Aldo Tonso

Drosophila melanogaster S2 cells for expression of heterologous genes: From gene cloning to bioprocess development

In the present review we discuss strategies that have been used for heterologous gene expression in Drosophila melanogaster Schneider 2 (S2) cells using plasmid vectors. Since the growth of S2 cells is not dependent on anchorage to solid substrates, these cells can be easily cultured in suspension in large volumes. The factors that most affect the growth and gene expression of S2 cells, namely cell line, cell passage, inoculum concentration, culture medium, temperature, dissolved oxygen concentration, pH, hydrodynamic forces and toxic metabolites, are discussed by comparison with other insect and mammalian cells. Gene expression, cell metabolism, culture medium formulation and parameters involved in cellular respiration are particularly emphasized. The experience of the authors with the successful expression of a biologically functional protein, the rabies virus glycoprotein (RVGP), by recombinant S2 cells is presented in the topics covered.

Recombinant rabies virus glycoprotein synthesis in bioreactor by transfected Drosophila melanogaster S2 cells carrying a constitutive or an inducible promoter

S2 cell populations (S2AcRVGP2K and S2MtRVGP-Hy) were selected after transfection of gene expression vectors carrying the cDNA encoding the rabies virus glycoprotein (RVGP) gene under the control of the constitutive (actin) or inductive (metallothionein) promoters. These cell populations were cultivated in a 1L bioreactor mimicking a large scale bioprocess. Cell cultures were carried out at 90 rpm and monitored/controlled for temperature (28 degrees C) and dissolved oxygen (10 or 50% air saturation). Cell growth attained approximately 1.5-3 x 10(7)cells/mL after 3-4 days of cultivation. The constitutive synthesis of RVGP in S2AcRVGP2K cells led to values of 0.76 microg/10(7) cells at day 4 of culture. The RVGP synthesis in S2MtRVGP-Hy cell fraction increased upon CuSO(4) induction attaining specific productivities of 1.5-2 microg/10(7) cells at days 4-5. RVGP values in supernatant as a result of cell lysis were always very low (<0.2 microg/mL) indicating good integrity of cells in culture. Overall the RVGP productivity was of 1.5-3mg/L. Our data showed an important influence of dissolved oxygen on RVGP synthesis allowing a higher and sustained productivity by S2MtRVGP-Hy cells when cultivated with a DO of 10% air saturation. The RVGP productivity in bioreactors shown here mirrors those previously observed for T-flasks and shaker bottles and allow the preparation of the large RVGP quantities required for studies of structure and function.

Effect of dissolved oxygen concentration on red pigment and citrinin production by Monascus purpureus ATCC 36928

The present study investigated the effects of agitation speed, N (200, 500, 600 or 700 rpm), and dissolved oxygen concentration, C (120, >70, 70, 60, 10 or < 10%), on red pigment and citrinin production by Monascus purpureus ATCC 36928, cultivated in liquid medium by a batch process. The gas flow rate was the same for all runs with C controlled by means of the incoming gas composition control (air/N2 or air/O2). From the response surface plots it can be verified that the effect of C was greater than that of N on the production of both metabolites. The absorbance for red pigments varied from 1.6 U (C< 10%; N=200 rpm) up to 3.3 U (C=60%; N=600 rpm), an increase of 106%, while citrinin concentration increased 257%, from 14.2 to 50.7 mg.L-1. The most appropriate conditions were C=60% and N=600rpm, under which the highest red pigment absorbance (3.3U) and half of the highest citrinin concentration were obtained.

Nomenclature and guideline to express the amount of a membrane protein synthesized in animal cells in view of bioprocess optimization and production monitoring.

Studies of a bioprocess optimization and monitoring for protein synthesis in animal cells face a challenge on how to express in quantitative terms the system performance. It is possible to have a panel of calculated variables that fits more or less appropriately the intended goal. Each mathematical expression approach translates different quantitative aspects. We can basically separate them into two categories: those used for the evaluation of cell physiology in terms of product synthesis, which can be for bioprocess improvement or optimization, and those used for production unit sizing and for bioprocess operation. With these perspectives and based on our own data of kinetic S2 cells growth and metabolism, as well as on their synthesis of the transmembrane recombinant rabies virus glycoprotein, here indicated as P, we show and discuss the main characteristics of calculated variables and their recommended use. Mainly applied to a bioprocess improvement/optimization and that mainly used for operation definition and to design the production unit, we expect these definitions/recommendations would improve the quality of data produced in this field and lead to more standardized procedures. In turn, it would allow a better and easier comprehension of scientific and technological communications for specialized readers.

The effect of dissolved oxygen concentration control on cell growth and antibiotic retamycin production in Streptomyces olindensis So20 fermentations

Cell growth and retamycin production in Streptomyces olindensis So20 were studied at different dissolved oxygen levels during fermentation. The profiles for cell growth and retamycin production in an experiment with dissolved oxygen control at 5% air saturation during production were similar to those in an experiment without dissolved oxygen control. However, despite slower cell growth, a twofold increase in the final retamycin concentration was achieved when dissolved oxygen was controlled at 100% during the growth phase, compared to a base experiment (no DO control). Therefore, the results revealed that retamycin production was dependent on dissolved oxygen concentration during the growth phase, even with sufficient oxygen. On the other hand, a large oxygen supply during the production phase did not contribute to an improvement in retamycin production.

