Activation of Soluble Polysaccharides with CDAP for Use in Protein–Polysaccharide Conjugate Vaccines and Immunological Reagents
In this article in Vaccines I address how to control the chemistry and obtain reproducible results.
CDAP (1-cyano-4-dimethylaminopyridine tetrafluoroborate) is employed in the synthesis of conjugate vaccines as a cyanylating reagent. In the published method, which used pH 9 activation at 20 °C (Vaccine, 14:190, 1996), the rapid reaction made the process difficult to control. Here, we describe optimizing CDAP activation using dextran as a model polysaccharide. CDAP stability and reactivity were determined as a function of time, pH and temperature. While the rate of dextran activation was slower at lower pH and temperature, it was balanced by the increased stability of CDAP, which left more reagent available for reaction. Whereas maximal activation took less than 2.5 min at pH 9 and 20 °C, it took 10–15 min at 0 °C. At pH 7 and 0 °C, the optimal time increased to >3 h to achieve a high level of activation. Many buffers interfered with CDAP activation, but DMAP could be used to preadjust the pH of polysaccharide solutions so that the pH only needed to be maintained. We found that the stability of the activated dextran was relatively independent of pH over the range of pH 1–9, with the level of activation decreased by 40–60% over 2 h. The use of low temperature and a less basic pH, with an optimum reaction time, requires less CDAP, improving activation levels while making the process more reliable and easier to scale up. READ HERE: Vaccines