Adsorption–desorption of recombinant hepatitis B surface antigen (r‐HBsAg) from P. pastoris on a diatomaceous earth matrix: Optimization of parameters for purification

Abstract

Recombinant hepatitis B surface antigen (r-HBsAg) produced in yeast is adsorbed on a diatomaceous earth matrix for purification purposes. A pH dependence in the adsorption-elution behavior was found. The capacity of celite (Hyflo Super Cei) for adsorbing r-HBsAg increased with decreasing pH. Nonspecific proteins were also adsorbed, but a low pH dependence was found. Elution from the matrix was performed using a basic pH buffer, in which r-HBsAg is more specifically adsorbed/desorbed than contaminant proteins, permitting the purification of the r-HBsAg. A pH of 4.0 was used for adsorption and pH 8.2 was used for desorption. The described protocol allows a purification factor between three- and fivefold with respect to contaminant proteins and sixfold with respect to contaminant DNA. Finally, the adsorption step was successfully scaled-up for production purposes.

A food-grade process for isolation and partial purification of bacteriocins of lactic acid bacteria that uses diatomite calcium silicate

“…The remaining bacteriocin still associated with the Micro-Cel at these extreme pH conditions would most likely be due to the hydrophobic interactions. In contrast, Agraz et al (1) reported that up to 80% of hepatitis B surface antigen was desorbed at pH 8.2 (in the presence of 0.02 M Tris-HCl, 0.003 M EDTA, and 100 g of sucrose per liter) after adsorption onto diatomaceous earth.…”

“…Diatomaceous earths and porous silica-derived compounds (e.g., calcium silicate and silicon dioxide) have been widely used as food-grade anticaking, clarifying, and filtration agents (2,9), for cell and enzyme immobilization (12,20), and for controlled-release formulations of biologically active molecules (21,27). In addition, Pillot et al (24) and Agraz et al (1) have reported the use of diatomaceous earth for purification of hepatitis B surface antigen. In the present study, bacteriocins were extracted from growth media by adsorption to Micro-Cel, a porous calcium silicate produced by the hydrothermal reaction of diatomite, hydrated lime, and water (4).…”

High recovery of intracellular recombinant HBsAg from Pichia pastoris via continuous pulsed laser cell disruption system optimized by response surface methodology

“…We further examined the protein concentration and cell disruption efficiency in pulsed laser system at 1,064 nm using different power outputs. The cell disruption efficiency and the percentage released rHBsAg was calculated, respectively, based on the ratio of total released protein and rHBsAg in the laser‐treated sample to the total released protein and rHBsAg in the well‐established conventional procedure—bead mill . Laser‐pulsed duration and cycle number were set to the optimum points in the selected range, which were 100 mSec and four cycles, respectively.…”

Optimisation of the coupled monoclonal antibody density for recombinant hepatitis B virus surface antigen immunopurification

“…The rHBsAg was recovered and submitted to initial purification steps as previously described [16] and optimised [17,18]. Briefly, the cells were harvested by centrifugation and disrupted on a bed mill (KDL type: WAB, Basel, Switzerland).…”