RESUMEN
PURPOSE: The purpose of this paper is to determine the significance of cyclic imide formation of an aspartic acid residue during storage on the pharmaceutical quality of a recombinant human glial cell line-derived neurotrophic factor (rhGDNF) formulation. METHODS: A combination of chromatography, peptide mapping, mass spectroscopy, and protein sequencing was used to purify and characterize the degradation product. Circular dichroism, 1,8-ANS and heparin binding, melting temperature determination, bioassays, and preclinical pharmacokinetic and toxicology testing were performed to examine its equivalence to native rhGDNF. RESULTS: The rhGDNF with cyclic imide at aspartic acid residue 96 showed identical activity, structure, pharmacokinetic profile, and toxicity profile to the native rhGDNF. CONCLUSIONS: Formation of cyclic imide at aspartic acid residue 96 does not affect the pharmaceutical quality of the rhGDNF formulation.
Asunto(s)
Proteínas de Drosophila , Imidas/química , Factores de Crecimiento Nervioso , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/farmacología , Animales , Ácido Aspártico/química , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Ciclización , Estabilidad de Medicamentos , Almacenaje de Medicamentos , Ganglios Simpáticos/citología , Ganglios Simpáticos/efectos de los fármacos , Factor Neurotrófico Derivado de la Línea Celular Glial , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial , Heparina/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Ratones , Mitógenos/farmacología , Proteínas del Tejido Nervioso/farmacocinética , Proteínas Proto-Oncogénicas/efectos de los fármacos , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-ret , Ratas , Proteínas Tirosina Quinasas Receptoras/efectos de los fármacos , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacología , Sustancia Negra/citología , Sustancia Negra/efectos de los fármacosRESUMEN
Human glial cell line-derived neurotrophic factor is a single polypeptide of 134 amino acids and functions as a disulfide-linked dimer. Incubation of the protein in pH 5.0 and at 37 degreesC for 1 week showed that 5% of the material was converted to a form that eluted after the major protein peak on a cation-exchange column. The modified component gave an average molecular mass of 30367.0 u (theoretical = 30384.8 u). Within measurement error, this 17.8-u decrease in mass indicated the loss of a water molecule. This observation, together with the protein's behavior on cation-exchange chromatography and the mode of incubation used to generate the modification, was consistent with cyclic imide (succinimide) formation at an aspartyl residue. Hence, only a monomer of the dimeric protein was modified. The modified monomer was purified and subjected to peptic degradation. By a combination of N-terminal analysis and mass spectrometry, the region containing Asp95-Lys96 was identified to be modified. This was further confirmed by carboxypeptidase Y digestion of the modified peptide where the modified region was found to be resistant to further enzymatic degradation. Furthermore, incubation of the modified monomer in pH 8. 5 for 2 h yielded two peaks, in agreement with the succinimide model where the cyclic imide was hydrolyzed into a mixture of isoaspartate and aspartate. Tryptic mapping of the isoaspartyl-containing protein showed that Asp95 was refractory to Edman degradation, confirming it was in the isoaspartate form. Hence, the modification observed was due to succinimide formation at Asp95. This is the first report of succinimide formation at an Asp-Lys linkage.