Study of pH-Dependent Kinetics of Filgrastim Pegylation with Succinimide Derivatives of Poly(Ethylene Glycol) and Comparison of the Properties of Pegylated Forms Obtained Under Various Conditions

Abstract

Filgrastim was pegylated by acylation with a linear 20-kDa pegylating agent (PEG agent) to search for conjugates characterized by biological activity comparable to or exceeding that of commercial drugs. The pegylation kinetics were studied at different pH values taking into account the contribution of the hydrolysis kinetics of the PEG agent. The optimal time for pegylation to a required protein conversion and the method of adding the PEG agent to the reaction mixture (once or fractionally) at a given pH value and ratio between the PEG agent and protein were calculated using a proposed equation based on previously obtained pegylation and hydrolysis rate constants. Various mixtures of pegylated positional isomers were obtained. Their contents in the mixtures depended on the pH value because of the different pK_a values of the amino-acid residues involved in the reaction. The estimated specific biological activity of the obtained monopegylated forms of Filgrastim appeared comparable with that of the product Neulastim.