressed in protein-free medium by BHK cells.

The production, purification and stability of quality (in terms of integrity and glycosylation) of an antibody/interleukin-2 fusion protein with potential application in tumour-targeted therapy expressed in BHK21 cells are described. Consistency of the product throughout time was determined by analysis of glycosylation of the fusion protein using MALDI-TOF mass spectroscopy and HPAEC-PAD combined with product integrity studies by SDS-PAGE and Western blotting. These investigations showed consistent expression in terms of integrity and of three major oligosaccharide structures of the fusion protein after 62 generations. The data obtained at this stage indicated the suitability of the cell line for production purposes. Different approaches for the production of this protein were subsequently carried out. The relative productivity of the recombinant fusion protein and general performance of the cells in two different protein-free medium (PFM) culture systems, continuous chemostat and continuous perfusion using a Centritech centrifuge as a cell retention device, were studied. The results indicate that the chemostat culture resulted in more stable and controllable nutrient environment, which could indicate better product consistency, in accordance with what has been observed under serum-containing conditions, in relation to the perfusion culture. Finally, product obtained from the chemostat culture was analysed and purified. The purification process was optimised with an increase in the overall yield from 38 to 70% being obtained, a significant improvement with important consequences for the implementation of an industrial-scale culture system. In conclusion, it was possible to produce and purify the recombinant antibody/interleukin-2 fusion protein assuring the quality and stability of the product in terms of integrity and glycosylation. Therefore, a candidate production process was established.

Influence of environmental factors on lipase production by Lactobacillus plantarum.

A strain of Lactobacillus plantarum, DSMZ 12028 (Deutsch Sammlung von Mikroorganismen und Zellkulturen), isolated from a Portuguese dry fermented sausage, "chouriço", was found to produce true lipase, producing free fatty acids from triolein (olive oil). This enzymatic activity was found in whole cells, but was negligible in comparison to lipolytic activity in culture supernatant. Therefore, only extracellular activity was studied. The effect of pH, temperature and glucose concentration on extracellular lipase production was studied in continuously stirred tank reactors, the first time this technology has been used to study the production of this enzyme in lactobacilli. Maximum lipase production was achieved at a pH of 5.5 and 30 degrees C and was kept at a significant level over a wide range of dilution rates (0.05-0.4 h-1); the production of lipase was still significant for low pH values, temperature and glucose concentration, conditions that are close to the ones present during chouriço ripening. The effect of glucose concentration was also studied in a batch system. The control of lipase production was found to be related both to glucose concentration in the medium and to the growth rate/dilution rate. Glucose concentration was found to be important for fast lipase production, although it did not influence the maximum lipase activity reached in a batch culture.