Abstract

The biochemical and biophysical characterization of recombinant protein is required, when they are developed for human clinical use. A number of techniques can be used to determine the biophysical properties of protein and to examine their biochemical and biological integrity. The results of these experiments are compared with those obtained using naturally occurring proteins in order to be confident that the recombinant protein has the desired characteristics of the naturally occurring one. In this study, the purified protein was characterized by using Neupogen® and PDgrastim as reference standards. This research investigates the characterization of final product of rh-GCSF as characterization analysis: Bacterial endotoxin test, CD measurement, Disulfide bond analysis, Analysis of monomer and aggregates form of rh-GCSF. Also purity was measured by SDS-PAGE, Western blotting and quantified by Bradford . An efficient, scalable and cost-effective procedure for production and purification of rh-GCSF in E. coli were used. The quantitative analysis shows that the purified protein yield was 400 mg from 1 g of cell dry mass (40%) by Bradford, SDS-PAGE (gel densitometry) and Western blotting and the purity was more than 99%. According to the inspection chromatogram, obtained peak conforms to molecular weight of rh-GCSF. Disulfide bonds are in correct position, rh-GCSF and reference standard chromatograms overlap with each other. The obtained results approved that the rh-GCSF protein isolated in this study was highly pure and comparable with the innovator products, Neupogen® and PD grastim. Based on the above results, the product has been found to be adequate for preclinical studies.