Purification of rabies virus glycoprotein produced in Drosophila melanogaster S2 cells: An efficient immunoaffinity method.

Most rabies vaccines are based on inactivated virus, which production process demands a high level of biosafety structures. In the past decades, recombinant rabies virus glycoprotein (RVGP) produced in several expression systems has been extensively studied to be used as an alternative vaccine. The immunogenic characteristics of this protein depend on its correct conformation, which is present only after the correct post-translational modifications, typically performed by animal cells. The main challenge of using this protein as a vaccine candidate is to keep its trimeric conformation after the purification process. We describe here a new immunoaffinity chromatography method using a monoclonal antibody for RVGP Site II for purification of recombinant rabies virus glycoprotein expressed on the membrane of Drosophila melanogaster S2 cells. RVGP recovery achieved at least 93%, and characterization analysis showed that the main antigenic proprieties were preserved after purification.

Purification of rabies virus glycoprotein produced in Drosophila melanogaster S2 cells: an efficient immunoaffinity method

Most rabies vaccines are based on inactivated virus, which production process demands a high level of biosafety structures. In the past decades recombinant Rabies Virus Glycoprotein (RVGP) produced in several expression systems, has been extensively studied to be used as an alternative vaccine. The immunogenic characteristics of this protein depend on its correct conformation, which is present only after the correct post‐translational modifications, typically performed by animal cells. The main challenge of using this protein as a vaccine candidate is to keep its trimeric conformation after the purification process. We describe here a new immunoaffinity chromatography method using a monoclonal antibody for RVGP site II for purification of recombinant rabies virus glycoprotein expressed on the membrane of Drosophila melanogaster S2 cells. RVGP recovery achieved at least 93%, and characterization analysis showed that the main antigenic proprieties were preserved after purification.

Adaptation of Mammalian Cells to Chemically Defined Media.

In order to circumvent ethical, technical, and economic drawbacks regarding the use of animal serum in cell culturing, it is possible to adapt mammalian cells to serum-free media. Nowadays, there are several serum-free formulations available, including fully animal derived-free and chemically defined media, and different adaptation techniques. This article focuses on the gradual adaptation of a mammalian suspension cell culture to a chemically defined medium. The first step is to transfer the cells cultured in medium supplemented with fetal bovine serum (FBS) to a chemically defined medium of your choice, containing the same amount of FBS. The next steps consist of progressively reducing the amount of FBS, while monitoring cell growth and viability up to the complete elimination of FBS. This protocol has been successfully used to adapt THP-1 cells to a chemically defined medium with similar maximum specific growth rate as those cultured with FBS. © 2019 by John Wiley & Sons, Inc. Basic Protocol: Gradual adaptation to chemically defined medium Alternate Protocol: Direct adaptation to chemically defined medium Support Protocol 1: Determining maximum specific growth rate of a cell culture Support Protocol 2: Cell freezing and thawing.

The Overt and Hidden Use of Animal-Derived Products in Alternative Methods for Skin Sensitisation: A Systematic Review.

In vitro methods that can replace animal testing in the identification of skin sensitisers are now a reality. However, as cell culture and related techniques usually rely on animal-derived products, these methods may be failing to address the complete replacement of animals in safety assessment. The objective of this study was to identify the animal-derived products that are used as part of in vitro methods for skin sensitisation testing. Thus, a systematic review of 156 articles featuring 83 different in vitro methods was carried out and, from this review, the use of several animal-derived products from different species was identified, with the use of fetal bovine serum being cited in most of the methods (78%). The use of sera from other animals, monoclonal antibodies and animal proteins were also variously mentioned. While non-animal alternatives are available and methods free of animal-derived products are emerging, most of the current methods reported used at least one animal-derived product, which raises ethical and technical concerns. Therefore, to deliver technically and ethically better in vitro methods for the safety assessment of chemicals, more effort should be made to replace products of animal origin in existing methods and to avoid their use in the development of new method protocols.

Adaptation of a skin sensitization assay to a chemically defined culture.

