Development of a size exclusion chromatography method for a determination of free polyribosyl-ribitol-phosphate (PRP) and conjugated to tetanus toxoid (PRP-TTD) in Haemophilus influenzae type B vaccines

Abstract

Haemophilus influenzae type b is an opportunistic pathogen responsible for invasive and severe infections such as bacteremia and acute bacterial meningitis in susceptible populations, mainly children under 5 years of age. Its capsular antigen, polyribosyl-ribitol-phosphate, is a specific polysaccharide of serotype b and has been used to produce vaccines, initially in isolation, with little success due to poor immune stimulation in the target population, and providing little sustained protection over time. Thus, arose the conjugated vaccines currently used, where the capsular polysaccharide binds to a carrier protein to generate a thymus-dependent immune response with immunological memory and suitable for use in children from 2 months of age. Polyribosyl-ribitol-phosphate is characterized by being a polymeric sequence of the phosphated sugar of variable length, forming a mixture with different abundances of various molecular weights. The length of the polysaccharide chains is a quality parameter of the production process of these vaccines, and is controlled by size-exclusion chromatography techniques.The presented work consisted in developing an analytical technique for the determination of polyribosyl-ribitol-phosphate in its free form and conjugated to tetanus toxoid present in vaccines against Haemophilus influenzae type b, using size-exclusion chromatography coupled to refractive index and UV detectors at wavelengths of 215 and 280 nm. The development was based on bibliographic backgrounds that use a similar methodology as a control of the conjugation step in the production process of the active pharmaceutical ingredient of anti-Haemophilus influenzae type b vaccines. In the procedure, international standards for free analytes and molecular weight standards were used, and the ability of the system to discriminate the vaccine components and to detect differences that could indicate a compromise in the quality of this vaccine was evaluated.