Polysaccharide purification for vaccine production using lytic enzymes, tangential flow filtration, and multimode chromatography
Inventors
Kapre, Subhash V. • Datta, Anup K.
Assignees
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Publication Number
US-10702596-B2
Publication Date
2020-07-07
Expiration Date
Abstract
An improved, cost effective and shortened process of purification of the capsular polysaccharide of S. pneumoniae, Group B Streptococcus, H. influenzae, S. typhoid and N. meningitidis is disclosed. The process includes a cocktail of enzyme treatment, tangential flow filtration, and multimodal chromatography purification. For Gram-negative bacteria, endotoxin removal process involves endotrap HD resin. This shortened process achieved the purity required by WHO/EP/BP for the use in human vaccine preparation, with simple steps and higher yield as compared to conventional processes. The steps of the process avoid use of organic solvents such as, for example, alcohol, phenol, and ultracentrifugation that are otherwise expensive and time consuming to perform, and/or health hazards for commercial use. This process disclosed is also simple, efficient, non-toxic, easy to scale-up, and environmentally friendly.
Core Innovation
The invention describes bacterial capsular-polysaccharide compositions treated with deoxycholate and antimicrobial enzymes to reduce residual nucleic acid and protein. The described purification uses clarification, enzyme treatment, filtration, and chromatography, with additional endotoxin removal for Gram-negative materials. [procedural detail omitted for safety]
The provided content states that the process avoids conventional alcohol and phenol precipitation and ultracentrifugation. It reports vaccine-grade purity and yields of about 65–78% in listed examples, with tables and NMR figures documenting low residual protein, nucleic acid, and endotoxin and removal of cell-wall polysaccharide. The background section itself is not included in the provided content.
Claims Coverage
The independent claims cover bacterial polysaccharide compositions treated with deoxycholate and antimicrobial enzymes, with specified limits on residual nucleic acid and protein, across two inventive features.
Bacterial polysaccharide composition with reduced impurities
Claim 1 covers a polysaccharide composition obtained or derived from Gram-positive or Gram-negative bacteria, treated with deoxycholate and an antimicrobial enzyme, containing no more than 0.35% nucleic acid and 0.85% protein after treatment.
Bacterial polysaccharide composition with specified enzyme components
Claim 30 covers a polysaccharide composition obtained or derived from Gram-positive or Gram-negative bacteria, treated with deoxycholate and an antimicrobial enzyme comprising Benzonase, Mutanolysin/lysozyme, β-D-N-acetyl glucosaminidase, and Proteinase K. The composition contains no more than 0.35% nucleic acid and 0.85% protein after treatment.
The independent claims center on bacterial polysaccharide compositions treated with deoxycholate and antimicrobial enzymes, and set limits for residual nucleic acid and protein. The second independent claim further specifies the enzyme components.
Stated Advantages
The described process avoids conventional alcohol and phenol precipitation and ultracentrifugation.
The process reports vaccine-grade purity and yields of about 65–78% in listed examples.
The reported analyses document low residual protein, nucleic acid, and endotoxin, and removal of cell-wall polysaccharide.
Documented Applications
The claims describe the polysaccharide composition as part of an immunological composition, with a dependent claim specifying an adjuvant.
The disclosed bacterial sources include Streptococcus pneumoniae, Group B Streptococcus, Haemophilus influenzae, and Neisseria meningitidis.
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