Rapid and sensitive anthrone-sulfuric acid assay in microplate format to quantify carbohydrate in biopharmaceutical products: method development and validation

Biologicals. 2008 Mar;36(2):134-41. doi: 10.1016/j.biologicals.2007.09.001. Epub 2007 Nov 26.


The need for an accurate, fast and reliable analysis of carbohydrate test is crucial for numerous biological processes. In that sense, anthrone-sulfuric acid assay is one of the most efficient quantification techniques successfully applied to carbohydrate determination. In this paper, a sensitive and accurate anthrone-sulfuric acid microplate assay was developed and validated for the quantitative estimation of yeast carbohydrates in the production of hepatitis B virus surface antigen, and the main component of the recombinant vaccine HEBERBIOVAC HB. A response surface methodology was applied to design and optimize the assay in order to maximize the differences on the expected effect and to minimize the number of experiments. The proposed method was linear over the concentration range from 10 to 120 microg/mL for glucose, with values for the coefficient of determination >0.99. Intra- and inter-assay variation coefficient ranged between 0.45-4.79% and 2.48-8.94%, respectively. The Student t-test used in the interference study, revealed good parallelism among curves (T(obs)< or =T(0.05)), which indicates the lack of interference in the working range. Yields obtained in accuracy test for two concentration levels varied between 90 and 105%, confirming the assay's reliability. In conclusion, the validated method, which has successfully been used for the process control monitoring of several samples generated from the production of hepatitis B vaccine, allows the quality and purity of the final product.

Publication types

  • Validation Study

MeSH terms

  • Anthracenes*
  • Biological Products / analysis*
  • Biological Products / chemistry
  • Carbohydrates / analysis*
  • Carbohydrates / chemistry
  • Pharmaceutical Preparations / analysis*
  • Pharmaceutical Preparations / chemistry
  • Pichia / chemistry
  • Pichia / metabolism
  • Sulfuric Acids*


  • Anthracenes
  • Biological Products
  • Carbohydrates
  • Pharmaceutical Preparations
  • Sulfuric Acids
  • anthrone
  • sulfuric acid