Aplicación genérica de sistemas de dos fases acuosas polietilénglicol–sal para el desarrollo de procesos de recuperación primaria de compuestos biológicos

Generic application of polyethylene glycol–salt aqueous two–phase systems for the development of processes to biological products primary recovery

J. Benavides y M. Rito–Palomares*

Departamento de Biotecnología e Ingeniería de Alimentos, Tecnológico de Monterrey Eugenio Garza Sada 2501 Sur, CP 64849, Monterrey NL, México. * Autor para la correspondencia. E–mail: mrito@itesm.mx.

Resumen

Se evaluó la aplicación genérica de los sistemas de dos fases acuosas (SDFA) polietilénglicol (PEG) – solución salina como una estrategia práctica para el desarrollo de bioprocesos que permiten la recuperación primaria de compuestos biológicos. Se utilizaron cuatro modelos experimentales: B–ficoeritrina (BFE) producida por Porphyridium cruentum, C–ficocianina (CFC) producida por Spirulina maxima, pseudo–partículas 2/6 de rotavirus (dlRLP) producidas por células de insecto High Five^TM y por último luteína producida por Chlorella protothecoides. Se estudió la influencia de los parámetros de sistema (peso molecular del polímero, PM PEG; longitud de línea de corte, LLC; relación de volumen, V_R; el pH del sistema) sobre el comportamiento de partición de los modelos experimentales seleccionados. Se establecieron condiciones de proceso para la recuperación primaria de los productos de interés utilizando SDFA. Dichas condiciones resultan en procesos con un reducido número de etapas, lo cual favorece el rendimiento obtenido de los productos. La influencia de los parámetros de sistema sobre el comportamiento de partición de los productos fue correlacionada con sus características fisicoquímicas, lo cual permitió establecer reglas generales que facilitan el desarrollo predictivo de procesos para la recuperación primaria de compuestos biológicos utilizando SDFA PEG – solución salina.

Palabras clave: sistemas de dos fases acuosas, recuperación primaria, compuestos biológicos.

Abstract

The generic application of Aqueous Two–Phase Systems (ATPS) polyethylene glycol (PEG) – salt was evaluated as a practical strategy for the development of bioprocesses for the primary recovery of biological products. Four experimental models were used: B–phycoerythrin (BFE) from Porphyridium cruentum, C–phycocyanin (CFC) from Spirulina maxima, double layered rotavirus–like particles (dlRLP) produced in High Five^TM insect cells, and lutein from Chlorella protothecoides. Influence of the system parameters (polymer molecular weight, MW PEG; tie line length, TLL; volume ratio, V_R; system pH) upon partition behavior of the selected experimental models. Process conditions for the primary recovery of the products of interest using ATPS were established. Such conditions result in processes with reduced number of stages, which favored high yields. The influence of system parameters upon partition behavior of the four experimental models was correlated with their physicochemical properties, which allowed establishing general rules that facilitate the predictive development of processes for the primary recovery of biological products using SDFA PEG – salt.

Keywords: aqueous two–phase systems, primary recovery, biological products.

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Referencias

Aguilar, O., Albiter, V., Serrano–Carreon, L., Rito–Palomares, M. (2006). Direct comparison between ion–exchange chromatography and aqueous two–phase processes for the partial purification of penicillin acylase produced by E. coli. Journal of Chromatography B 835, 77–83.         [ Links ]

Albertsson, PÅ. (1956). Chromatography and partition of cells and cell fragments. Nature 177, 771–774.         [ Links ]

Albertsson, PÅ., Johansson, G., Tjerneld, F. (1990). Aqueous Two–Phase Separations. In: Separation in Biotechnology (Asenjo, JA., ed.). Pp 287–327. Dekker, New York, USA.         [ Links ]

Andrews, BA., Huang, RB., Asenjo JA. (1995). Purification of virus like particles from yeast cells using aqueous two–phase systems. Bioseparation 5, 105–112.         [ Links ]

Benavides, J., Mena, JA., Cisneros–Ruiz, M., Ramírez, OT., Palomares, LA., Rito–Palomares, M. (2006). Rotavirus–like particles primary recovery from insect cells in aqueous two–phase systems. Journal of Chromatography B 842, 48–57.         [ Links ]

Benavides, J., Rito–Palomares, M. (2004). Bioprocess intensification: a potential aqueous two–phase process for the primary recovery of B–phycoerythrin from Porphyridium cruentum. Journal of Chromatography B 807, 33–38.         [ Links ]

Benavides, J., Rito–Palomares, M. (2005). Potential Aqueous Two–Phase Processes for the Primary Recovery of Colored Protein from Microbial Origin. Engineering in Life Sciences 5, 259–266.         [ Links ]

Benavides, J., Rito–Palomares, M. (2006). Simplified two–stage method to B–phycoerythrin recovery from Porphyridium cruentum. Journal of Chromatography B 844, 39–44.         [ Links ]

Bermejo–Román, R., Alvárez–Pez, JM., Acién Fernández, FG., Molina Grima, E. (2002). Recovery of pure B–phycoerythrin from the microalga Porphyridium cruentum. Journal of Biotechnology 93, 73–85.         [ Links ]

Cisneros, M., Benavides, J., Brenes, CH., Rito–Palomares, M. (2004). Recovery in aqueous two–phase systems of lutein produced by the green microalga Chlorella protothecoides. Journal of Chromatography B 807, 105–110.         [ Links ]

Cunha, MT., Aires–Barros, MR. (2002). Large–scale extraction of proteins. Molecular Biotechnology 20, 29–40        [ Links ]

