Purpose : Recombinant human epidermal growth factor (rhEGF) is a 6045-Da peptide that promotes the cell growth process, and it is also used for cosmetic purposes as an anti-aging compound. However, its penetration into skin is limited by its large molecular size. This study aimed to prepare rhEGF-loaded transfersomal emulgel with enhanced skin penetration compared with that of non-transfersomal rhEGF emulgel. Methods: Three transfersome formulations were prepared with different ratios between the lipid vesicle (phospholipid and surfactant) and rhEGF (200:1, 133:1, and 100:1) using a thin-film hydration-extrusion method. The physicochemical properties of these transfersomes and the percutaneous delivery of the transfersomal emulgel were evaluated. Long-term and accelerated stability studies were also conducted. Results: The 200:1 ratio of lipid to drug was optimal for rhEGF-loaded transfersomes, which had a particle size of 128.1 ± 0.66 nm, polydispersity index of 0.109 ± 0.004, zeta potential of −43.1 ± 1.07 mV, deformability index of 1.254 ± 0.02, and entrapment efficiency of 97.77% ± 0.09%. Transmission electron microscopy revealed that the transfersomes had spherical and unilamellar vesicles. The skin penetration of rhEGF was enhanced by as much as 5.56 fold by transfersomal emulgel compared with that of non-transfersomal emulgel. The stability study illustrated that the rhEGF levels after 3 months were 84.96–105.73 and 54.45%–66.13% at storage conditions of 2°C–8°C and 25°C ± 2°C/RH 60% ± 5%, respectively. Conclusion: The emulgel preparation containing transfersomes enhanced rhEGF penetration into the skin, and skin penetration was improved by increasing the lipid content.
We immobilized urokinase (UK) by covalent attachment to activated Sepharose 6B-CL through multi-point amine coupling and evaluated its performance in cleaving a fusion protein, which consisted of recombinant human growth hormone (hGH) and a fragment of glutathione S-transferase that was linked by a tetrapeptide of a UK-specific recognition sequence. Packing densities of aldehyde groups on the activated agarose surface could be controlled in a gel range of 7-60 micromol/ml aldehyde by the amount of glycidol used. The immobilization yield was nearly 100% at pH 10.5, and the specific activity of the immobilized UK was equivalent to about 80% of soluble UK under the assay conditions. The immobilized UK showed an improvement in pH and thermal stability, probably due to the structural rigidity imparted by multi-point linkages to the matrix. The cleavage rate by the immobilized UK was lower than that of the soluble enzyme but the side reaction of cryptic cleavage was significantly decreased, which might suggest that the enzyme's specificity was altered by the immobilization. Cleavage yield in the column packed with immobilized UK was dependent on the feed rate, and the yield was approx. 80% of that of the soluble UK. The monomeric hGH could be obtained by selectively precipitating the uncleaved fusion protein and the GST fragments at an acidic pH.
Human epidermal growth factor (hEGF) secreted by recombinant Escherichia coli was purified from culture broth by expanded-bed adsorption (EBA) chromatography, strong anion-exchange chromatography and finally preparative reversed-phase HPLC (RP-HPLC). The EBA chromatography step simultaneously captured the hEGF by cationic exchanger and removed the cellular biomass from the diluted culture broth. This step was carried out at high throughput, and resulted in a high yield (>90%) and a purification factor of approx. 20-fold to >80% purity. Its process performance was well maintained during a 16-fold scale-up. After the successive purification steps of anion-exchange chromatography and RP-HPLC, the overall yield was approx. 84% and the purity was satisfactory (>99.5%). It was concluded that the purification process was very efficient and scaleable, warranting its implementation in large-scale manufacturing.
A fusion protein, consisting of a human epidermal growth factor (hEGF) as the recognition domain and human angiogenin as the toxin domain, can be used as a targeted therapeutic against breast cancer cells among others. The fusion protein was expressed as inclusion body in recombinant E. coli, and when the conventional, solution-phase refolding process was used the refolding yield was very low due to severe aggregation. It was probably because of the opposite electric charge at a neutral pH resulting from the vastly different pI values of each domain. The solidphase refolding process that exploited the ionic interactions between ionic exchanger surface and the fusion protein was tried, but the adsorption yield was also very low, below 30%, regardless of the resins and pH conditions used. Therefore, to provide a higher ionic affinity toward the solid matrix, six lysine residues were tagged to the N-terminus of the hEGF domain. When heparin-Sepharose was used as the matrix, the adsorption capacity increased 2.5-3 times to about 88%. Besides the intrinsic affinity of angiogenin to heparin, the poly-lysine tag provided additional ionic affinity. And the subsequent refolding yield increased nearly 13-fold, from ca. 4.8% in the conventional refolding of the untagged fusion protein to 63.6%. The process was highly reproducible. The refolded protein in the column eluate retained RNase bioactivity of angiogenin.
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