Multivalent and Flexible PEG-Nitrilotriacetic Acid Derivatives for Non-covalent Protein Pegylation

Purpose. A new approach for non-covalent protein PEGylation is translated from immobilized metal ion affinity chromatography, and based on metal coordination bonds between a chelating agent linked to PEG, nitrilotriacetic acid (NTA), and the ring nitrogen of histidines in a protein. Methods. PEG-NTA conjugates were synthesized differing in the number of NTA unit and in the polymer structure. Three derivatives were investigated in association experiments with five model proteins. The most promising complex, PEG8-(NTA)8–Cu2+–G-CSF (granulocyte colony stimulating factor), was thoroughly characterized and the pharmacokinetic profile evaluated in rats. Results. The experiments demonstrated that only PEG8-(NTA)8, bearing eight NTA molecules on flexible PEG arms, associated strongly with those proteins having several histidines. The protein secondary structure was not affected in the complex.. PEG8-(NTA)8–Cu2+–G-CSF showed a KD of 4.7 nM, as determined by surface plasmon resonance, but the association was not stable in vivo. Conclusions. PEG8-(NTA)8 is the first derivative able to associate native proteins and forming soluble complexes with a nanomolar KD. The study highlights the need of a multivalent and flexible coordination and encourages further investigations to increase the stability of PEG8-(NTA)8 complexes in vivo either through the use of protein mutants or His-tag proteins.