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Keywords:

  • Binding site;
  • Ion-exchange chromatography;
  • Mono-PEGylation;
  • PEG chain length;
  • PEGylation

Abstract

The mechanisms behind protein PEGylation are complex and dictated by the structure of the protein reactant. Hence, it is difficult to design a reaction process which can produce the desired PEGylated form at high yield. Likewise, efficient purification processes following protein PEGylation must be constructed on an ad hoc basis for each product. The retention and binding mechanisms driving electrostatic interaction-based chromatography (ion-exchange chromatography) of PEGylated proteins (randomly PEGylated lysozyme and mono-PEGylated bovine serum albumin) were investigated, based on our previously developed model Chem. Eng. Technol. 2005, 28, 1387–1393. PEGylation of each protein resulted in a shift to a smaller elution volume compared to the unmodified molecule, but did not affect the number of binding sites appreciably. The shift of the retention volume of PEGylated proteins correlated with the calculated thickness of PEG layer around the protein molecule. Random PEGylation was carried out on a column (solid-phase PEGylation) and the PEGylated proteins were separated on the same column. Solid-phase PEGylation inhibited the production of multi-PEGylated forms and resulted in a relatively low yield of selective mono-PEGylated form. Pore diffusion may play an important role in solid-phase PEGylation. These results suggest the possibility of a reaction and purification process development based on the mechanistic model for PEGylated proteins on ion exchange chromatography.