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Updated: Jul 24, 2026
Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction
Published on: February 24, 2014
Development and characterization of an efficient, organic solvent-free capsular polysaccharide purification process
Shital S Jain1, Vikas K Singh2, Rajesh Kumar Kante2
1Serum Institute of India Pvt. Ltd., Hadapsar, Pune, Maharashtra, 411028, India; Savitribai Phule Pune University, Department of Biotechnology, Pune, Maharashtra, 411007, India.
Abstract:
Streptococcus pneumoniae serotype 22F is a significant concern in causing pneumococcal disease, and it is associated with increased rates of invasive pneumococcal disease (IPD) like septicaemia and meningitis. Capsular polysaccharide (CPS) of S. pneumoniae is the main causative agent for virulence, hence it is considered for vaccine development. The development of effective vaccine requires a high-purity CPS. Current purification methods utilize organic solvents, which raises the vaccine efficacy and safety concerns. In current study, we have established a novel, multi-step purification process for serotype 22F CPS that is entirely free of organic solvents. It integrates the treatment of fermentation harvest culture to deoxycholate (DOC), nuclease digestion, pH precipitation, and hydrophobic-interaction chromatography (HIC) using Octyl Sepharose to effectively remove major impurities viz. nucleic acids, proteins, and cell wall common polysaccharides (C-Ps). This optimized process yielded high capsular polysaccharide recovery of approximately 65 % with molecular weight 840 kDa. The final CPS structural identity was confirmed by 1H NMR and it meets stringent purity criteria as defined by WHO, with protein levels at 0.4 %, nucleic acids below 0.1 %, and cell wall polysaccharide (C-Ps) at 2.29 mol%. This solvent-free method provides a safe and scalable platform for the industrial production of pneumococcal polysaccharide antigens.
Insights
A new, solvent-free method purifies Streptococcus pneumoniae 22F capsular polysaccharide (CPS) for vaccines. This process enhances safety and efficacy by removing impurities, yielding high-purity CPS for industrial production.
Area of Science:
- Biochemistry
- Vaccine Development
- Microbiology
Background:
- Streptococcus pneumoniae serotype 22F causes significant invasive pneumococcal disease (IPD).
- Capsular polysaccharide (CPS) is key to S. pneumoniae virulence and a target for vaccine development.
- Current CPS purification methods use organic solvents, posing safety and efficacy concerns.
Purpose of the Study:
- To develop a novel, multi-step purification process for serotype 22F CPS.
- To eliminate the use of organic solvents in CPS purification.
- To ensure high purity and recovery of CPS for vaccine applications.
Main Methods:
- A solvent-free purification process integrating deoxycholate (DOC) treatment, nuclease digestion, pH precipitation, and hydrophobic-interaction chromatography (HIC).
- Octyl Sepharose was used for HIC to remove nucleic acids, proteins, and cell wall polysaccharides (C-Ps).
- Purified CPS was analyzed for purity, molecular weight, and structural identity using 1H NMR.
Main Results:
- Achieved high capsular polysaccharide recovery of approximately 65% with a molecular weight of 840 kDa.
- Demonstrated stringent purity criteria: protein levels at 0.4%, nucleic acids below 0.1%, and C-Ps at 2.29 mol%.
- Confirmed final CPS structural identity via 1H NMR.
Conclusions:
- The novel solvent-free purification method effectively removes major impurities from serotype 22F CPS.
- This process yields high-purity CPS meeting WHO standards, suitable for vaccine development.
- The method offers a safe and scalable platform for industrial production of pneumococcal polysaccharide antigens.
