Capillary Electrophoresis of PEGylated Proteins

Capillary electrophoresis has become a well-established technique for evaluating the composition and concentrations of a wide range of protein samples. In addition, capillary electrophoresis can be used to study a variety of protein properties including the net protein charge, the extent of ion binding, and the presence of specific biomolecular interactions. This wide range of applications is possible, at least in part, because of the strong theoretical foundation for describing the electrophoretic mobility of globular proteins. Smoluchowski's initial analysis of the electrophoretic mobility is valid for arbitrary particle shape under conditions where the Debye length (double layer thickness) is much smaller than the characteristic particle size. Subsequent studies have accounted for the finite size and relaxation of the electrical double layer, the effects of non-spherical particle geometry, and the influence of particle porosity. The resulting theoretical expressions for the electrophoretic mobility are in good agreement with experimental results. In contrast, much less is known about the electrophoretic mobility of more complex biomolecules. For example, there is considerable interest in the use of PEGylated therapeutic proteins, formed by the covalent attachment of one or more polyethylene glycol chains to the native protein. PEGylation can significantly increase the biological half-life by reducing the rate of kidney clearance, and it can also minimize adverse immunological reactions, dramatically improving the overall efficacy of the therapeutic protein. Fundamental studies of PEGylated proteins have clearly demonstrated that the attachment of the PEG chain(s) increases the effective protein size as determined by size exclusion chromatography 20 and membrane sieving. PEGylation also increases protein hydrophobicity, as determined by reverse phase chromatography, and it significantly reduces dynamic binding capacity in ion exchange chromatography , although no detailed models have been presented for either of these effects. PEGylated proteins do retain their electrical charge, with the resulting electrostatic interactions leading to a significant increase in retention during ultrafiltration through electrically-charged membranes with like polarity. In addition, there is evidence that the polyethylene glycol chains on PEGylated proteins can elongate under flow, reducing the effective protein size at high flow rates. Several investigators have used capillary electrophoresis for analysis of PEGylated protein samples. Semi-aqueous capillary electrophoresis has been used to resolve the different PEGylated forms of ribonuclease A, lysozyme, and recombinant TNF-α. Li et al. used capillary electrophoresis to measure the extent of aggregation of PEGylated ribonuclease A under different storage conditions. More recently, Na et al. used SDS capillary gel electrophoresis to check the purity of PEGylated αinterferon with both single and multiple PEG chains. In addition, Sharma and Carbeck used capillary electrophoresis to calculate the effective size of PEGylated lysozyme, implicitly assuming that the electrical charge on the native protein is displaced to the outer radius of a PEGylated sphere. The calculated values of this effective radius were in good agreement with results from partitioning in size exclusion chromatography, although the authors provided no physical basis for the use of this model. The objective of this study was to obtain quantitative data for the electrophoretic mobility and resolution of PEGylated α-lactalbumin using capillary electrophoresis. Data were also obtained with an acetylated form of α-lactalbumin, which was generated by covalent modification of the lysine amino groups but without any significant change in the protein molecular weight due to the small size of the