Prediction of the Viscosity Radius and the Size Exclusion Chromatography Behavior of PEGylated Proteins

Abstract

Size exclusion chromatography (SEC) was used to determine the viscosity radii of equivalent spheres for proteins covalently grafted with poly(ethylene glycol) (PEG). The viscosity radius of such PEGylated proteins was found to depend on the molecular weight of the native protein and the total weight of grafted PEG but not on PEG molecular weight, or PEG-toprotein molar grafting ratio. Results suggest grafted PEG's form a dynamic layer over the surface of proteins. The geometry of this layer results in a surface area to volume ratio approximately equal to that of a randomly coiled PEG molecule of equivalent total molecular weight. Two simple methods are given to predict the viscosity radius of PEGylated proteins.Both methods accurately predicted (3% absolute error) the viscosity radii of various PEGproteins produced using three native proteins, α-lactalbumin (14.2 kDa MW), β−lactoglobulin dimer (37.4 kDa MW) and bovine serum albumin (66.7 kDa MW), three PEG reagents (2400, 5600, and 22500 MW), and molar grafting ratios of 0 to 8. Accurate viscosity radius prediction allows calculation of the distribution coefficient, K av , for PEGproteins in SEC. The suitability of a given SEC step for the analytical or preparative fractionation of different PEGylated protein mixtures may therefore be assessed mathematically. The methods and results offer insight to several factors related to the production, purification, and uses of PEGylated proteins.

Interaction mechanism of mono‐PEGylated proteins in electrostatic interaction chromatography

“…In this study we focused on electrostatic interaction-based separation, i.e., IEC.The elution order is basically similar to that observed by Seely and Richey [6] and others [9,[11][12][13].…”

“…From the GH-I R curve the number of binding sites (charges) involved in electrostatic interaction, B value, was determined according to the following equation [8][9][10]. A is a parameter that includes the equilibrium coefficient, the binding site and the ion-exchange capacity [8][9][10].…”

“…PEGylated proteins can be separated by various types of chromatography, e.g., size exclusion chromatography (SEC), hydrophobic interaction chromatography and electrostatic interaction chromatography (ion-exchange chromatography, IEC) [5][6][7][8][9][10][11][12][13]. However, the separation mechanism is still not fully understood.…”

“…Due to the attached PEG molecule, PEGylated proteins behave like much larger molecules than the native protein as the hydrodynamic size increases [5,8,9]. IEC is usually recommended for PEGylated protein separations [5,7].…”

High affinity soluble ILT2 receptor: a potent inhibitor of CD8+ T cell activation

“…The choice of bis-maleimide substituted PEGs (Roberts et al, 2002) provided the advantage of producing uniform, chemically defined ILT2 dimers, with only a minor contamination by PEGylated ILT2 monomer. Due to the non-structured nature of PEG, the gain in the protein molecule's apparent size (diffusion radius) exceeds that calculated directly from the increase in molecular mass (Fee and Van Alstine, 2004). The effect could easily be observed during size-exclusion chromatography where the ILT2 dimer made with 3.4 kDa PEG (combined MW 48.4 kDa) behaved similarly to a much larger protein of approximately 80 kDa (data not shown).…”