Semi - Preparative Purifi cation and Characterization of Lysozyme Modifi ed with Poly Ethylene Glycol ( PEG ) The Agilent 1260
نویسنده
چکیده
This Application Note used hen egg white lysozyme as a model protein for purifi cation and biophysical characterization of PEGylated proteins. PEGylation to the N-terminus of lysozyme in a site-specifi c manner was achieved using PEG-propionaldehyde derivatives with a molecular weight of Mw 20,000. A semi-preparative RP-HPLC method for the purifi cation of PEGylated lysozyme was developed using an Agilent 1260 Infi nity Bio-inert LC System and an Agilent ZORBAX Semi-Preparative 300 SB-C18 column. An analytical-scale fraction collector with peak-based fraction collection was used to collect the purifi ed PEG lysozyme. The PEG lysozyme was characterized for its purity using the Agilent 2100 Bio-analyzer, intact mass was determined using an Agilent 1290 Infi nity LC System coupled to an Agilent 6530 Accurate-Mass Q-TOF LC/MS. The biological activity of lysozyme and its conjugate were determined by a turbidometric method using an Agilent Cary 60 spectrophotometer and Micrococcus lysodecticus cells as substrate. Semi-Preparative Purifi cation and Characterization of Lysozyme Modifi ed with Poly Ethylene Glycol (PEG) The Agilent 1260 Infi nity Bio-Inert Quaternary LC System, Agilent 2100 BioAnalyzer, Agilent 6530 Accurate-Mass Q-TOF LC/MS and Agilent Cary 60 UV-Vis
منابع مشابه
Preparative Chromatography From Analytical Scouting to Fraction Reanalysis
The purifi cation of a valuable product or product library after a long and complex synthesis is a very important process. The Agilent Automated Purifi cation Software introduces an Open-Access-based solution for isolating substances, with automated focused gradient to simplify the preand post-purifi cation steps. This Technical Overview describes the easy steps to follow, from crude sample ana...
متن کاملDocetaxel-Loaded Mixed Micelles and Polymersomes Composed of Poly (caprolactone)-Poly (ethylene glycol) (PEG-PCL) and Poly (lactic acid)-Poly (ethylene glycol) (PEG-PLA): Preparation and In-Vitro Characterization
Microwave irradiation was used to synthesize PEG-PCL and PEG-PLA copolymers that are composed of biodegradable polymers including PEG, PLA, and PCL. These copolymers were used for loading docetaxel in nanoparticles. Single emulsion-solvent evaporation technique was applied for preparing the PEG-PLA and PEG-PCL mixed nanoparticles (micelles and polymersomes) with different proportions, including...
متن کاملDrug Metabolite Purifi
Drug metabolism and pharmacokinetics (DMPK) studies require the identifi cation of drug metabolites usually by mass spectrometry (MS), and in later stages of drug development, confi rmation of metabolite structures by NMR. To achieve this, purifi cation of metabolites may be required. In this Application Note, purifi cation of bupropion drug metabolites was achieved using Agilent Automated Puri...
متن کاملDocetaxel-Loaded Mixed Micelles and Polymersomes Composed of Poly (caprolactone)-Poly (ethylene glycol) (PEG-PCL) and Poly (lactic acid)-Poly (ethylene glycol) (PEG-PLA): Preparation and In-Vitro Characterization
Microwave irradiation was used to synthesize PEG-PCL and PEG-PLA copolymers that are composed of biodegradable polymers including PEG, PLA, and PCL. These copolymers were used for loading docetaxel in nanoparticles. Single emulsion-solvent evaporation technique was applied for preparing the PEG-PLA and PEG-PCL mixed nanoparticles (micelles and polymersomes) with different proportions, including...
متن کاملEffect of different mass ratio of PLA: PEG segments in PLA-PEG-PLA copolymers on the physicochemical characterization and DNA release profile
Background: Adapting controlled release technologies to the delivery of DNA has the great potential to overcome extracellular barriers that limit gene delivery. This study investigates the effect of different mass ratio of PLA: PEG in the various tri block poly (lactic acid)-poly (ethylene glycol) - Poly (lactic acid) copolymer (PLA-PEG-PLA) on the properties of the resulting nanoparticles. Me...
متن کامل