Efficient Process Development for High-level Production, Purification, Formulation, and Characterization of Recombinant Mecasermin in E. coli.

Over-production recombinant Mecasermin was achieved by investigation effect of three factors, temperature, inducer amount and culture media in three levels according to Taguchi statistical design in E. coli in a bench-scale bioreactor. In optimal conditions (induction temperature 28°C, TB+G medium, with 0.1 mM IPTG as inducer) 0.84 g/L Mecasermin with expression levels of 38% of total protein and 4.13 g/L final dry cell biomass was produced, that is one of the highest values recombinant protein has been reported in the batch system. The cell disruption was done by lysozyme pretreatment with sonication to the efficient purification of Mecasermin. The isolated and washed inclusion bodies were solubilized in Gdn-HCl at pH 5.4 and folded with glutathione and purified with gel filtration. The purified rhIGF-1 (Mecasermin) was formulated with arginine. Mecasermin protein kept stability at 4°C for up to 2 years. The quantitative and qualitative control indicated that Mecasermin is expressed correctly (without the initial methionine by Mass spectrometry), pure (without endotoxin and other protein impurities), correct folding (FTIR, RF-HPLC), monomer form (SEC-HPLC) and active (bioactivity test). Also, the purification results revealed that expression in low temperature results the efficient purification of the over-produced Mecasermin with a high quantity and quality. This article is protected by copyright. All rights reserved.