title: Simple high-cell density fed-batch technique for high-level recombinant protein production with Pichia pastoris: Application to intracellular production of Hepatitis B surface antigen.
abstract: BACKGROUND
  Hepatitis B is a serious global public health concern. Though a safe and efficacious recombinant vaccine is available, its use in several resource-poor countries is limited by cost. We have investigated the production of Hepatitis B virus surface antigen (HBsAg) using the yeast Pichia pastoris GS115 by inserting the HBsAg gene into the alcohol oxidase 1 locus.
  
  
  RESULTS
  Large-scale production was optimized by developing a simple fed-batch process leading to enhanced product titers. Cells were first grown rapidly to high-cell density in a batch process using a simple defined medium with low salt and high glycerol concentrations. Induction of recombinant product synthesis was carried out using rather drastic conditions, namely through the addition of methanol to a final concentration of 6 g L-1. This methanol concentration was kept constant for the remainder of the cultivation through continuous methanol feeding based on the on-line signal of a flame ionization detector employed as methanol analyzer in the off-gas stream. Using this robust feeding protocol, maximum concentrations of ~7 grams HBsAg per liter culture broth were obtained. The amount of soluble HBsAg, competent for assembly into characteristic virus-like particles (VLPs), an attribute critical to its immunogenicity and efficacy as a hepatitis B vaccine, reached 2.3 grams per liter of culture broth.
  
  
  CONCLUSIONS
  In comparison to the highest yields reported so far, our simple cultivation process resulted in an ~7 fold enhancement in total HBsAg production with more than 30% of soluble protein competent for assembly into VLPs. This work opens up the possibility of significantly reducing the cost of vaccine production with implications for expanding hepatitis B vaccination in resource-poor countries.
authors.affiliation: Helmholtz Centre for Infection Research, Braunschweig, Germany. ursula.rinas@helmholtz-hzi.de.
authors.forename: Chandrasekhar
authors.initials: C
authors.lastname: Gurramkonda
authors.forename: Ahmad
authors.initials: A
authors.lastname: Adnan
authors.forename: Thomas
authors.initials: T
authors.lastname: Gäbel
authors.forename: Heinrich
authors.initials: H
authors.lastname: Lünsdorf
authors.forename: Anton
authors.initials: A
authors.lastname: Ross
authors.forename: Satish Kumar
authors.initials: SK
authors.lastname: Nemani
authors.forename: Sathyamangalam
authors.initials: S
authors.lastname: Swaminathan
authors.forename: Navin
authors.initials: N
authors.lastname: Khanna
authors.forename: Ursula
authors.initials: U
authors.lastname: Rinas