Determining buffer conditions for downstream processing of VLP-based recombinant hepatitis B surface antigen using multimodal resins in bind-elute and flow-through purification modes.

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. In this study, optimization of rHBsAg (recombinantly-expressed in Pichia pastoris) DSP was performed using selection of buffering conditions in the semi-purification step. In the semi-purification optimization step, up to 73% of the protein impurities were eliminated and the utmost increase in rHBsAg purity (ca. 3.6-fold) was achieved using 20 mM sodium acetate, pH 4.5. 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. 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. The purification performance of the resin was constantly retained (97-100%) and no significant resin damage took place after 10 adsorption-elution-cleaning cycles. 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. This process may be also employable for purification of both non-VLP- and VLP- based target proteins expressed in the yeast.

Role of Intravesical BCG as a Therapeutic Vaccine for Treatment of Bladder Carcinoma

Bacterial products have attracted much attention as potential antitumor agents, with the ability to provide direct tumoricidal effects, leading to the inhibition of tumor growth. Treatment of superficial bladder cancer with intravesical Bacillus Calmette-Guérin (BCG) has a more reduction potential than surgery in tumor recurrence rate. BCG, the gold standard for nonmuscle invasive bladder cancer, is manufactured from different strains and produced commercially with varied strengths. There are a few countries known as the manufacturer of this strategic biopharmaceutical product, and Iran as a member of the Eastern Mediterranean Region plays a vital role in supplying this vaccine. Studies have failed to uncover the exact mechanism of action of the intravesical; however, evidence points toward an immunogenic mechanism that proficiently modifies a biologic response and provokes the immune cells in order to kill and suppress tumors. Among various underlying mechanisms, BCG bacillus attachment to fibronectin through its fibronectin attachment protein is a pivotal mechanism for BCG tumoricidal activity.

Challenges to Improve the Stability and Efficacy of an Intravesical BCG Product.

The aim of this investigation was to improve the storage stability and survival rate of an intravesical BCG product, manufactured with an attenuated strain of Mycobacterium bovis (Pasteur strain 1173P2 of BCG) in the presence of sodium glutamate. Formulations with various concentrations of trehalose (a known protectant) were developed as liquid and lyophilized forms. Formulations were evaluated by different methods, including optical density measurement, safety assessment, skin reaction test, moisture content determination, viability assay, bacterial and fungal contaminations and the results were compared with those obtained for sodium glutamate-containing formulations. The stability tests were also carried out in various storage durations and different temperatures. To develop the lyophilization protocol, glass transition temperatures in the presence of both stabilizers were determined using differential scanning calorimetry. In general, results showed that trehalose could considerably increase the stability of the product against freezing and drying processes, increase the survival rate even in the liquid formulations, as well as the production of an acceptable cake. However, further studies are required to optimize the product characteristics.