title: Separation of pegylated recombinant proteins and isoforms on CIM ion exchangers.
abstract: Protein pegylation is a process of covalent attachment of a polyethylene glycol (PEG) group to the protein tertiary structure that can "mask" the agent from the immune system and also increases the hydrodynamic size of the agent. Usually the pegylation prolongs the protein stability in the organism due to reduced renal clearance and provides superior water solubility to hydrophobic molecules. The mono-pegylated form of protein is usually prefered for medical applications. Different conditions with different PEG reagents have to be tested to find optimal pegylation procedure with specific protein. The goal of this study was to prepare screening method for separation of random mono-pegylated protein. Cytochrome C and beta lactoglobulin were pegylated with four reagents and a complete screening of several chromatographic monoliths in ion exchange mode with different buffers was performed to optimaly separate each mono-pegylated protein. The screening method was developed that produces optimal separation of target pegylated protein on CIM monoliths. Because of short chromatographic run time, CIM monoliths are perfect candidates to test alot of parameters. The results obtained show that each protein has its own unique separation parameters (pH, ionexchange ligand, buffer type). Two biopharmaceuticals were isolated using protocol: super human leptin antagonist (SHLA) was purified from inclusion bodies and mono-pegylated super mouse leptin antagonist (SMLA) from pegylated mixture. During study it was observed that the convective interaction media (CIM) monoliths additionally discriminate between protein isoforms pegylated on different sites in 3D structure of the protein.
authors.affiliation: BIA Separations d.o.o., Mirce 21, 5270 Ajdovščina, Slovenia. Electronic address: jernej.gaspersic@monoliths.com.
authors.forename: Jernej
authors.initials: J
authors.lastname: Gašperšič
authors.affiliation: FKKT, Večna Pot 113, 1000 Ljubljana, Slovenia.
authors.forename: Aleš
authors.initials: A
authors.lastname: Podgornik
authors.affiliation: BIA Separations d.o.o., Mirce 21, 5270 Ajdovščina, Slovenia.
authors.forename: Petra
authors.initials: P
authors.lastname: Kramberger
authors.affiliation: BIA Separations d.o.o., Mirce 21, 5270 Ajdovščina, Slovenia.
authors.forename: Marko
authors.initials: M
authors.lastname: Jarc
authors.affiliation: BIA Separations d.o.o., Mirce 21, 5270 Ajdovščina, Slovenia.
authors.forename: Janez
authors.initials: J
authors.lastname: Jančar
authors.affiliation: BIA Separations d.o.o., Mirce 21, 5270 Ajdovščina, Slovenia.
authors.forename: Mirjan
authors.initials: M
authors.lastname: Žorž
authors.affiliation: BIA Separations d.o.o., Mirce 21, 5270 Ajdovščina, Slovenia.
authors.forename: Nika Lendero
authors.initials: NL
authors.lastname: Krajnc
mesh_terms: D000818:Animals; D002852:Chromatography, Ion Exchange / Q000379:methods*; D057927:Hydrophobic and Hydrophilic Interactions; D051379:Mice; D011092:Polyethylene Glycols / Q000737:chemistry*; D020033:Protein Isoforms / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification*
chemical_list: D020033:Protein Isoforms; D011994:Recombinant Proteins; D011092:Polyethylene Glycols
keywords: Ion exchange; Monolith; Pegylating; Purification