Pharmaceutical significance of the cyclic imide form of recombinant human glial cell line derived neurotrophic factor.

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.

Formation of an active dimer during storage of interleukin-1 receptor antagonist in aqueous solution.

The degradation products of recombinant human interleukin-1 receptor antagonist (rhIL-1ra) formed during storage at 30 degrees C in aqueous solution were characterized. Cationic exchange chromatography of the stored sample showed two major, new peaks eluting before (P1) and after (L2) the native protein, which were interconvertible. Size-exclusion chromatography and electrophoresis documented that both the P1 and L2 fractions were irreversible dimers, formed by noncovalent interactions. A competition assay with interleukin-1 indicated that on a per monomer basis the P1 and L2 dimers retained about two-thirds of the activity of the native monomer. Infrared and far-UV circular dichroism spectroscopies showed that only minor alterations in secondary structure arose upon the formation of the P1 dimer. However, alteration in the near-UV circular dichroism spectrum suggested the presence of disulfide bonds in the P1 dimer, which are absent in the native protein. Mass spectroscopy and tryptic mapping, before and after carboxymethylation, demonstrated that the P1 dimer contained an intramolecular disulfide bond between Cys-66 and Cys-69. Although conversion of native protein to the P1 dimer was irreversible in buffer alone, the native monomer could be regained by denaturing the P1 dimer with guanidine hydrochloride and renaturing it by dialysis, suggesting that the intramolecular disulfide bond does not interfere with refolding. Analysis of the time course of P1 formation during storage at 30 degrees C indicated that the process followed first-order, and not second-order, kinetics, suggesting that the rate-limiting step was not dimerization. It is proposed that a conformational change in the monomer is the rate-limiting step in the formation of the P1 dimer degradation product. Sucrose stabilized the native monomer against this process. This result can be explained by the general stabilization mechanism for this additive, which is due to its preferential exclusion from the protein surface.

Genetic and biochemical characterization of the thymidine kinase gene from herpesvirus of turkeys.

The thymidine kinase gene encoded by herpesvirus of turkeys has been identified and characterized. A viral mutant (ATR0) resistant to 1-beta-D-arabinofuranosylthymine was isolated. This mutant was also resistant to 1-(2-fluoro-2-deoxy-beta-D-arabinofuronosyl)-5-methyluracil and was unable to incorporate [125I]deoxycytidine into DNA. The mutant phenotype was rescued by a cloned region of the turkey herpesvirus genome whose DNA sequence was found to contain an open reading frame similar to that for known thymidine kinases from other viruses. When expressed in Escherichia coli, this open reading frame complemented a thymidine kinase-deficient strain and resulted in thymidine kinase activity in extracts assayed in vitro.