There are currently three in vitro methods adopted by the Organization for the Economic Co-operation and Development for testing chemicals based on the third key event of the skin sensitization adverse outcome pathway, the activation of dendritic cells. All of them use culture medium supplemented with fetal bovine serum (FBS), which brings technical disadvantages and animal welfare concerns. The objective of this study was to analyze the possibility of eliminating the use of FBS in the human Cell Line Activation Test (h-CLAT). After successful implementation of the h-CLAT using THP-1 cells cultured in FBS-containing medium, several attempts to adapt THP-1 cells to four different serum-free media were made. The best results were obtained with gradual adaptation to RPMI-1640 medium with HL-1™ Supplement and to X-VIVO™ 10. Adapted cells were cryopreserved and submitted to the reactivity check. After being approved, they were used in dose finding and proficiency assays. Despite minor adjustments in the original protocol, it was possible to correctly predict the sensitizing potential of the ten proficiency substances using THP-1 cells adapted to X-VIVO™ 10, which indicates that it is possible to eliminate the use of FBS in the h-CLAT, using a chemically defined medium.

Culturing Drosophila melanogaster (S2) in a chemostat.

Insect cells are used for the expression of complex proteins in products such as vaccines and biopharmaceuticals. Physiology of a Drosophila melanogaster (lineage S2), transfected to stably express rabies virus glycoprotein (RVGP), was studied in batch culture and in a chemostat with serum-free medium. In batch mode, the system reached 3 × 10(7) cells mL(-1) with specific growth rate of 1.5 d(-1) with RVGP at 2.50 µg L(-1). When operated continuously, three well-defined steady states were achieved at dilution rates (D) of 0.8, 0.5 and 0.2 d(-1). The residual glucose and glutamine concentrations and the cell yields on glucose and glutamine decreased at lower D. High values of substrate consumption for maintenance may explain this variation in yields. The results indicated that glucose is not the limiting substrate of this process.

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.

Adaptation of the "Dynamic Method" for measuring the specific respiration rate in oxygen transfer systems through diffusion membrane.

Monitoring the specific respiration rate (Q(O2)) is a valuable tool to evaluate cell growth and physiology. However, for low Q(O2) values the accuracy may depend on the measurement methodology, as it is the case in animal cell culture. The widely used "Dynamic Method" imposes serious difficulties concerning oxygen transfer cancellation, especially through membrane oxygenation. This paper presents an improved procedure to this method, through an automated control of the gas inlet composition that can minimize the residual oxygen transfer driving force during the Q(O2) measurement phase. The improved technique was applied to animal cell cultivation, particularly three recombinant S2 (Drosophila melanogaster) insect cell lines grown in a membrane aeration bioreactor. The average measurements of the proposed method reached 98% of stationary liquid phase balance method, taken as a reference, compared to 21% when the traditional method was used. Furthermore, this methodology does not require knowledge of the volumetric transfer coefficient k(L)a, which may vary during growth.

Behavior of wild-type and transfected S2 cells cultured in two different media.

An animal protein-free medium composed of IPL-41 containing 6 g L(-1) yeastolate ultrafiltrate, 10 g L(-1) glucose, 2 g L(-1) lactose, 5 g L(-1) glutamine, 1% lipid emulsion, and 0.1% Pluronic F-68 was used for producing recombinant proteins in batch mode employing two cell lines, S2AcRVGP2k expressing the G glycoprotein from rabies virus (RVGP) and S2AcHBsAgHy-9C expressing the surface antigen of hepatitis B virus (HBsAg), both obtained from Drosophila melanogaster S2 cells. Growth of wild-type S2 cells was also evaluated in the same medium. Cell behavior in the tested medium was compared to that verified in Sf900 II®. The results show that in shake flasks, S2AcRVGP2k and S2AcHBsAgHy-9C cells reached around 2 × 10(7) cells mL(-1) in both media. In supplemented IPL-41 and Sf900 II® media, S2AcRVGP2k cells produced 367 ng RVGP mL(-1) and 638 ng RVGP mL(-1), respectively, while S2AcHBsAgHy-9C cells correspondently produced 573 ng HBsAg mL(-1) and 322 ng HBsAg mL(-1) in the mentioned media. In stirred tanks, S2AcRVGP2k cells reached 3 × 10(7) cells mL(-1) and produced up to 758 ng RVGP mL(-1). In general, glucose was consumed by cells, while lactate and ammonia were produced.

