Kinevac stability after reconstitution with sodium chloride injection USP, 0.9%.

UNLABELLED: Our objective was to compare the stability of Kinevac when reconstituted with sodium chloride injection, USP, 0.9%, versus the manufacturer's recommended sterile water for injection, USP, and to determine the effects on stability of deviating from the manufacturer's recommended methods of product preparation. METHODS: Kinevac was reconstituted with either sterile water or 0.9% sodium chloride. Triplicate high-performance liquid chromatography was performed on each vial of reconstituted sample at time zero and at time zero plus 8 h. The concentration of each sample, as measured by the peak area, was recorded at each time point. The process was repeated over 4 consecutive days. RESULTS: Kinevac reconstituted with sterile water resulted in the recovery of 89.73% of the time zero concentration after 8 h. Kinevac reconstituted with 0.9% sodium chloride resulted in chemical stability of the injection, with 80.05% recovery of the time zero value after 8 h. CONCLUSION: Kinevac is more stable when reconstituted with sterile water than when reconstituted with 0.9% sodium chloride. Kinevac should be reconstituted with sterile water for injection as per the manufacturer's instructions.

Defining the role of arginine 96 in green fluorescent protein fluorophore biosynthesis.

Aequoria victoria green fluorescent protein (GFP) is a revolutionary molecular biology tool because of its spontaneous peptide backbone cyclization and chromophore formation from residues Ser65, Tyr66, and Gly67. Here we use structure-based design, comprehensive targeted mutagenesis, and high-resolution crystallography to probe the significant functional role of conserved Arg96 (R96) in chromophore maturation. The R96M GFP variant, in which the R96M side chain is similar in volume but lacks the R96 positive charge, exhibits dramatically slower chromophore maturation kinetics (from hours to months). Comparison of the precyclized conformation of the chromophore-forming residues with the mature R96M chromophore reveals a similar Y66 conformer, contrary to the large Y66 conformational change previously defined in the slowly maturing R96A variant [Barondeau, D. P., Putnam, C. D., Kassmann, C. J., Tainer, J. A., and Getzoff, E. D. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 12111-12116]. Comprehensive R96 mutagenesis and fluorescent colony screening indicate that only the R96K substitution restores wild-type maturation kinetics. Further, we show that the slowly maturing R96A variant can be complemented with a Q183R second-site mutation designed to restore the missing R96 positive charge and rapid fluorophore biosynthesis. Moreover, comparative structural analysis of R96M, R96K, R96A/Q183R, and wild-type GFP reveals the importance of the presence of positive charge, rather than its exact position. Together, these structural, mutational, and biochemical results establish a pivotal role for the R96 positive charge in accelerating the GFP post-translational modification, with implications for peptide backbone cyclization in GFP, its homologues, and related biological systems.