Effect of PEG molecular weight and PEGylation degree on the physical stability of PEGylated lysozyme.
During production, purification, formulation, and storage proteins for pharmaceutical or biotechnological applications face solution conditions that are unfavorable for their stability. Such harmful conditions include extreme pH changes, high ionic strengths or elevated temperatures. The characterization of the main influencing factors promoting undesired changes of protein conformation and aggregation, as well as the manipulation and selective control of protein stabilities are crucially important to biopharmaceutical research and process development. In this context PEGylation, i.e. the covalent attachment of polyethylene glycol (PEG) to proteins, represents a valuable strategy to improve the physico-chemical properties of proteins. In this work, the influence of PEG molecular weight and PEGylation degree on the physical stability of PEGylated lysozyme is investigated. Specifically, conformational and colloidal properties were studied by means of high-throughput melting point determination and automated generation of protein phase diagrams, respectively. Lysozyme from chicken egg-white as a model protein was randomly conjugated to 2kDa, 5kDa and 10kDa mPEG-aldehyde and resulting PEGamer species were purified by chromatographic separation. Besides protein stability assessment, residual enzyme activities were evaluated employing a Micrococcus lysodeikticus based activity assay. PEG molecules with lower molecular weights and lower PEGylation degrees resulted in higher residual activities. Changes in enzyme activities upon PEGylation have shown to result from a combination of steric hindrance and molecular flexibility. In contrast, higher PEG molecular weights and PEGylation degrees enhanced conformational and colloidal stability. By PEGylating lysozyme an increase of the protein solubility by more than 11-fold was achieved.
['Morgenstern|Josefine|J|', 'Baumann|Pascal|P|', 'Brunner|Carina|C|', 'Hubbuch|Jürgen|J|']
D002626:Chemistry, Pharmaceutical D003102:Colloids D008970:Molecular Weight D009113:Muramidase D011092:Polyethylene Glycols D011487:Protein Conformation D055550:Protein Stability D011506:Proteins D012995:Solubility
PEGylation Melting point determination Protein phase diagram Enzyme activity Molecular flexibility