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

Abstract

Conjugate vaccines are remarkable advances in vaccinology. For the preparation of polysaccharide conjugate vaccines, the polysaccharides can be conveniently functionalized and linked to vaccine carrier proteins using 1-cyano-4dimethylaminopyridine tetrafluoroborate (CDAP), an easy-to-handle cyanylating reagent. CDAP activates polysaccharides by reacting with carbohydrate hydroxyl groups at pH 7-9. The stability and reactivity of CDAP are highly pH-dependent. The pH of the reaction also decreases during activation due to the hydrolysis of CDAP, which makes good pH control the key to reproducible activation. The original CDAP activation protocol was performed at room temperature in unbuffered pH 9 solutions. Due to the rapid reaction under this condition (<3 min) and the accompanying fast pH drop from the rapid CDAP hydrolysis, it was challenging to quickly adjust and maintain the target reaction pH in the short time frame. The improved protocol described here is performed at 0 °C, which slows CDAP hydrolysis and extends the activation time from 3 min to ~15 min. Dimethylaminopyridine (DMAP) was also used as a buffer to pre-adjust the polysaccharide solution to the target activation pH before adding the CDAP reagent. The longer reaction time, coupled with the slower CDAP hydrolysis and the use of DMAP buffer, makes it easier to maintain the activation pH for the entire duration of the activation process. The improved protocol makes the activation process less frenetic, more reproducible, and more amenable to scaling up.

Genetically detoxified tetanus toxin as a vaccine and conjugate carrier protein

“…Primary amines are often used for conjugation via NHS ester labeling and so the number of accessible ε lysine amines on a carrier protein were of interest. TNBS was used to estimate the number of primary amines on 8MTT [ 29 ]. Forty-seven primary amines per mole 8MTT were detected out of a possible 108.…”

“…Detection was at 280 nm. Primary amine concentrations were determined using trinitrobenzene sulfonic acid (TNBS) [ 29 ]. Lf assay was performed at the National Institute for Biological Standards and Control (United Kingdom), using the 3rd International Standard tetanus toxoid for use in flocculation test as the reference standard and 66/021 Equine tetanus antitoxin as the flocculation antibody.…”

“…The amino-PRP was then concentrated to 3 mL, using an Amicon Ultra 15 device (30 kDa cutoff). The amine concentration was determined using the TNBS assay with glycine as the amine standard [ 29 ] and a resorcinol/sulfuric acid assay with ribose as the standard [ 29 ], with a conversion factor of 331g PRP/- mole ribose. 8MTT and monomeric TTxd were thiolated with SPDP (Covachem, LLC, Loves Park, IL) using 15x molar excess.…”

Conjugation Mechanism for Pneumococcal Glycoconjugate Vaccines: Classic and Emerging Methods

“…Moreover, CDAP-activated polysaccharides can be functionalized with a diamine or a dihydrazide to make amino- or hydrazide-derivatized polysaccharides. Further optimization of CDAP polysaccharide activation with pH control and buffer optimization was later described [ 41 , 45 ].…”

Modeling 1-Cyano-4-Dimethylaminopyridine Tetrafluoroborate (CDAP) Chemistry to Design Glycoconjugate Vaccines with Desired Structural and Immunological Characteristics

“…The use of DMAP (with a pKa of 9.7) still requires a pH adjustment to maintain the reaction at the target pH of 9 by promptly adding aliquots of 0.1 M NaOH after CDAP addition [13,22]. Almost all buffers having a pKa close to 9 contain a nucleophilic group like a primary amine that can react directly with the activated PS or hydroxyl groups that can be activated by CDAP.…”