The pathogenic bacteria classified as Streptococcus pneumoniae (pneumococci, Pn) have been subdivided into 84 antigenic serotypes, based on the capsular polysaccharide (PnPs) of the organism. Disease states attributable to these organisms include pneumonia, meningitis, otitis media, bacteremia and acute exacerbations of chronic bronchitis, sinusitis, arthritis, and conjunctivitis. The preponderance of these diseases, however, are caused by a limited subset of the 84 known isolates. Thus, a polyvalent vaccine containing the PnPs from the most prevalent and pathogenic isolates of the organism can provide protection against a very high percentage of the most frequently reported pathogens of this class.
The PnPs utilized for preparing conjugate vaccines are always associated with a substantial amount of a common impurity/contaminant, called C-Polysaccharide (C-Ps). Although the presence of the C-polysaccharide contaminant does not interfere with the immune responses against the type specific antigens, production of anti-C-polysaccharide antibodies may correlate with the tissue destruction observed in some unresolved pneumococcal infections. The C-Ps content could compromise the efficacy of a pneumococcal conjugate vaccine.
The C-Ps has also been classified as a polysaccharide impurity by WHO, refer WHO TRS 19-23 Oct. 2009 (Recommendations to assure the quality, safety and efficacy of pneumococcal conjugate vaccines).
Thus a need for improving purity of the polysaccharides i.e. reduction in contamination with group-specific C-polysaccharide always exist in the industry.
U.S. Pat. No. 4,242,501 claims ammonium sulphate based polysaccharide precipitation, wherein C-Polysaccharide content is reduced to less than 0.5%. The purification process however consists of 12 steps and utilizes alcohol for precipitation also.
U.S. Pat. No. 5,623,057 claims C-polysaccharide content less than 3% by utilizing Isopropyl alcohol based PnPs purification and also employs ion exchange chromatography before or after partial hydrolysis (Sonic or thermal) of polysaccharide.
U.S. Pat. No. 5,714,354 claims an “alcohol free” PnPs purification process, utilizes CTAB (1-4%), hydroxyapatite chromatography and ion exchange chromatography. Here CTAB step removes majority of C-polysaccharide.
U.S. Pat. No. 5,847,112 claims C-Ps reduction by 3-20 fold, However here PnPs purification comprises of multiple isopropyl alcohol and cetavlon steps.
1572/MUM/2010 claims deoxycholate & Hydrophobic interaction chromatography based (i.e. alcohol and CTAB free) purification. Here C-Ps content is 4-14%.
The prior art methods utilize CTAB, alcohol and are multi-step. CTAB has been previously utilized for selectively precipitation of polysaccharide, however CTAB is a hazardous chemical.
Further removal of CTAB from precipitated PnPs requires ethanol. Use of ethanol during pneumococcal polysaccharide purification is associated with following operational problems: a) Use of ethanol requires a flame proof facility; b) designing such facility is very costly, c) ethanol is under custom/Govt. Regulation, d) ethanol is hazardous, e) effluent treatment is very difficult f) amount of ethanol requirement is huge and it's is almost ˜4-6 L of ethanol per liter of process material and g) requires costly charcoal filtration and lyophilization steps.
The prior art methods that utilize ethanol and CTAB steps require 77-90 hrs to achieve the C-Ps-reduced polysaccharide preparation and hence are laborious.
Surprisingly we have found that when a preferred single or multiple step chromatography is utilized for preparation of pneumococcal polysaccharides, a substantial reduction of group specific C-Ps content is observed. The said process being alcohol and CTAB free is easily scalable, cost efficient and less laborious.