Application of a rapid HILIC-UV method for synthesis optimization and stability studies of immunogenic neo-glycoconjugates

Proteins and glycoproteins with therapeutic activity are susceptible to environmental factors, which can cause their degradation and the loss of their activity. Thus, the maintenance of their stability during the production process is a critical factor. In this work, a simple and rapid hydrophilic interaction liquid chromatography HILIC-UV method was validated in terms of accuracy, precision, linearity, LOD, LOQ and specificity and applied to the investigation of the stability of intact proteins and their neo-glycoconjugates with antigenic activity against tuberculosis. The method proved to be suitable for the estimation of the degradation of the proteins under critical conditions (i.e. freeze-thaw cycles) and for the monitoring of their coupling reaction with saccharidic moieties, without the need of sample preparation. In addition, the chromatographic analysis allowed to calculate the yields of the protein glycosylation reaction.