Large-scale production of recombinant hepatitis B surface antigen from Pichia pastoris.

The ability of the Pichia pastoris-based technology for large-scale production of recombinant hepatitis B virus surface antigen (HBsAg) and both reproducibly purify HBsAg and remove most of the relevant contaminants was ascertained by evaluating ten industrial production batches, five in 1993 and five in 1998. At an early stage, the clarification of mechanically disrupted yeast cells by acid precipitation renders HBsAg with a purity as low as 3.8 +/- 0.6%. However, by adsorption/desorption from diatomaceous earth matrix, the purity of HBsAg rapidly increases to 18.8 +/- 5%, which is suitable for chromatographic processing. This step also eliminates non-particulated forms of HBsAg, significantly lowers the amount of carbohydrates and lipids, and concentrates the HBsAg 4.8-fold. Finally, a sequential purification procedure that includes large-scale immunoaffinity, ion-exchange, and size-exclusion chromatographies further purifies the preparation, resulting in a product (HBsAg at a concentration of 1.3 +/- 0.2 g l-1) with a purity of 95% or more. Furthermore, each of the other contaminants measured reaches the following low levels per 20 micrograms HBsAg: host deoxyribonucleic acid (< 10 pg), carbohydrates (1.2 +/- 0.02 micrograms), lipids (14 +/- 0.28 micrograms), immunopurification-released immunoglobulin G (less than 100 ppm), and endotoxins (106.7 +/- 19.3 pg). These values are below those specified for recombinant DNA hepatitis B vaccines according to World Health Organization (WHO) guidelines.