title: Dielectrophoretic behavior of PEGylated RNase A inside a microchannel with diamond-shaped insulating posts.
abstract: Ribonuclease A (RNase A) has proven potential as a therapeutic agent, especially in its PEGylated form. Grafting of PEG molecules to this protein yields mono-PEGylated (mono-PEG) and di-PEGylated (di-PEG) RNase A conjugates, and the unreacted protein. Mono-PEG RNase A is of great interest. The use of electrokinetic forces in microdevices represents a novel alternative to chromatographic methods to separate this specie. This work describes the dielectrophoretic behavior of the main protein products of the RNase A PEGylation inside a microchannel with insulators under direct current electric fields. This approach represents the first step in route to design micro-bioprocesses to separate PEGylated RNase A from unreacted native protein. The three proteins exhibited different dielectrophoretic behaviors. All of them experienced a marked streaming pattern at 3000 V consistent with positive dielectrophoresis. Native protein was not captured at any of the conditions tested, while mono-PEG RNase A and di-PEG RNase A were captured presumably due to positive dielectrophoresis at 4000 and 2500 V, respectively. Concentration of mono-PEG RNase A with a maximal enrichment efficiency of ≈9.6 times the feed concentration was achieved in few seconds. These findings open the possibility of designing novel devices for rapid separation, concentration, and recovery of PEGylated RNase A in a one-step operation.
authors.affiliation: Centro de Biotecnología-FEMSA, Tecnológico de Monterrey, Monterrey, Mexico.
authors.forename: Marco A
authors.initials: MA
authors.lastname: Mata-Gómez
authors.affiliation: School of Engineering and Sciences, Tecnológico de Monterrey, Monterrey, Mexico.
authors.forename: Roberto C
authors.initials: RC
authors.lastname: Gallo-Villanueva
authors.affiliation: Centro de Biotecnología-FEMSA, Tecnológico de Monterrey, Monterrey, Mexico.
authors.forename: José
authors.initials: J
authors.lastname: González-Valdez
authors.affiliation: School of Engineering and Sciences, Tecnológico de Monterrey, Monterrey, Mexico.
authors.forename: Sergio O
authors.initials: SO
authors.lastname: Martínez-Chapa
authors.affiliation: Centro de Biotecnología-FEMSA, Tecnológico de Monterrey, Monterrey, Mexico.
authors.forename: Marco
authors.initials: M
authors.lastname: Rito-Palomares
mesh_terms: D000818:Animals; D002417:Cattle; D003198:Computer Simulation; D018130:Diamond; D004586:Electrophoresis / Q000295:instrumentation* / Q000379:methods; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation* / Q000379:methods; D011092:Polyethylene Glycols / Q000737:chemistry*; D012259:Ribonuclease, Pancreatic / Q000737:chemistry*
chemical_list: D011092:Polyethylene Glycols; D018130:Diamond; D012259:Ribonuclease, Pancreatic
keywords: Dielectrophoresis; Microchannel; Microfluidics; PEGylation; Ribonuclease A