Development Of An Integrated Recovery Process For Recombinant Hepatitis B Core Antigen

Hepatitis B core antigen (HBcAg) expressed intracellularly in Escherichia coli (E. coli) has great potential and high demand in pharmaceutical market. The main objective of this study was to develop an efficient and cost effective protocol for the recovery of recombinant HBcAg. Cell disruption is the prerequisite step to release the intracellular HBcAg to the surrounding medium prior to subsequent purification processes. In the current study, E. coli was disrupted by mechanical method of bead milling. Bead milling can be operated either in batch or continuous recycling mode. The performance of bead milling is affected by parameters such as impeller tip speed, biomass concentration, bead loading and feed flow rate. Thus, in the first chapter of the study, the effects of these parameters on different modes of operation were investigated. The effect of these operation modes on the subsequent downstream processing was also examined. In the second chapter of the study, a purification protocol for the recovery of HBcAg was developed. The HBcAg was initially purified by the integrated operation of batch mode bead milling and expanded bed adsorption (EBA), and subsequently purified by size exclusion chromatography (SEC). The results of the cell disruption study show that the optimum disruption of E. coli and release of intracellular HBcAg in batch mode bead milling was achieved at impeller tip speed of 14 m/s, biomass concentration of 20% (w/v) and bead loading of 80% (v/v), whilst the performance of continuous recycling bead milling was peak at impeller tip speed of 14 m/s, biomass concentration of 10% (w/v) and feed flow rate of 15 L/h. Batch mode was ideal for the batch anion-exchange adsorption, in which a HBcAg yield of 34.3%, a HBcAg purity of 65% and a purification factor of 2.86 was achieved. In the purification study, the product yield, product purity and purification factor achieved in the integrated EBA process was 55%, 42.3% and 1.96, respectively. The partially purified HBcAg was then further purified by SEC, in which a product yield of 42.4% was obtained. The SEC purification has also yielded a HBcAg purity of 88.2%, which corresponded to a purification factor of 4.08. The purified HBcAg was confirmed to be functionally active and hence, can be used in the development of hepatitis B virus (HBV) diagnostic kits

The release of hepatitis B core antigen from Escherichia coli by batch mode bead milling

The performance of a batch model bead mill on the release of hepatitis B core antigen (HBcAg) from Escherichia coli was investigated in this study. The operating parameters examined were impeller tip speed (8–14 m/s), biomass concentration [5–20% (w/v)] and bead loading [65–80% (v/v)]. The highest yield (24.3 mg/g cell) and rate constant (0.471 l/min) of HBcAg release were achieved at impeller tip speed of 14 m/s. However, the high-shear stress under these operating conditions caused damage of the HBcAg. The highest yield (22.7 mg/g cell) and rate constant (0.344 l/min) of HBcAg release were observed at biomass concentration of 20% (w/v). There was no significant effect of bead loading on the performance of bead milling being observed. In conclusion, the optimal operating condition for the release of HBcAg was at bead loading of 75% (v/v), biomass concentration of 20% (w/v) and impeller tip speed of 10 m/s

The direct recovery of recombinant hepatitis B core antigen from disruptate derived from continuous-flow bead milling

HBcAg (hepatitis B core antigen) is a nanoplex bioproduct that has a great potential in the development of therapeutic drugs and vaccines. In the present study, a continuous-flow bead milling for the disruption of Escherichia coli was optimized and a direct recovery protocol to isolate the recombinant HBcAg from the unclarified E. coli disruptate was developed. The optimal condition for continuous-flow bead milling for the release of HBcAg from E. coli was achieved at a feed flow rate of 15 litres/h, biomass concentration of 10% [ww/v (wet weight/vol.)] and impeller tip speed of 14 m/s. The sucrose-density-gradient analysis showed that the particulate form of the HBcAg released by this optimal condition is still preserved. In the direct purification of HBcAg from the unclarified disruptate, the AE-EBAC (anion-exchange expanded-bed adsorption chromatography) technique was employed. A 54% adsorption and 50.7% recovery of HBcAg were achieved in this direct recovery process. The purity of HBcAg recovered was 49.8%, which corresponds to a purification factor of 2.0. ELISA showed that the HBcAg recovered is functionally active

A preparative purification process for recombinant Hepatitis B core antigen using online capture by expanded bed adsorption followed by size-exclusion chromatography

Hepatitis B core antigen (HBcAg) is an important serological marker used in the diagnosis of hepatitis B virus (HBV) infections. In the current study, a fast and efficient preparative purification protocol for truncated HBcAg from Escherichia coli disruptate was developed. The recombinant HBcAg was first captured by anion exchange expanded bed adsorption chromatography integrated with a cell disruption process. This online capture process has shortened the process time and eliminated the “hold-up” period that may be detrimental to the quality of target protein. The eluted product from the expanded bed adsorption chromatography was subsequently purified using size-exclusion chromatography. The results showed that this novel purification protocol achieved a recovery yield of 45.1% with a product purity of 88.2%, which corresponds to a purification factor of 4.5. The recovered HBcAg is still biologically active as shown by ELISA test

Comparative evaluation of different cell disruption methods for the release of recombinant hepatitis B core antigen from Escherichia coli

A comparative evaluation of five different cell-disruption methods for the release of recombinant hepatitis B core antigen (HBcAg) from Escherichia coli was investigated. The cell disruption techniques evaluated in this study were high-pressure homogenization, batch-mode bead milling, continuous-recycling bead milling, ultrasonication, and enzymatic lysis. Continuous-recycling bead milling was found to be the most effective method in terms of operating cost and time. However, the highest degree of cell disruption and amounts of HBcAg were obtained from the high-pressure homogenization process. The direct purification of HBcAg from the unclarified cell disruptate derived from high-pressure homogenization and bead milling techniques, using batch anion-exchange adsorption methods, showed that the conditions of cell disruption have a substantial effect on subsequent protein recovery steps