OPTIMIZATION OF HEPATITIC B SURFACE ANTIGEN (HBsAg) PURIFICATIONS TO IMPROVING THE PRODUCTION OF HEPATITIS B VACCINES ON Pichia pastoris

• Main Author: Kusuma Cahyani, Rizky
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/35869
• Institution: Institut Teknologi Bandung

Hepatitis B is a liver inflammatory disease caused by hepatitis B virus (HBV). This infection can be prevented by vaccination which contains HBV surface protein (SHBsAg). However vaccine supply is limited. Several attempts have been conducted to produce local SHBsAg. However the purity degree and protein yield are still inadequate. Therefore optimization of HBsAg purification steps is required to obtain high yield with better purification fold. In this study, optimization of purification was done in 2 steps, precipitation using variation of NaCl concentration (0,3 M; 0,5 M; 0,7 M) and PEG (3%, 5%, 7%); ion exchange chromatography (IEC) using NaCl 300-500 mM elution buffer concentration. To determine HBsAg protein, BCA and ELISA assay was used in this study. Visualization of HBsAg protein was done by SDS-PAGE analysis. Based on quantitative analysis, optimal condition for precipitation step were obtained in 0,3 M NaCl and PEG 3%, while in ion exchange chromatography step, the optimum condition for protein eluting was at NaCl 500 mM. SDS PAGE analysis indicates that the presence of protein HBsAg with molecular wieght of 25 kDa (monomer) and 50 kDa (dimer). The optimum condition for purification of SHBsAg produced in Pichia pastoris gave yield of 47% with specific activity of HBsAg 0.283 ng, so that it would increase the production of hepatitis B vaccine to be more optimal.