title: Preferential Interactions and the Effect of Protein PEGylation.
abstract: BACKGROUND
  PEGylation is a strategy used by the pharmaceutical industry to prolong systemic circulation of protein drugs, whereas formulation excipients are used for stabilization of proteins during storage. Here we investigate the role of PEGylation in protein stabilization by formulation excipients that preferentially interact with the protein.
  
  
  RESULTS
  The model protein hen egg white lysozyme was doubly PEGylated on two lysines with 5 kDa linear PEGs (mPEG-succinimidyl valerate, MW 5000) and studied in the absence and presence of preferentially excluded sucrose and preferentially bound guanine hydrochloride. Structural characterization by far- and near-UV circular dichroism spectroscopy was supplemented by investigation of protein thermal stability with the use of differential scanning calorimetry, far and near-UV circular dichroism and fluorescence spectroscopy. It was found that PEGylated lysozyme was stabilized by the preferentially excluded excipient and destabilized by the preferentially bound excipient in a similar manner as lysozyme. However, compared to lysozyme in all cases the melting transition was lower by up to a few degrees and the calorimetric melting enthalpy was decreased to half the value for PEGylated lysozyme. The ratio between calorimetric and van't Hoff enthalpy suggests that our PEGylated lysozyme is a dimer.
  
  
  CONCLUSIONS
  The PEGylated model protein displayed similar stability responses to the addition of preferentially active excipients. This suggests that formulation principles using preferentially interacting excipients are similar for PEGylated and non-PEGylated proteins.
authors.affiliation: Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Louise Stenstrup
authors.initials: LS
authors.lastname: Holm
authors.affiliation: Department of Chemistry, Faculty of Science, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Peter W
authors.initials: PW
authors.lastname: Thulstrup
authors.affiliation: Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Marina R
authors.initials: MR
authors.lastname: Kasimova
authors.affiliation: Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Marco
authors.initials: M
authors.lastname: van de Weert
mesh_terms: D000818:Animals; D002626:Chemistry, Pharmaceutical / Q000379:methods; D002645:Chickens; D005079:Excipients / Q000737:chemistry*; D009113:Muramidase / Q000737:chemistry; D011108:Polymers / Q000737:chemistry; D055550:Protein Stability; D011506:Proteins / Q000737:chemistry*; D013816:Thermodynamics
chemical_list: D005079:Excipients; D011108:Polymers; D011506:Proteins; C421864:hen egg lysozyme; D009113:Muramidase