Molecular size distribution assessment of Haemophilus influenzae vaccine containing lactose by HPAEC-PAD and colorimetric assays.

Molecular size distribution of Haemophilus influenzae type b (Hib) conjugate vaccine is an important indicator for its immunogenicity and stability. Molecular size distribution was evaluated by High-Performance Protein Chromatography on Sepharose CL-4B column, and fractions were pooled. The use of high flow rate, incorporation of a calibration standard with the injected buffer and pooling method yielded a superior assay compared to conventional pharmacopeial method. The pools were analyzed for determination of distribution coefficient (KD) of 0.2 and 0.5 using two validated techniques: High Performance Anion Exchange Chromatography with pulsed amperometric detection (HPAEC-PAD) for ribitol determination and an optimized colorimetric assay for phosphorus determination. Linearity was achieved over range of 0.10-10.0 µg/mL and 1.0-8.0 µg/mL with LOD of 0.03 and 0.28 µg/mL for HPAEC and colorimetric assays, respectively. The developed assays were successfully applied in quality control monitoring of Hib conjugate vaccine. The optimized colorimetric method had shortened the analysis time to 25 min compared to 3.5 h for the European pharmacopeial assay by modifying the burning process. HPAEC stability results revealed 40% decrease in MSD after stressed storage conditions. The proposed assays offer a reliable and economic platform for monitoring the quality attributes of Hib for biopharma industry.

Effects of Algal Extracellular Polysaccharides on the Formation of Filamentous Manganese Oxide Particles in the Near-Bottom Layer of Lake Biwa.

Filamentous manganese (Mn) oxide particles, which occur in the suboxic zone of stratified waterbodies, are important drivers of diverse elemental cycles. These particles are considered to be bacteriogenic; despite the importance of biogeochemical implications, however, the environmental factor responsible for their formation has not been identified. The aim of this study was to demonstrate the involvement of algal extracellular polysaccharides in Mn oxide particle formation. Based on this study of laboratory cultures of a model Mn(II)-oxidizing bacterium, the supply of algal extracellular mucilage was shown to stimulate Mn(II) oxidation and thus the production of filamentous Mn oxide particles. This observation was consistent with the results obtained for naturally occurring particles collected from a near-bottom layer (depth of approximately 90 m) in the northern basin of Lake Biwa, Japan, that is, most Mn particles resembling δ-MnO2 were associated with an extracellular mucilage-like gelatinous matrix, which contained dead algal cells and was lectin-stainable. In the lake water column, polysaccharides produced by algal photosynthesis sank to the bottom layer. The analysis of the quality of water samples, which have been collected from the study site for 18 years, reveals that the annual average total phytoplankton biovolume in the surface layer correlates with the density of filamentous Mn particles in the near-bottom layer. Among different phytoplankton species, green algae appeared to be the key species. The results of this study suggest that algal extracellular polysaccharides serve as an important inducer for the formation of filamentous Mn oxide particles in the near-bottom layer of the northern basin of Lake Biwa.

Validation of a spectrophotometric methodology for the quantification of polysaccharides from roots of Operculina macrocarpa (jalapa)

The roots from Operculina macrocarpa (L.) Urb., Convolvulaceae, are widely used in Brazilian traditional medicine as a laxative and purgative. The biological properties of this drug material have been attributed to its polysaccharides content. Thus, the aim of this study was to evaluate the polysaccharide content in drug material from O. macrocarpa by spectrophotometric quantitative analysis. The root was used as plant material and the botanical identification was performed by macro and microscopic analysis. The plant material was used to validate the spectrophotometric procedures at 490 nm for the quantification of the reaction product from drug polysaccharides and phenol-sulfuric acid solution. The analytical procedure was evaluated in order to comply with the necessary legal requirements by the determination of the following parameters: specificity, linearity, selectivity, precision, accuracy and robustness. This study provides with a simple and valid analytical procedure (linear, precise, accurate and reproducible), which can be satisfactorily used for quality control and standardization of herbal drug from O. macrocarpa.