title: High-resolution, preparative purification of PEGylated protein using a laterally-fed membrane chromatography device.
abstract: We discuss the use of a laterally-fed membrane chromatography (or LFMC) device for single-step purification of mono-PEGylated lysozyme. Recent studies have shown such LFMC devices to be suitable for high-resolution, multi-component separation of proteins in the bind-and-elute mode. The device used in this study contained a stack of rectangular cation-exchange membranes having 9.25mL bed volume. PEGylation of lysozyme was carried out in batch mode using 5kDa methoxy-polyethyleneglycol propionaldehyde (or m-PEG propionaldehyde) in the presence of sodium cyanoborohydride as reducing agent. Membrane chromatographic separation was carried out at 1.62 membrane bed volumes per minute flow rate, in the bind-and-elute mode. When a salt gradient was applied, the higher PEGylated forms of lysozyme (i.e. the byproducts) eluted earlier than mono-PEGylated lysozyme (the target product), while lysozyme eluted last. Under elution conditions optimized for resolution and speed, the separation could be carried out in less than 15 membrane bed volumes. High purity and recovery of mono-PEGylated lysozyme was obtained. The resolution of separation of mono-PEGylated lysozyme obtained under the above condition was comparable to that reported in the literature for equivalent cation-exchange resin columns while the flow rate expressed in bed volumes/min was 21.7 times higher. Also, the number of theoretical plates per meter was significantly higher with the LFMC device. Therefore the LFMC based purification process discussed in this paper combined high-productivity with high-resolution.
authors.affiliation: Department of Chemical Engineering, McMaster University, 1280 Main St W, Hamilton, Ontario, L8S 4L8, Canada.
authors.forename: Pedram
authors.initials: P
authors.lastname: Madadkar
authors.affiliation: Department of Chemical Engineering, McMaster University, 1280 Main St W, Hamilton, Ontario, L8S 4L8, Canada.
authors.forename: Sergio Luna
authors.initials: SL
authors.lastname: Nino
authors.affiliation: Department of Chemical Engineering, McMaster University, 1280 Main St W, Hamilton, Ontario, L8S 4L8, Canada. Electronic address: rghosh@mcmaster.ca.
authors.forename: Raja
authors.initials: R
authors.lastname: Ghosh
mesh_terms: D002845:Chromatography / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D011092:Polyethylene Glycols / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*
chemical_list: D011506:Proteins; D011092:Polyethylene Glycols
keywords: Device; Laterally-fed membrane chromatography; Membrane chromatography; PEGylated protein; Protein purification