Edahiro, JI., Yamada, M., Seike, S., Kakigi, Y., Miyanaga, K., Nakamura, M., Kanamori, T., Seki, M. (2005). Separation of Cultured Strawberry Cells Producing Anthocyanins in Aqueous Two–Phase System. Journal of Bioscience and Bioengineering 100(4), 449454.         [ Links ]

Esmanhoto, E., Vahan Kilikian, B. (2004). ATPS applied to extraction of small molecules –polycetides – and simultaneous clarification of culture media with filamentous microorganisms. Journal of Chromatography B 807,139–144.         [ Links ]

Frerix, A., Geilenkirchen, P., Muller, M., Kula, MR., Hubbuch, J. (2007). Separation of genomic DNA, RNA, and open circular plasmid DNA from supercoiled plasmid DNA by combining denaturation, selective renaturation and aqueous two–phase extraction. Biotechnology and Bioengineering 96, 57–66.         [ Links ]

Hernández–Mireles, T., Benavides, J., Rito–Palomares, M. (2006). Proceso para la purificación del colorante proteico B–ficoeritrina producido por Porphyridium cruentum. Revista Mexicana de Ingenieria Química 5, 131–143.         [ Links ]

Huddleston, J., Veide, A., Köhler, K., Flanagan, J., Enfors, SO., Lyddiat, A. (1991). The molecular basis of partitioning in aqueous two–phase systems. Trends in Biotechnology 9, 381–388.         [ Links ]

Iriti, M., Faoro, F. (2006). Grape phytochemicals: A bouquet of old and new nutraceuticals for human health. Medical Hypotheses 67, 833838.         [ Links ]

Johansson, G., Walter, H. (2000). Partitioning and concentrating biomaterials in aqueous phase systems. International Review of Cytology 192, 33–60.         [ Links ]

Kepka, C., Collet, E., Persson, J., Ståhl, Å., Lagerstedt, T., Tjerneld, F. and Veide, A. (2003). Pilot–scale extraction of an intracellular recombinant cutinase from E. coli cell homogenate using a thermoseparationg aqueous two–phase system. Journal of Biotechnology 103, 165181.         [ Links ]

Liu, CL., Kamei, DT., King, JA., Wang, DI., Blankschtein, D. (1998). Separation of proteins and viruses using two–phase aqueous micellar systems. Journal of Chromatography B 711, 127–138.         [ Links ]

MacColl, R., Lee, JJ., Berns, DS. (1971). Protein aggregation in C–Phycocyanin. Studies at very low concentrations with the photoelectric scanner of the ultracentrifuge. Biochemical Journal 122, 421–426.         [ Links ]

Mena, JA., Ramirez, OT., Palomares, LA. (2005). Quantification of rotavirus–like particles by gel permeation chromatography. Journal of Chromatography B 824, 267–276.         [ Links ]

Morre, DM., Morre DJ. (2000). Aqueous two–phase partition applied to the isolation of plasma membranes and Golgi apparatus from cultured mammalian cells. Journal of Chromatography B 743, 377–387.         [ Links ]

Nam, KH., Chang, WJ., Hong, H., Lim, SM., Kim, DI., Koo, YM. (2005). Continuous–flow fractionation of animal cells in microfluidic device using aqueous two–phase extraction. Biomed Microdevices 7, 189–195.         [ Links ]

Pico, G., Romanini, D., Nerli, B., Farruggia, B. (2006). Polyethyleneglycol molecular mass and polydispersivity effect on protein partitioning in aqueous two–phase systems. Journal of Chromatography B 830, 286–292.         [ Links ]

Rahimpour, F., Feyzi, F., Maghsoudi, S., Hatti–Kaul, R. (2006). Purification of plasmid DNA with polymer–salt aqueous two–phase system: optimization using response surface methodology. Biotechnology and Bioengineering 95, 627–637.         [ Links ]

Rao, AV., Rao, LG. (2007). Carotenoids and human health. Pharmacological Research 55, 207216.         [ Links ]

Ribeiro, SC., Monteiro, GA., Cabral, JM., Prazeres, DM. (2002). Isolation of plasmid DNA from cell lysates by aqueous two–phase systems. Biotechnology and Bioengineering 78, 376384.         [ Links ]

Rito–Palomares, M. (2002). The Practical Application of Aqueous Two–Phase Processes for the Recovery of Biological Products. Journal of Microbiology and Biotechnology 12 (4), 535–543.         [ Links ]

Rito–Palomares, M. (2004). Practical application of aqueous two–phase partition to process development for the recovery of biological products. Journal of Chromatography B 807, 3–11.         [ Links ]

Schindler, J., Nothwang, HG. (2006). Aqueous polymer two–phase systems: effective tools for plasma membrane proteomics. Proteomics 6 (20), 5409–5417.         [ Links ]

Shibusawa, Y., Takeuchi, N., Tsutsumi, K., Nakano, S., Yanagida, A., Shindo, H., Ito, Y. (2007). One–step purification of histone deacetylase from Escherichia coli cell–lysate by counter–current chromatography using aqueous two–phase system. Journal of Chromatography A 1151, 158–163.         [ Links ]

Solano–Castillo, C., Rito–Palomares, M. (2000). Kinetics of phase separation under different process and design parameters in aqueous two–phase systems. Journal of Chromatography B 743, 195–201.         [ Links ]

Spelzini, D., Pico, G., Farruggia, B. (2006). Dependence of chymosin and pepsin partition coefficient with phase volume and polymer pausidispersity in polyethyleneglycol–phosphate aqueous two–phase system. Colloids Surf B Biointerfaces 51, 80–85.         [ Links ]

Vonshak, A. (1990). Recent advances in microalgal biotechnology. Biotechnology Advances 8, 709–727.         [ Links ]