Method for Simultaneous Quantitation of Free Carrier Protein and Free Polysaccharide in Glycoconjugate Vaccines by High Performance Liquid Chromatography

Abstract

Disease caused by bacterial infections affect children and adults worldwide can be potentially life-threatening. Bacterial strains, such as Staphylococcus aureus and Streptococcus pnueumoniae (pneumococcus), express capsular polysaccharides on their surface, which act as a virulence factor for evading the immune system and are structurally unique depending on the serotype. Vaccines designed against the capsular polysaccharides have shown to be effective in reducing the incident rate of disease caused by bacterial injections. Glycoconjugate vaccines comprise large molecular mass bacterial capsular polysaccharides conjugated to a carrier protein, which helps activate T cells that trigger immunological memory functions. Un conjugated (free) carrier protein and polysaccharide are critical attributes typically included in analytical testing for glycoconjugate vaccines and must be monitored throughout the vaccine development process. High levels of free polysaccharide or carrier protein may be indicative of poor conjugation efficiency or product degradation caused by manufacturing inconsistencies, formulation, or storage conditions. In this work, we will present the development of an HPLC method to simultaneously quantitate free carrier protein and free polysaccharide in a glycoconjugate vaccine drug substance. This method would be developed for two S. aureus glycoconjugate drug substances, CP5-CRM197 and CP8-CRM197. Initial work focused on using hydrophobic interaction chromatography (HIC) as the mode of separation by utilizing the often-ignored separation space prior to the void volume. However, the separation proved to be difficult in a 1D format due to the complexity of the sample mixture and the chromatographic behavior of the glycoconjugate. Therefore, a 2D-LC approach was taken to separate and quantitate the free carrier protein and free polysaccharide in the two S. aureus glycoconjugate drug substances. This work focused on the development of SEC and RPLC methods, 2D-LC instrumentation design, 2D-LC method development, and assay performance. Results were then compared to results from current technologies in place based on capillary electrophoresis (CE) for the quantitation of free carrier protein and free polysaccharide to assess the feasibility of supporting manufacturing process and formulation development studies. In addition, this methodology was explored as a potential platform technology for monitoring free carrier protein and polysaccharide in other glycoconjugate vaccine projects in the development pipeline.