Automation of the anthrone assay for carbohydrate concentration determinations.

Reported is the adaptation of a manual polysaccharide assay applicable for glycoconjugate vaccines such as Prevenar to an automated liquid handling system (LHS) for improved performance. The anthrone assay is used for carbohydrate concentration determinations and was scaled to the microtiter plate format with appropriate mixing, dispensing, and measuring operations. Adaptation and development of the LHS platform was performed with both dextran polysaccharides of various sizes and pneumococcal serotype 6A polysaccharide (PnPs 6A). A standard plate configuration was programmed such that the LHS diluted both calibration standards and a test sample multiple times with six replicate preparations per dilution. This extent of replication minimized the effect of any single deviation or delivery error that might have occurred. Analysis of the dextran polymers ranging in size from 214 kDa to 3.755 MDa showed that regardless of polymer chain length the hydrolysis was complete, as evident by uniform concentration measurements. No plate positional absorbance bias was observed; of 12 plates analyzed to examine positional bias the largest deviation observed was 0.02% percent relative standard deviation (%RSD). The high purity dextran also afforded the opportunity to assess LHS accuracy; nine replicate analyses of dextran yielded a mean accuracy of 101% recovery. As for precision, a total of 22 unique analyses were performed on a single lot of PnPs 6A, and the resulting variability was 2.5% RSD. This work demonstrated the capability of a LHS to perform the anthrone assay consistently and a reduced assay cycle time for greater laboratory capacity.

An integrity assay for a meningococcal type B conjugate vaccine.

The development of an analytical procedure for the evaluation of a conjugate vaccine's structural wholeness or integrity is described. The principle component of the vaccine was the N-propionylated group B meningococcal polysaccharide (NPr-GBMP) covalently attached to a carrier protein. The goal of the procedure was to determine whether any whole polysaccharide, oligosaccharide, or monosaccharide, from minute to moderate levels, became detached off the conjugate. Free saccharide was isolated from the formulation, which included an aluminum hydroxide adjuvant for analysis. Due to its linkage, the NPr-GBMP did not release sialic acid efficiently with acid hydrolysis to the extent necessary for accurate quantitation. To accomplish depolymerization, the NPr-GBMP was subjected to methanolysis, 3N hydrochloric acid in methanol for 16h at 80 degrees C. The main product of the methanolysis reaction was a de-N-acylated methyl glycoside of sialic acid. N-acetylneuraminic acid oligomers and colominic acid were used to confirm the methanolysis depolymerization efficiency of the alpha(2 --> 8) saccharides; with the treatment all oligomers produced a common methyl glycoside. For this determination anion exchange chromatography and size exclusion chromatography were both interfaced to an integrated pulsed amperometric detector. Sensitivity and linearity were demonstrated to be sufficient for the application with vaccine dose formulations with low total saccharide concentrations.