Ultrafiltration of PEGylated Proteins

Open Access
Author:
Molek, Jessica R.
Graduate Program:
Chemical Engineering
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
July 28, 2008
Committee Members:
  • Andrew Zydney, Committee Chair
  • Wayne Roger Curtis, Committee Member
  • Darrell Velegol, Committee Member
  • Erwin A Vogler, Committee Member
Keywords:
  • membrane transport
  • capillary electrophoresis
  • size exclusion chromatography
  • PEGylation
  • diafiltration
  • ultrafiltration
  • polyethylene glycol
  • alpha-lactalbumin
Abstract:
There is considerable clinical interest in the use of "second-generation" therapeutics produced by conjugation of a native protein with various polymers including polyethylene glycol (PEG). PEG–protein conjugates, so-called PEGylated proteins, can exhibit enhanced stability, half-life, and bioavailability. One of the challenges in the commercial production of PEGylated proteins is the purification required to remove unreacted polymer, native protein, and in many cases PEGylated proteins with non-optimal degrees of conjugation. The overall objective of this thesis was to examine the use of ultrafiltration for the purification of PEGylated proteins. This included: (1) analysis of size-based separation of PEGylated proteins using conventional ultrafiltration membranes, (2) use of electrically-charged membranes to exploit differences in electrostatic interactions, and (3) examination of the effects of PEGylation on protein fouling. The experimental results were analyzed using appropriate theoretical models, with the underlying physical properties of the PEGylated proteins evaluated using size exclusion chromatography, capillary electrophoresis, dynamic light scattering, and reverse phase chromatography. PEGylated proteins were produced by covalent attachment of activated PEG to a protein (alpha-lactalbumin, ovalbumin, or bovine serum albumin) via primary amines on the lysine residues. In contrast to the partition coefficient evaluated by size exclusion chromatography, the sieving coefficient of the PEGylated proteins in ultrafiltration depended upon both the number and size of the attached PEG chains due to the elongation or deformation of the PEG associated with the filtrate flux. Transmission of PEGylated proteins through charged membranes was dramatically reduced at low ionic strength due to strong electrostatic interactions, despite the presence of the neutral PEG. The results from small-scale ultrafiltration experiments were used to develop a two-stage diafiltration process to purify PEGylated alpha-lactalbumin. This process provided a purification factor greater than 1000 with respect to the unreacted protein and greater than 20-fold with respect to the PEG with an overall yield of PEGylated alpha-lactalbumin of 78%. These results provide the first demonstration of the potential of using ultrafiltration for the purification of protein-polymer conjugates.