Activation of Soluble Polysaccharides with 1-Cyano-4-Dimethylaminopyridine Tetrafluoroborate (CDAP) for Use in Protein–Polysaccharide Conjugate Vaccines and Immunological Reagents. III Optimization of CDAP Activation

Abstract

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.

Activation and Conjugation of Soluble Polysaccharides using 1-Cyano-4-Dimethylaminopyridine Tetrafluoroborate (CDAP)

“…We found that while many common buffers interfere with the CDAP activation reaction, DMAP could successfully be used as the buffer to manage the pH 8 . Furthermore, DMAP is already a reaction byproduct of CDAP activation.…”

“…CDAP is a convenient reagent to derivatize and conjugate polysaccharides. This article describes the general method to use CDAP to derivatize polysaccharides with hydrazides (PS-ADH) and incorporates recently published improvements 8 .…”

“…The same process can be used to derivatize polysaccharides with primary amines by substituting the ADH for a diamine. It is recommended to use hexane diamine to derivatize polysaccharides with amines 8 . The aminated polysaccharide (PS-NH 2 ) can be conjugated using reagents developed for protein conjugation 11 .…”

“…DMAP has a pKa of 9.5 and thus has good buffering power around pH 9, and unlike many other buffers, DMAP was not found to promote CDAP hydrolysis 8 . Furthermore, DMAP is already a process intermediate and therefore does not add a new component to the reaction mixture.…”

“…Hydrazide-derivatized polysaccharides are often coupled to proteins using a carbodiimide reagent (e.g., 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC)) 7 . Further optimization of CDAP polysaccharide activation has been described by Lees et al 8 and is incorporated into the protocol described here.…”