Quantitative high throughput analytics to support polysaccharide production process development.

The rapid development of purification processes for polysaccharide vaccines is constrained by a lack of analytical tools current technologies for the measurement of polysaccharide recovery and process-related impurity clearance are complex, time-consuming, and generally not amenable to high throughput process development (HTPD). HTPD is envisioned to be central to the improvement of existing polysaccharide manufacturing processes through the identification of critical process parameters that potentially impact the quality attributes of the vaccine and to the development of de novo processes for clinical candidates, across the spectrum of downstream processing. The availability of a fast and automated analytics platform will expand the scope, robustness, and evolution of Design of Experiment (DOE) studies. This paper details recent advances in improving the speed, throughput, and success of in-process analytics at the micro-scale. Two methods, based on modifications of existing procedures, are described for the rapid measurement of polysaccharide titre in microplates without the need for heating steps. A simplification of a commercial endotoxin assay is also described that features a single measurement at room temperature. These assays, along with existing assays for protein and nucleic acids are qualified for deployment in the high throughput screening of polysaccharide feedstreams. Assay accuracy, precision, robustness, interference, and ease of use are assessed and described. In combination, these assays are capable of measuring the product concentration and impurity profile of a microplate of 96 samples in less than one day. This body of work relies on the evaluation of a combination of commercially available and clinically relevant polysaccharides to ensure maximum versatility and reactivity of the final assay suite. Together, these advancements reduce overall process time by up to 30-fold and significantly reduce sample volume over current practices. The assays help build an analytical foundation to support the advent of HTPD technology for polysaccharide vaccines. It is envisaged that this will lead to an expanded use of Quality by Design (QbD) studies in vaccine process development

Competitive Inhibition of Heparinase by Persulfonated Glycosaminoglycans: A Tool to Detect Heparin Contamination

Heparin and the low molecular weight heparins are extensively used as medicinal products to prevent and treat the formation of venous and arterial thrombi. In early 2008, administration of some heparin lots was associated with the advent of severe adverse effects, indicative of an anaphylactoid-like response. Application of orthogonal analytical tools enabled detection and identification of the contaminant as oversulfated chondroitin sulfate (OSCS) was reported in our earlier report. Herein, we investigate whether enzymatic depolymerization using the bacterially derived heparinases, given the structural understanding of their substrate specificity, can be used to identify the presence of OSCS in heparin. We also extend this analysis to examine the effect of other persulfonated glycosaminoglycans (GAGs) on the action of the heparinases. We find that all persulfonated GAGs examined were effective inhibitors of heparinase I, with IC50 values ranging from approximately 0.5–2 μg/mL. Finally, using this biochemical understanding, we develop a rapid, simple assay to assess the purity of heparin using heparinase digestion followed by size-exclusion HPLC analysis to identify and quantify digestion products. In the context of the assay, we demonstrate that less than 0.1% (w/w) of OSCS (and other persulfonated polysaccharides) can routinely be detected in heparin