DOE-based process optimization for development of efficient methods for purification of recombinant hepatitis B surface antigen from Pichia pastoris feedstock using Capto adhere resin

Abstract

BackgroundThe multimodal chromatography resins, such as Capto adhere, are considered good candidates to be utilized in downstream processing due to their high capacity and selectivity; however, their multimodal interactions lead to an intricacy in the adsorption-desorption patterns and systematic characterization of conditions for process steps is necessary. MethodsCapto adhere, a strong ion exchanger with multimodal functionality, was used in this study for the final aim of recombinant hepatitis B surface antigen (rHBsAg) purification from Pichia pastoris (P. pastoris) industrial feedstock. Optimization of various parameters was done using the design of experiments (DOE) approach to determine the best binding and non-binding conditions. ResultsMaximum rHBsAg binding on Capto adhere occurred in 20 mM sodium acetate, pH 4.5, and a binding capacity of about 0.75 mg/ml was achieved, which was much higher than rHBsAg binding capacity of other resins reported so far. In elution optimization investigations, it was revealed that 1 M arginine (buffered in 50 mM sodium phosphate, pH 6.5) was the most efficient eluting agent. The binding and elution optimal conditions were utilized for further purification of rHBsAg from P. pastoris industrial feedstock in bind-elute mode, and the recovery and purity of the obtained rHBsAg were about 60% and 100%, respectively. Following optimization in the flow-through purification mode, the target protein recovery was significantly increased (up to 97%) and the target protein purity of more than 95% was achievable. SEC-HPLC analysis showed that the obtained retention times for the purified rHBsAg were similar to those reported previously. ConclusionsThese results suggest that Capto adhere under such optimized conditions can be considered as a good candidate for efficient purification of rHBsAg from P. pastoris industrial feedstock in downstream processing.