title: Site-specific conjugation of diethylenetriaminepentaacetic acid to recombinant human granulocyte-colony-stimulating factor: preservation of protein structure and function.
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, demonstrating preservation of protein bioactivity. These results reveal a simple and efficient method for conjugating DTPA to protein, via reaction with the dianhydride, to yield a homogeneous and well-defined product. The procedure may prove to be a useful method of labeling growth factors and related proteins while preserving structural and functional integrity.
authors.affiliation: Department of Pharmaceutics and Drug Delivery, Amgen, Inc., Thousand Oaks, California 91320, USA.
authors.forename: L D
authors.initials: LD
authors.lastname: Ralph
authors.forename: C M
authors.initials: CM
authors.lastname: Ransone
authors.forename: M
authors.initials: M
authors.lastname: Treuheit
authors.forename: S
authors.initials: S
authors.lastname: Lauren
authors.forename: V
authors.initials: V
authors.lastname: Katta
authors.forename: D C
authors.initials: DC
authors.lastname: Litzinger
mesh_terms: D000818:Animals; D001665:Binding Sites; D002942:Circular Dichroism; D006224:Cricetinae; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D004369:Pentetic Acid / Q000378:metabolism*; D010449:Peptide Mapping; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor; D004369:Pentetic Acid