QUANTITATIVE-ANALYSIS OF POLYETHYLENE-GLYCOL (PEG) IN PEG-MODIFIED PROTEINS CYTOKINES BY AQUEOUS 2-PHASE SYSTEMS/

Covalent attachment of poly(ethylene glycol) (PEG) to proteins produce s conjugates with altered/improved physicochemical and biological prop erties which depend upon the number of PEG chains linked. Quantificati on of the attached PEG is however not a trivial issue. The partition c oefficient, K, of the PEG-protein conjugate in PEG/dextran two-phase s ystems provides a quantitative measure for the degree of modification. A linear relationship between log K and the number of PEG chains was observed in fractionated PEG-modified-granulocyte-macrophage colony st imulating factor conjugates having 1 to 3 substitutions. Furthermore, in mixtures of PEG-bovine-serum-albumin conjugates with increasing deg rees of modification, a linear relationship was found between log K an d n, the average substitution. The increment in log K per PEG chain ad ded is protein specific and this suggests that the interactions betwee n the PEG-protein conjugate and the polymers in the phase system are m ore complex than just a simple affinity of the PEG for the PEG-rich to p phase. Increasing the polymer concentration in the phase system prod uces larger increments in log K per PEG molecule attached and the prop ortionality between log K and number of PEG molecules is only compromi sed for conjugates with high degree of substitution when partitioned i n biphasic systems of high concentration of polymers.