ABSTRACT
The dynamic binding capacity [DBC] of a chromatography matrix in protein purification is the amount of the total protein absorbed into the matrix, before occurrence of a significant break in the breakthrough curve. Optimization of the process criteria for maximum DBC avoids extra process scale-up and reduces the processing time, costs and protein loss. Taguchi method is a simple useful tool in experimental design to estimate the optimal condition with minimum experiments. In this research, linear flow rate, pH and protein concentration of the feed were checked according to an L9 orthogonal Taguchi array, to estimate the best conditions for maximum DBC of Q-sepharose fast flow [QSFF] resin in recombinant human erythropoietin purification process. A crud sample containing human recombinant erythropoietin was harvested from a cell culture of Chinese hamster ovary [CHO] cell line. Desalted harvests with different total protein concentrations [30, 40 and 50 microg.mL[-1]] and pH values [5, 6 and 7] were loaded into a packed column of QSFFwith different linear flow rates [60, 120 and 280 cm.h[-1]] up to 10% of the breakthrough curve. The total protein loading to the column was checked by UV absorbance and Lowry method, and erythropoietin concentration was measured by ELISA. Analysis of variance [ANOVA] was applied to determine the optimum condition. Finally, total protein concentration of 50 microg.mL[-1], pH of 5 and flow rate of 120 cm.h[-1], were anticipated as the optimal process conditions with 5.85 mg.mL[-1]of resin as the dynamic binding capacity. Experiments with anticipated optimal criteria were performed three times and no significant difference was observed [p = 0.136, and 6.06 mg/mL as the average dynamic binding capacity]
ABSTRACT
To increase the production level of heterologous proteins in plants, strategies such as choice of stronger promoters, optimization of codon usage and specific localization of foreign proteins are of major concern. Calcitonin [CT], a 32 amino acid polypeptide is a powerful and specific inhibitor of bone resorption and is used to treat several human diseases. Calcitonin activity is not species-specific which make it possible to produce in various animal sources, however, antibody formation in the prolonged application of animal CT leads to gradual decrease or loss of activity. That is why the long term treatment of human patients with CT requires homologous calcitonin. In this study, a human calcitonin [hCT] gene, driven by two different promoters [granule bound starch synthase I and Cauliflower mosaic virus 35S] was expressed in potato plants, using Agrobacterium-mediated transformation. Molecular analysis, including PCR, RT-PCR, Northern dot blot hybridizations showed that hCT could be successfully transcribed in transgenic potato plants. The immunoassay results showed that tissue specific expression in potato, led to almost five-fold more hCT accumulation than constitutively expression in all plant tissues
Subject(s)
Solanum tuberosum , Plant Tubers , Promoter Regions, GeneticABSTRACT
One of the most important aspects in recombinant biologic production, based on GMP rules, is the accuracy of final product quality control, especially assessment of host cell macromolecules contamination rate in final product. The purification requirement can be eliminated when the yeast cell containing the recombinant protein is used as a host cell. It is possibile that the final product contaminated to the host cell protein during purification stages of HBsAg [HBV vaccine]. The protein purification costs depend on the purification procedures required. Nowadays several companies produce commercial kits for identification and assessment of host cell protein contamination based on ELISA and Western blotting methods. But high prices, difference in sensitivity and lack of easy access to these kits sometimes create problems. So, in this study, two methods of Ammonium sulphate and caprilic acid precipitation technique were used separately for IgG purification. The results showed that IgG purification increased by 97% in caprylic acid method, compared with only a 77% increase in ammonium sulphate method. There were also significant differences in specificity and sensitivity between our standardized ELISA technique and using commercial kit [Cygnus CHO HCP]