Abstract
The Streptococcus pneumoniae bacteria has over 100 known serotypes that display a continuous change in prevalence by patients’ age and geographical location and therefore necessitate continued efforts toward development of new vaccines with broader protection. Glycoconjugate vaccines have been instrumental in reducing global morbidity and mortality caused by Streptococcus pneumoniae infections. In these vaccines, the bacterial polysaccharide is conjugated to a carrier protein to enhance immunogenicity. To ensure well defined immunogenicity and stability of conjugated vaccines, reliable quantification of non-conjugated (free) polysaccharide is a critical, albeit challenging step during vaccine clinical dosing, release and stability monitoring. Multivalent preparations of Cross-reactive material 197 (CRM197)- conjugated pneumococcal polysaccharide materials often contain only nanogram levels of each individual free polysaccharide at final container concentrations. We have developed a novel method for the separation of free polysaccharides from conjugated material that requires no sample derivatization, employing instead an approach of quantitative immunoprecipitation of CRM197 with 3 different monoclonal antibodies and magnetic beads. A mix of antibodies against both linear and conformational epitopes enables successful removal of conjugates regardless of the protein folded state. The remaining free polysaccharide is subsequently measured in a serotype-specific ELISA.
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Acknowledgments
Funding of this work was provided entirely by Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, USA. The authors gratefully acknowledge Thorsten Verch, Gabriela Tamassia, Liming Guan, and Qiana Wilson for their help in ELISA assay planning and execution. Our gratitude goes toward our colleagues in the antibody reagent and formulation development groups for providing the materials used in this study.
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for this research was provided by Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Rahway, NJ, USA. The authors are employees of Merck & Co., Inc., and may be eligible for stock options or have stock ownership. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
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All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by MG and RS. The first draft of the manuscript was written by MG and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
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Funding for this research was provided by Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, USA. The authors are employees of Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, USA, and may be eligible for stock options or have stock ownership in Merck & Co., Inc., Rahway, NJ, USA. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
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Grozdanovic, M., Samuel, R., Grau, B. et al. Serotype-specific quantification of residual free polysaccharide in multivalent pneumococcal conjugate vaccines. Glycoconj J 41, 47–55 (2024). https://doi.org/10.1007/s10719-023-10143-6
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DOI: https://doi.org/10.1007/s10719-023-10143-6