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

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ABSTRACT

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 units 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 was 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 K D 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 with native proteins and form soluble complexes with a nanomolar K D. 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.

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ACKNOWLEDGMENTS

The authors were supported in part by MIUR project CPDA085841 and BioKer srl (IT).

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Author notes

  1. Tetsuya Ishino

    Present address: Pfizer Global Research & Development, Saint Louis, Missouri, USA

Authors and Affiliations

  1. Department of Pharmaceutical Sciences, University of Padua, via F. Marzolo 5, 35131, Padua, Italy

    Anna Mero, Francesco M. Veronese & Gianfranco Pasut

  2. Drexel University College of Medicine, Philadelphia, Pennsylvania, USA

    Tetsuya Ishino & Irwin Chaiken

Authors
  1. Anna Mero
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  2. Tetsuya Ishino
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  3. Irwin Chaiken
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  4. Francesco M. Veronese
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  5. Gianfranco Pasut
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Correspondence to Gianfranco Pasut.

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Mero, A., Ishino, T., Chaiken, I. et al. Multivalent and Flexible PEG-Nitrilotriacetic Acid Derivatives for Non-covalent Protein Pegylation. Pharm Res 28, 2412–2421 (2011). https://doi.org/10.1007/s11095-011-0468-8

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  • DOI: https://doi.org/10.1007/s11095-011-0468-8

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