Targeting the hydrophilic regions of recombinant proteins by MS via in-solution buffer-free trypsin digestion.

A desalting step using reversed phase chromatography is a common practice prior to mass spectrometry analysis of proteolytic digests in spite of the detrimental exclusion of the hydrophilic peptides. The detection of such peptides is also important for the complete coverage of protein sequences and the analysis of posttranslational modifications as inquired by regulatory agencies for the commercialization of biotechnological products. The procedure described here, named in-solution buffer-free digestion, simplifies the sample processing and circumvents the above-mentioned limitations by allowing the detection of tryptic hydrophilic peptides via direct ESI-MS analysis. Two DNA recombinant proteins such as HBcAg (hepatitis B core antigen) and fusion VEGF (vascular endothelial growth factor) were analyzed with the proposed in-solution buffer-free digestion allowing the detection of extremely hydrophilic di-, tri- and tetra-peptides, C-terminal His-tail peptide, as well as disulfide-containing peptides. All these molecular species are hardly seen in mass spectrometric analysis using a standard digestion that includes a C18-desalting step. The procedure was also successfully tried on hydrophilic tetra- and hexa-peptides of Ribonuclease B carrying an N-glycosylation site occupied with "high-mannose" N-glycan chains. The in-solution buffer-free digestion constitutes a simple and straightforward approach to analyse the hydrophilic proteolytic peptides which are commonly elusive to the detection by conventional mass spectrometric analysis.

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

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.