Spectrophotometric analysis for determining the average number of poly(ethylene) glycol molecules on PEGylated proteins utilizing a protein digestion step

Academic Article

Abstract

  • The use of PEG to modify proteins has produced a need for quick and simple yet sensitive methods for the determination of the average number of PEG molecules attached to a protein. Existing methods for the determination of the average number of PEGs bound to a protein are limited to proteins with small numbers of available lysines, high degrees of PEGylation often exceeding 10-15% modification of available lysines (TNBS and fluorescamine) or small proteins (<100 kDa) with small degrees of PEGylation, <5 PEG/protein (CE). The inclusion of a pronase digestion step to the biphasic assay provides a simple and sensitive method for the determination of the average number of PEGs bound to a protein regardless of the protein size or the degree of PEGylation, based on PEG partitioning in a two-phase system. Additionally the assay is straightforward, allowing a large number of samples to be measured within a short period with inexpensive standard laboratory equipment.
  • Authors

    Published In

    Digital Object Identifier (doi)

    Author List

  • Barker TH; Klinger MM; Feldman DS; Fuller GM; Hagood JS
  • Start Page

  • 382
  • End Page

  • 385
  • Volume

  • 290
  • Issue

  • 2