title: PEGylated protein separation using different hydrophobic interaction supports: Conventional and monolithic supports.
abstract: Protein hydrophobicity can be modified after a PEGylation process. However, hydrophobic interaction chromatography (HIC) has been used to separate PEGylation reaction products less frequently than other techniques. In this context, chromatographic monoliths represent a good alternative to continue exploring the separation of PEGylated proteins with HIC. In this work, the separation of PEGylated proteins using C4 A monolith as well as Toyopearl Butyl 650C and Butyl Sepharose was analyzed. Three proteins were used as models: RNase A, β-lactoglobulin, and lysozyme. All proteins were PEGylated in the N-terminal amino groups with 20 kDa methoxy poly(ethylene glycol) propionaldehyde. The concentration of ammonium sulfate (1 M) used was the same for all stationary phases. The results obtained demonstrated that the C4 A monolith could better resolve all protein PEGylation reaction mixtures, since the peaks of mono- and di-PEGylated proteins can be clearly distinguished in the chromatographic profiles. On the contrary, while using Butyl Sepharose media only the PEGylation reaction mixtures of RNase A could be partially separated at 35 and 45 CVs. PEGylated proteins of β-lactoglobulin and lysozyme could not be resolved when Toyopearl Butyl 650C and Butyl Sepharose were used. It is then clear that monoliths are an excellent choice to explore the purification process of PEGylated proteins exploiting the advantages of HIC. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:702-707, 2016.
authors.affiliation: Centro de Biotecnología-FEMSA, Escuela de Ingeniería y Ciencias, Tecnológico de Monterrey. Campus Monterrey, Monterrey, NL, 64849, México.
authors.forename: Karla
authors.initials: K
authors.lastname: Mayolo-Deloisa
authors.affiliation: Centro de Biotecnología-FEMSA, Escuela de Ingeniería y Ciencias, Tecnológico de Monterrey. Campus Monterrey, Monterrey, NL, 64849, México.
authors.forename: José
authors.initials: J
authors.lastname: González-Valdez
authors.affiliation: Centro de Biotecnología-FEMSA, Escuela de Ingeniería y Ciencias, Tecnológico de Monterrey. Campus Monterrey, Monterrey, NL, 64849, México.
authors.forename: Marco
authors.initials: M
authors.lastname: Rito-Palomares
mesh_terms: D002853:Chromatography, Liquid; D057927:Hydrophobic and Hydrophilic Interactions; D007782:Lactoglobulins / Q000737:chemistry* / Q000302:isolation & purification*; D009113:Muramidase / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D011092:Polyethylene Glycols / Q000737:chemistry*; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D007782:Lactoglobulins; D011092:Polyethylene Glycols; D012259:Ribonuclease, Pancreatic; D009113:Muramidase
keywords: Butyl Sepharose; CIM C4 A monolith; HIC; PEGylated proteins; Toyopearl Butyl 650C