![]() Like other virus-like particles (VLPs), rHBsAg has also the potential to be used as vaccine carrier and gene therapy vector 8, 9, 10. Purification of hepatitis B surface antigen (HBsAg) from the plasma of the virus carriers was the initial common source of HBV vaccines however, the inadequate supply of human plasma plus increased risk of viral transmission were the main motives to the production of recombinant HBsAg 4, 5, 6, 7. Hepatitis B virus (HBV) accounts for around 80% of the global burden of hepatocellular carcinoma (HCC) and finally 820,000 deaths annually 1, 2, 3. This process may be also employable for purification of both non-VLP- and VLP- based target proteins expressed in the yeast. The new DSP developed for production of rHBsAg in this study can substitute the conventional one with granting satisfactory target protein quality, long-lasting resin efficacy, shorter and less expensive process. The purification performance of the resin was constantly retained (97–100%) and no significant resin damage took place after 10 adsorption–elution–cleaning cycles. Following assessment of critical quality attributes (i.e., purity, particle size distribution, host cell DNA, host cell protein, secondary structures, specific activity and relative potency), it was indicated that the characteristics of rHBsAg purified by the new DSP were similar or superior to the ones obtained from conventional DSP. By using rHBsAg binding and nonbinding situations obtained from the response surface plot in design of experiments (DOE), additional bind-elute and flow-through purification mode experiments were conducted and rHBsAg with high purity (near 100%) and recovery (> 83%) was achieved. 3.6-fold) was achieved using 20 mM sodium acetate, pH 4.5. In the semi-purification optimization step, up to 73% of the protein impurities were eliminated and the utmost increase in rHBsAg purity (ca. In this study, optimization of rHBsAg (recombinantly-expressed in Pichia pastoris) DSP was performed using selection of buffering conditions in the semi-purification step. The difficulties in purification of VLP-based recombinant hepatitis B surface antigen (rHBsAg) are mainly emerged from inefficient semi-purification step plus proteins physicochemical properties and these issues make the downstream processing (DSP) very lengthy and expensive.
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