Site-specific conjugation of diethylenetriaminepentaacetic acid to recombinant human granulocyte-colony-stimulating factor: preservation of protein structure and function

Biochemistry
L D RalphD C Litzinger

Abstract

The chelating agent diethylenetriaminepentaacetic acid (DTPA) was conjugated site-specifically to the N-terminus of recombinant human granulocyte-colony-stimulating factor (rhG-CSF) by reaction of the protein with DTPA dianhydride at an initial pH of 6.0. The reaction was efficient in that 84% of the starting rhG-CSF was N-terminally modified and could be purified to homogeneity by cation-exchange chromatography. Chelation of 111In by the DTPA-rhG-CSF conjugate was demonstrated by cation-exchange HPLC and thin-layer chromatography. Metal contamination of conjugate preparations, as well as metal-loading onto the conjugate, could be monitored by either cation-exchange HPLC or isoelectric focusing. The 1:1 stoichiometric molar ratio of DTPA to protein for the DTPA-rhG-CSF conjugate was determined by thin-layer chromatography and mass spectrometry, and the localization of the conjugated DTPA moiety was resolved using a peptide mapping procedure. The secondary structure (i.e., alpha-helicity) of the protein was unmodified following conjugation as revealed by circular dichroism. Furthermore, the conjugate induced a similar induction of peripheral WBC counts as unmodified rhG-CSF when injected subcutaneously into hamsters, demonstrati...Continue Reading

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Citations

May 23, 2002·The Journal of Pharmacology and Experimental Therapeutics·T J McCarthyM J Welch
Jun 15, 1996·Analytical Chemistry·A L BurlingameS J Gaskell

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