CONJUGATION OF A SHIGELLA FLEXNERI 2A DERIVED SYNTHETIC OLIGOSACCHARIDE TO TETANUS TOXOID

Here we describe the process steps of the manufacturing of an experimental conjugate vaccine against S. flexneri with a synthetic oligosaccharide. Modification of tetanus toxoid (TTd) with N-gamma-(maleimidobutyryloxy)succinimide ester (GMBS), followed by conjugation of a synthetic pentadecasaccharide, containing a (masked) thiol-spacer, was investigated^[1]. The oligosaccharide is derived from the O-antigen of the lipopolysaccharide of S. flexneri type 2a.

TTd itself already contained a significant amount of aggregates. During initial modification experiments, additional aggregation was observed. Individual modification of the TTd monomers, dimers, trimers and larger entities showed that aggregation induction could only be observed for monomeric TTd. Furthermore, different ratios of the linker to TTd had an effect on the aggregation and the use of a HEPES buffer induced less aggregation.

Using design of experiments (DoE), the modification was thoroughly studied. The primary goal was to have a maximum amount of modified primary amino groups (> 22) without inducing additional aggregation (< 7.5%). The variables time, pH, temperature, TTd concentration and ratio TTd to linker were investigated. Through statistical analysis of the DoE a model was constructed which showed that the concentration of TTd, the ratio of TTd to the linker, the pH and temperature all had an effect on the aggregation and amount of linkers coupled per TTd. From these analyses optimum reaction conditions were defined, and confirmed in a robustness DoE.

Conjugation of the pentadecasaccharide to the modified TTd yielded reproducible results, as measured by HPSEC and colorimetric assays. The final conjugated pentadecasaccharide can be predicted from the saccharide to TTd ratio applied during the conjugation reaction, giving complete control of each of the reaction steps.