Behavior of wild-type and transfected S2 cells cultured in two different media

An animal protein-free medium composed of IPL-41 containing 6 g L−1 yeastolate ultrafiltrate, 10 g L−1 glucose, 2 g L−1 lactose, 5 g L−1 glutamine, 1% lipid emulsion, and 0.1% Pluronic F-68 was used for producing recombinant proteins in batch mode employing two cell lines, S2AcRVGP2k expressing the G glycoprotein from rabies virus (RVGP) and S2AcHBsAgHy-9C expressing the surface antigen of hepatitis B virus (HBsAg), both obtained from Drosophila melanogaster S2 cells. Growth of wild-type S2 cells was also evaluated in the same medium. Cell behavior in the tested medium was compared to that verified in Sf900 II®. The results show that in shake flasks, S2AcRVGP2k and S2AcHBsAgHy-9C cells reached around 2 × 107 cells mL−1 in both media. In supplemented IPL-41 and Sf900 II® media, S2AcRVGP2k cells produced 367 ng RVGP mL−1 and 638 ng RVGP mL−1, respectively, while S2AcHBsAgHy-9C cells correspondently produced 573 ng HBsAg mL−1 and 322 ng HBsAg mL−1 in the mentioned media. In stirred tanks, S2AcRVGP2k cells reached 3 × 107 cells mL−1 and produced up to 758 ng RVGP mL−1. In general, glucose was consumed by cells, while lactate and ammonia were produced.

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.

Growth of recombinant Drosophila melanogaster Schneider 2 cells producing rabies virus glycoprotein in bioreactor employing serum-free medium.

Drosophila melanogaster Schneider 2 (S2) cells have been increasingly used as a suitable expression system for the production of different recombinant proteins, and the employment of bioreactors for large-scale culture is an important tool for this purpose. In this work, Drosophila S2 cells producing the rabies virus glycoprotein RVGP were cultivated in bioreactor, employing a serum-free medium, aiming an improvement in cell growth and in glycoprotein production. To overcome cell growth limitation commonly observed in stirred flasks, different experiments in bioreactor were performed, in which some system modifications were carried out to attain the desired goal. The study showed that this cell line is considerably sensitive to hydrodynamic forces, and a high cell density (about 16.0 x 10(6) cells mL(-1)) was only obtained when Pluronic F68((R)) percentage was increased to 0.6% (w/v). Despite ammonium concentration affected RVGP production, and also cell growth, an elevated amount of the target protein was obtained, attaining 563 ng 10(-7) cells.

Analytical approach for the extraction of recombinant membrane viral glycoprotein from stably transfected Drosophila melanogaster cells.

Parameters for storage, lysis and concentration of Drosophila melanogaster Schneider 2 (S2AcRVGP) cells expressing the recombinant rabies virus glycoprotein (RVGP) were studied with regard to RVGP quantification by ELISA, for productivity evaluation and future purification. Lysis buffers were formulated with Tris, NaCl, glycerol, EDTA, KCl, Na(2)PO(4), MgCl(2), PMSF and NP-40 or CHAPS. S2AcRVGP cells (10(7) cells at the exponential growth phase) were frozen at -20 degrees C as a dry pellet, suspended in buffer (B) formulations or after treatment with lysis buffer (LB) formulations. They were then thawed as cell pellets or with B formulations or PBS at 4 degrees C or at room temperature and then lysed with LB formulations. For RVGP quantification by ELISA, a protocol was chosen of cell preparation including cell freezing as dry pellet, cell thawing at 4 degrees C with B4 (Tris, NaCl, MgCl(2), PMSF) and cell lysis with the LB4 (B4 + NP-40) since it fulfilled requirements of high RVGP detection, and was easily performed with mixtures freezing quickly, and a cost-saving LB formulation could be used. Using these established conditions, we examined the optimal cell concentration for RVGP quantification by ELISA. Results showed that an increase in the RVGP detection (from 62.5 to 1083 ng/10(7) cells) paralleled a decrease in the cell number (3 x 10(7) - 10(5) cells) used. The NP-40 concentration present in the LB4 was further investigated as a function of the cell number used for sample preparation. Previous results were confirmed indicating that higher NP-40 concentrations led to a decreased detection of RVGP. Altogether our data clearly pointed out the crucial effects of cell freeze, thaw, lysis and concentration on immune detection of recombinant membrane glycoproteins and can be useful as a guideline for sample preparation for this purpose.

