High expression of methylotrophic yeast-derived recombinant human erythropoietin in a pH-controlled batch system

To accomplish the worldwide demand for recombinant human erythropoietin [rHuEpo] as a therapeutic, application of cost-efficient expression system of methylotrophic yeast Pichia pastoris [P. pastoris] rather than mammalian cells is indispensable. Herein, a report on high levels secreted-expression of Pichia-derived rHuEpo by batch fermentation in a pH stabilized format is presented. The full length cDNA of rHuEpo was inserted into pPICZ alpha A vector under control of AOX[1] promoter, downstream of the secretion-alpha-factor and electroporated into P. pastoris strain X33. The highest expression transformant was selected by screening among the colonies surviving high concentration of Zeocin [1.0 mg/ml], followed by comparative small scale expression analysis by ELISA. Stabilization of pH around 6.0 by adding phosphoric acid into the culture media during induction period, improved the yield of expression to 150 mg/l of the media. Single-step Nickel-affinity chromatography was employed for purification of rHuEpo-6xHis to 80% purity. Analyses by SDSPAGE, Western blot and N-terminal protein sequencing confirmed the authenticity of the 33 kDa expressed rHuEpo with a native N-terminal indicating the proper cleavage of secretion-signal. Results of this study, further confirmed the possibility of employing methylotrophic yeast for scaled up production aims of rHuEpo as a cost-efficient expression system when provided evidence for higher expression yields through application of pH-controlled systems

Design and evaluation of a new dry powder inhaler.

Three versions of a new dry powder inhaler [DPI], RG-haler, were designed using two kinds of grid inserts. Salbutamol sulfate/lactose blend [Ventolin Rotacaps[R]] was selected as a model formulation to analyze the performance of all inhalers and compare their efficiency with three marketed devices [Rotahaler[R], Spinhaler[R] and ISF inhalator[R]] using the twin impinger [TI]. Deposition of the drug in device was significantly [P<0.05] lower for ISF inhalator[R] and all kinds of RG-halers in comparison with those of Rotahaler[R] and Spinhaler[R]. The amount of drug deposited in the stage 2 and the respirable dose for RG-halers were similar to those of ISF inhalator[R] and significantly [P<0.05] higher than those of Rotahaler[R] and Spinhaler[R]. The results suggest efficient aerosol generating capability of the RG-haler