Effect of yeast extract on glucoamylase synthesis byAspergillusawamori NRRL 3112

SummaryGlucoamylase synthesis is strongly affected by yeast extract concentration (CE). An eight fold increase in CE caused a two fold increase in the maximum glucoamylase activity value (Am) for the cultivations conducted with an initial glucose concentration (GO) of about 20 g/l, and a four fold increase in Am in the runs with a Go value of about 40 g/l. Five mathematical correlations are presented, showing a very good adjustment to the experimental results.

A multivariate calibration procedure for UV/VIS spectrometric monitoring of BHK-21 cell metabolism and growth

Monitoring mammalian cell culture with UV–vis spectroscopy has not been widely explored. The aim of this work was to calibrate Partial Least Squares (PLS) models from off‐line UV–vis spectral data in order to predict some nutrients and metabolites, as well as viable cell concentrations for mammalian cell bioprocess using phenol red in culture medium. The BHK‐21 cell line was used as a mammalian cell model. Spectra of samples taken from batches performed at different dissolved oxygen concentrations (10, 30, 50, and 70% air saturation), in two bioreactor configurations and with two strategies to control pH were used to calibrate and validate PLS models. Glutamine, glutamate, glucose, and lactate concentrations were suitably predicted by means of this strategy. Especially for glutamine and glucose concentrations, the prediction error averages were lower than 0.50 ± 0.10 mM and 2.21 ± 0.16 mM, respectively. These values are comparable with those previously reported using near infrared and Raman spectroscopy in conjunction with PLS. However, viable cell concentration models need to be improved. The present work allows for UV–vis at‐line sensor development, decrease cost related to nutrients and metabolite quantifications and establishment of fed‐batch feeding schemes.

Use of uniform designs in combination with neural networks for viral infection process development

This work aimed to compare the predictive capacity of empirical models, based on the uniform design utilization combined to artificial neural networks with respect to classical factorial designs in bioprocess, using as example the rabies virus replication in BHK‐21 cells. The viral infection process parameters under study were temperature (34°C, 37°C), multiplicity of infection (0.04, 0.07, 0.1), times of infection, and harvest (24, 48, 72 hours) and the monitored output parameter was viral production. A multilevel factorial experimental design was performed for the study of this system. Fractions of this experimental approach (18, 24, 30, 36 and 42 runs), defined according uniform designs, were used as alternative for modelling through artificial neural network and thereafter an output variable optimization was carried out by means of genetic algorithm methodology. Model prediction capacities for all uniform design approaches under study were better than that found for classical factorial design approach. It was demonstrated that uniform design in combination with artificial neural network could be an efficient experimental approach for modelling complex bioprocess like viral production. For the present study case, 67% of experimental resources were saved when compared to a classical factorial design approach. In the near future, this strategy could replace the established factorial designs used in the bioprocess development activities performed within biopharmaceutical organizations because of the improvements gained in the economics of experimentation that do not sacrifice the quality of decisions.

Semliki forest virus as a vector: pros and cons for its use in biopharmaceuticals production

The number of biopharmaceuticals for medical and veterinarian use produced in mammalian cells is increasing year after year. All of them are obtained by stable recombinant cell lines. However, it is recognized that transient gene expression produces high level expression in a short time. In that sense, viral vectors have been extensively used for producing recombinant proteins on lab-scale. Among them, Semliki Forest virus is commonly employed for this purpose. This review discusses the main aspects related to the use of Semliki Forest virus technology as well as its advantages and drawbacks which limit currently its utilization in biopharmaceutical industry on large-scale.

Immobilization of primary cultures of insulin-releasing human pancreatic cells.

Transplantation of pancreatic islets isolated from organ donors constitutes a promising alternative treatment for type1 Diabetes, however, it is severely limited by the shortage of organ donors. Ex-vivo islet cell cultures appear as an attractive but still elusive approach for curing type 1 Diabetes. It has recently been shown that, even in the absence of fibrotic overgrowth, several factors, such as insufficient nutrition of the islet core, represent a major barrier for long-term survival of islets grafts. The use of immobilized dispersed cells may contribute to solve this problem due to conceivably easier nutritional and oxygen support to the cells. Therefore, we set out to establish an immobilization method for primary cultures of human pancreatic cells by adsorption onto microcarriers (MCs). Dispersed human islets cells were seeded onto Cytodex1 microcarriers and cultured in bioreactors for up to eight days. The cell number increased and islet cells maintained their insulin secretion levels throughout the time period studied. Moreover, the cells also presented a tendency to cluster upon five days culturing. Therefore, this procedure represents a useful tool for controlled studies on islet cells physiology and, also, for biotechnological applications.