Separation And Moderate-purity Purification Of Recombinant Human Epidermal Growth Factor (rhEGF) And Its Application In Cosmetics

The objectives of this thesis are: to design a process of purification of recombinant human epidermal growth factor(rhEGF) with low cost, easy operation and meeting the request of purity used in daily chemicals; to manufacture the cosmetics containing an active additive of rhEGF, and to evaluate the curative effect of cosmetics on the skin regeneration and senescence.The separation and purification process of rhEGF from fermentation broth including following steps: centrifuging fermentation broth to get supernatant containing rhEGF, salting-out of rhEGF from supernatant, desalting, separating rhEGF by ion exchange chromatography, desalting again, decoloring, and vacuum freeze-drying. The factors affecting separation and purificntion were evaluated rind the optimal operation conditions were obtained.Fermentation broth was centrifugated at 40癈, 6000 rpm, for 25 min, to obtain supernatant, which was salted-out by 361 g( NH4)2 SO4 solid/ L(60% saturated degree) under pH6.0 and low agitation speed(60 rpm) for 2 hours and standing for 4 hours. Then, centrifuged sedimentation was dissolved with PBS at pH7.4. Obtained rhFGF solution was dialyzed by dialysis bag, in which, the rhEGF solution was 2/3 in full and the ratio of rhEGF to water was 1:40, each dialysis lasted for 2 hours and the operation was repeated for 5 times. After dialysis, solution was ion-exchanged in a DEAE Sephadex?A50 column with a height of 10 cm( XK16/20). The column was operated with volume velocity of 150mL/h, volume injection of 75mL, and \\as eluted by PBS solution containing 0.5 mol/L NaCl.. The eluant contained 4.42-5.44 g/L of rhEGF and was decolored by a D314 resin column with the height of 7.5 cm( XK16/20), and operated at pH5.0. volume velocity of 60 mL/h. and volume injection of 120 mL. The collection of rhEGF (3.54-4.35 g rhEGF/L) was freeze-dried in Freezone?4.5 apparatus to obtain rhEGF powder with the purity of above 32%. The overall yield of rhEGF was above 53%.The experimental results indicate that the addition of 10%mannitol, 10%silk protein and 1%HA helps not only to reduce the loss of activity of rhEGF up to 50% (25 癈,after 30 days), but also to multiply the effect of beautying. The effective concentration of rhEGF is l00ug/mL with 1% HA and 1% silk protein. The statistic analysis showed that freezing-dried powder of rhEGF demonstrated good effect to cure acne, to reduce wrinkle and caducity, and the distinct difference was observed between with and without treatment of rhEGF( P<0.05-0.01). The optimizedABSTRACTconcentration of rhEGF in cosmetics was 200jig/mL, which should added when temperature of the neutral liquid or ointment was decreased to about 40 癈. The average ratio of effectiveness by using of cosmetics containing rhFGF was 84.9% after 30 days from the time of use.The freezing-dried powder of rhEGF with moderate-purity met the requirement of daily chemicals used in cosmetic industry. Cosmetics containing rhEGF. together with manntiol, HA and silk protein, had the anticipative effect of anti-acne, anti-wrinkle, and whitening.