Quantitative analysis of polyethylene glycol (PEG) in PEG-modified proteins/cytokines by aqueous two-phase systems.
Covalent attachment of poly(ethylene glycol) (PEG) to proteins produces conjugates with altered/improved physicochemical and biological properties which depend upon the number of PEG chains linked. Quantification of the attached PEG is however not a trivial issue. The partition coefficient, K, of the PEG-protein conjugate in PEG/dextran two-phase systems 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 stimulating factor conjugates having 1 to 3 substitutions. Furthermore, in mixtures of PEG-bovine-serum-albumin conjugates with increasing degrees of modification, a linear relationship was found between log K and n, the average substitution. The increment in log K per PEG chain added is protein specific and this suggests that the interactions between the PEG-protein conjugate and the polymers in the phase system are more complex than just a simple affinity of the PEG for the PEG-rich top phase. Increasing the polymer concentration in the phase system produces larger increments in log K per PEG molecule attached and the proportionality between log K and number of PEG molecules is only compromised for conjugates with high degree of substitution when partitioned in biphasic systems of high concentration of polymers.
Delgado
C
C
Molecular Cell Pathology Laboratory, Royal Free Hospital School of Medicine, London, UK.
Malik
F
F
Selisko
B
B
Fisher
D
D
Francis
G E
GE
D055598:Chemical Phenomena; D002627:Chemistry, Physical; D016207:Cytokines / Q000032:analysis / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005240:Feasibility Studies; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000032:analysis* / Q000737:chemistry; D007457:Iodine Radioisotopes; D007700:Kinetics; D011092:Polyethylene Glycols / Q000032:analysis* / Q000737:chemistry; D012710:Serum Albumin, Bovine / Q000032:analysis* / Q000737:chemistry; D014867:Water / Q000737:chemistry
D016207:Cytokines; D007457:Iodine Radioisotopes; D014867:Water; D012710:Serum Albumin, Bovine; D011092:Polyethylene Glycols; D016178:Granulocyte-Macrophage Colony-Stimulating Factor