Determination of free carrier protein in glycoconjugate vaccines by size exclusion chromatography–high performance liquid chromatography with fluorescence detector

Abstract

AbstractPolysaccharide‐protein conjugates elicit a protective immune response in children below 2 years. The conjugated antigens using different carrier proteins generate T‐cell dependent antibody response in children and increased antibody titers in adults than unconjugated polysaccharide vaccines. Different marketed conjugate vaccines, contain carrier proteins such as cross‐reactive material, Tetanus Toxoid, Diphtheria toxoid and Protein D (derived from non‐typeable Haemophilus influenzae). Quantification of unconjugated polysaccharide (free poly) and protein (free protein) in the purified conjugates (drug substance) and formulated drug product are important quality control parameters. In addition, the bulk conjugates are monitored for free protein as stability indicating parameter. Since the purified conjugates contain low amounts of free protein, highly sensitive methods are needed. We developed a method using size‐exclusion chromatography with high‐performance liquid chromatography with fluorescence detection for free protein quantification and validated. The method can be used for quantifying the free carrier protein in Pneumococcal, Typhoid and Hib polysaccharides conjugated with different carrier proteins such as cross‐reactive material, Tetanus Toxoid and pneumococcal surface adhesin A. The method we report is sensitive, accurate, and precise with a linear range of 0.625 to 40 μg/mL and the limit of quantification is 0.12, 1.74, and 13.2 μg/mL for CRM197, PsaA, and Tetanus Toxoid, respectively. Spike recoveries were between 80 and 120% for all the conjugates using different carrier proteins. Reported method is simple, direct and can be used for quantitative determination of any free carrier protein in polysaccharide conjugates with greater application in polysaccharide‐protein conjugate manufacturing and release in the vaccine industry.