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Keywords:

  • RNase A;
  • α-Lactalbumin;
  • PEGylation;
  • aqueous two-phase systems

Abstract

BACKGROUND: PEGylation reactions often result in a heterogeneous population of conjugated species and unmodified proteins that presents a protein separations challenge. Aqueous two-phase systems (ATPS) are an attractive alternative for the potential fractionation of native proteins from their PEGylated conjugates. The present study characterizes the partition behaviors of native RNase A and α-Lac and their mono and di-PEGylated conjugates on polyethylene glycol (PEG)—potassium phosphate ATPS.

RESULTS: A potential strategy to separate unreacted native protein from its PEGylated species was established based upon the partition behavior of the species. The effect of PEG molecular weight (400–8000 g mol−1), tie-line length (15–45% w/w) and volume ratio (VR; 0.33, 1.00 and 3.00) on native and PEGylated proteins partition behavior was studied. The use of ATPS constructed with high PEG molecular weight (8000 g mol−1), tie-line lengths of 25 and 35% w/w, and VR values of 1.0 and 3.0 allowed the selective fractionation of native RNase A and α-Lactalbumin, respectively, from their PEGylated conjugates on opposite phases. Such conditions resulted in an RNase A bottom phase recovery of 99%, while 98% and 88% of mono and di-PEGylated conjugates, respectively were recovered at the top phase. For its part, α-Lac had a bottom phase recovery of 92% while its mono and di-PEGylated conjugates were recovered at the top phase with yields of 77% and 76%, respectively.

CONCLUSIONS: The results reported here demonstrate the potential application of ATPS for the fractionation of PEGylated conjugates from their unreacted precursors. Copyright © 2010 Society of Chemical Industry