Development, validation and application of a new ELISA for process control of the production of recombinant Hepatitis B surface antigen

Abstract

The present work describes the development and validation of a sandwich-type enzyme-linked immunosorbent assay (ELISA) for quantifying the surface antigen of hepatitis B virus (HBsAg), obtained from a recombinant strain of the methylotrophic yeast Pichia pastoris. It is based on monoclonal antibody CB.Hep-1, normally employed as a ligand for the purification of HBsAg. Validation followed the guidelines of ICH Q2 and CECMED regulation No. 41 from 2007. Parameters such as linear working range, specificity, precision and accuracy were analyzed. The assay has a lower quantification limit of 11.9 ng/mL. Monoclonal antibody CB.Hep-1, used both for coating and as a conjugate during the ELISA, specifically bound recombinant HBsAg with excellent accuracy. An analysis of variance for the interference study yielded a probability, for each process control sample type/buffer combination, higher than 0.1, for a confidence level of 99%. Intra-assay variability ranged from 0.77 to 7.47%, and inter-assay variability ranged from 1.19 to 19.41%, always staying, for each sample, below 10 and 20% respectively. Recovery ranged from 98.18 to 100.31%, with a variation coefficient under 20%. The ELISA is specific for this monoclonal antibody within the range of studied concentrations, and has a linear response for antigen concentrations from 191.7 to 11.9 ng/mL. Given its precision, specificity and accuracy, this ELISA is a powerful tool for process control during the production of the recombinant vaccine against HBV.

Keywords

• HBsAg
• validation
• Process control
• ELISA