Separation of PEGylated α-lactalbumin from unreacted precursors and byproducts using ultrafiltration

Jessica R. Molek, Andrew L. Zydney

Research output: Contribution to journalArticlepeer-review

38 Scopus citations


There is considerable clinical interest in the use of "second- generation" therapeutic proteins produced by conjugation of the native protein with various polymers including poly(ethylene glycol) (PEG). One of the challenges in the production of polymer-protein conjugates is the need to remove residual polymer, native (unreacted) protein, and any reaction byproducts from the final therapeutic formulation. The overall objective of this study was to evaluate the possibility of using ultrafiltration for the purification of a model PEGylated protein. Sieving data were obtained using PEGylated α-lactalbumin, the native protein, and the poly(ethylene glycol) over a range of pH, ionic strength, and filtrate flux using both neutral and charge-modified composite regenerated cellulose membranes. Purification of the PEGylated protein was achieved using a two-stage diafiltration process. The first stage used a neutral membrane to remove the unreacted protein and any small reaction byproducts while retaining the large PEGylated product. The second stage used a negatively charged membrane to remove the neutral poly(ethylene glycol) while retaining the PEGylated α-lactalbumin as a result of strong electrostatic interactions. These results clearly demonstrate the potential of using membrane-based separations for the purification of second-generation therapeutic proteins.

Original languageEnglish (US)
Pages (from-to)1417-1424
Number of pages8
JournalBiotechnology progress
Issue number6
StatePublished - Nov 2007

All Science Journal Classification (ASJC) codes

  • Biotechnology


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