Evaluation of immobilized metal membrane affinity chromatography for purification of an immunoglobulin G1 monoclonal antibody.

The large scale production of monoclonal antibodies (McAbs) has gaining increased relevance with the development of the hybridoma cell culture in bioreactors creating a need for specific efficient bioseparation techniques. Conventional fixed bead affinity adsorption commonly applied for McAbs purification has the drawback of low flow rates and colmatage. We developed and evaluated a immobilized metal affinity chromatographies (IMAC) affinity membrane for the purification of anti-TNP IgG(1) mouse McAbs. We immobilized metal ions on a poly(ethylene vinyl alcohol) hollow fiber membrane (Me(2+)-IDA-PEVA) and applied it for the purification of this McAbs from cell culture supernatant after precipitation with 50% saturation of ammonium sulphate. The purity of IgG(1) in the eluate fractions was high when eluted from Zn(2+) complex. The anti-TNP antibody could be eluted under conditions causing no loss of antigen binding capacity. The purification procedure can be considered as an alternative to the biospecific adsorbent commonly applied for mouse IgG(1) purification, the protein G-Sepharose.

Nitrogenase activity of Beijerinckia derxii is preserved under adverse conditions for its growth

In order to evaluate the response of Beijerinckia derxii ICB-10 to different environmental factors, growth curves and specific nitrogenase activity were studied. Tested conditions were as follows: a) media with different pH values (2.5, 2.8, 4.2 and 5.7); b) medium supplemented with 230 æM aluminium sulphate; c) media with two different potassium phosphate concentrations (50 mM and 100 mM); d) shaken or still cultures; e) medium supplemented with 40 mM sodium thiosulphate. Growth curves and specific nitrogenase activity at pH 4.2 were closely similar to those for cultures at pH 5.7 (standard condition), whereas no growth occurred at pH 2.5. Changes in growth curves and/or specific nitrogenase activity were observed under the following conditions: I) pH 2.8 (decrease in initial CFU number, reduction of maximum specific growth rate, reduced number of generations and stimulation of nitrogenase activity), II) presence of aluminium (early death phase), III) 50 mM PO4(3-) (reduction of maximum specific growth rate), IV) 100 mM PO4(3-) (reduction of both number of generations and maximum specific growth rate as well as early death phase), V) low O2 availability (increasing nitrogenase activity), and VI) presence of thiosulphate (reduction of maximum specific growth rate; early death phase and high stimulation of nitrogenase activity). The data obtained showed the high variability of the cell growth response to environmental factors. Nitrogenase activity was always preserved even when population growth was affected

A study of the influence of pH and of dissolved oxygen concentration on BHK-21 cells growth

In a joint project of IPT (Instituto de Pesquisas Tecnológicas do Estado de São Paulo) and Vallée S.A., the growth of BHK-21 cells, used for the production of a vaccine against foot-and-mouth disease, was studied. The present work aimed the evaluation of the influence of and dissolved oxygen concentration on this growth. Assays were performed in a 2 L stirred bioreactor, with a BHK-21 cell line supplied by Vallée S.A. The culture medium contained glucose, several amino acids, vitamins and bovine serum. Seven experiments were performed, in which pH varied between 6.8 and 7.4, and the dissolved oxygen concentration between 2 and 60(per cent) of air saturation. The definition of optimal values was based on analysis of cell productivity and amounts of cells produced at the point at which the cultures attained maximal cell concentration. Optimal values for these paramenters were observed at pH 7.2 and 10 per cente dissolved oxygen concentration.