title: Study on the thermal stability of green fluorescent protein (GFP) in glucose parenteral formulations.
abstract: Large volume parenteral solutions (LVPS) that are widely used in the healthcare system must be processed by moist-heat treatment to an assured sterility level in which the efficacy is measured by a bioindicator (BI) that provides fast, accurate and reliable results. This study evaluated the thermal stability of green fluorescent protein (GFP) into glucose-based LVPS (1.5-50%) solutions to determine its utility as a BI for thermal processes. GFP, expressed by Escherichia coli, isolated/purified by TPP/HIC, was diluted in buffered (each 10mM: Tris-EDTA, pH 8; phosphate, pH 6 and 7; acetate, pH 5) and in water for injection (WFI; pH 6.70+/-0.40) glucose solutions (1.5-50%) and exposed to constant temperatures from 80 degrees C to 95 degrees C. The thermal stability was expressed in decimal reduction time (D-value, time required to reduce 90% of the GFP fluorescence intensity). At 95 degrees C, the D-values for GFP in 1.5-50% glucose were: (i) 1.63+/-0.23 min (pH 5); (ii) 2.64+/-0.26 min (WFI); (iii) 2.50+/-0.18 min (pH 6); (iv) 3.24+/-0.28 min (pH 7); (v) 2.89+/-0.44 min (pH 8). By the convenient measure of fluorescence intensity and its thermal stability, GFP has the potential as a BI to assay the efficacy of moist-heat processing of LVPS at temperatures < or =100 degrees C.
authors.affiliation: Department of Pharmaceutical Technology, School of Pharmaceutical Science, University of São Paulo, Av. Prof. Lineu Prestes, 580 Bloco 16, Ed. Semi Industrial, Butantã, 05508-900 São Paulo, SP, Brazil. marishii@usp.br
authors.forename: Marina
authors.initials: M
authors.lastname: Ishii
authors.forename: Juliana S
authors.initials: JS
authors.lastname: Kunimura
authors.forename: Thereza Christina Vessoni
authors.initials: TC
authors.lastname: Penna
authors.forename: Olivia
authors.initials: O
authors.lastname: Cholewa
mesh_terms: D002021:Buffers; D003001:Cloning, Molecular; D004340:Drug Contamination / Q000517:prevention & control*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005947:Glucose / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007263:Infusions, Parenteral; D007700:Kinetics; D049408:Luminescent Agents / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D019999:Pharmaceutical Solutions; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013242:Sterilization / Q000379:methods*
chemical_list: D002021:Buffers; D049408:Luminescent Agents; D019999:Pharmaceutical Solutions; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D005947:Glucose