Application Note: Application Solutions for Purifying and Analyzing PEGylated Proteins Using Various Chromatography Products.
18 November 2008

Three application notes for purifying and analyzing PEGylated proteins by reversed phase and gel filtration chromatography are available from Phenomenex®. The application notes feature Phenomenex’s market-leading Jupiter™ and BioSep™ HPLC product lines and provide detailed method development guidance.

PEGylation (protein modification using polyethylene glycol) of proteins has become a widespread method for improving the pharmacokinetic properties of protein and peptide therapeutics. Unfortunately, upon modification such protein-PEG conjugates are difficult to purify and analyze using traditional chromatography methods. The research presented in the application notes uses different PEG modifiers to look at the effect such modifiers had on the chromatography of several different proteins and peptides. Separation methods were optimized for various PEGylation reagents and proteins using Phenomenex’s HPLC protein purification products Jupiter 300 and BioSep.

Conditions for purifying PEGylated proteins from their unmodified protein counterparts using reversed phase chromatography featuring Phenomenex’s Jupiter 300 3µm C18 and Jupiter 300 5µm HPLC columns are presented in two different technical notes. Operating conditions were optimized using various mobile phases, temperatures, gradients, and chromatography phases; such conditions are discussed in detail for these technical notes and provide suggested starting conditions for developing reversed-phase purification methods for PEGylated-proteins. The third technical note discusses methods for purifying PEGylated proteins from unmodified proteins using gel filtration chromatography featuring Phenomenex’s BioSep 2000 HPLC column, and discusses the strengths and weaknesses of each separation method.