Method for removal of impurities from bacterial capsular polysaccharide based preparations
Inventors
DHERE, Rajeev Mhalasakant • Pisal, Sambhaji Shankar • ANNAMRAJU, Dattatreya Sarma
Assignees
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Publication Number
US-11739356-B2
Publication Date
2023-08-29
Expiration Date
Abstract
The present invention relates to an improved process for purification of bacterial capsular polysaccharides, more specifically capsular polysaccharides of gram negative bacteria. The process comprises of concentration and dia filtration of harvest, treatment with anionic detergent and strong alkali followed by centrifugation, diafiltration and cationic detergent based precipitation of bacterial polysaccharides. The process results in significant reduction of endotoxin, protein and nucleic acid impurities thereby providing higher recovery of capsular polysaccharide with the desired O-acetyl levels. Said process is scalable, non-enzymatic, and employs fewer purification steps.
Core Innovation
The invention relates to a method for isolation of polysaccharide from Neisseria meningitidis selected from serotype A and serotype X. The method uses a detergent comprising sodium dodecyl sulfate added to a crude polysaccharide solution at a final concentration of 0.1 to less than 1%, followed by addition of EDTA and sodium acetate. The crude solution is then subjected to alcohol precipitation, centrifugation, and retention of the supernatant.
A key part of the workflow is removal of detergent present in the supernatant by precipitation of detergent using a potassium salt selected from potassium chloride, potassium acetate, potassium sulfate, potassium carbonate, potassium bicarbonate, potassium phosphate, potassium hydrogen phosphate, potassium dihydrogen phosphate, potassium nitrate, and combinations thereof. After detergent removal, the polysaccharide is concentrated and diafiltrated.
The obtained purified polysaccharide from Neisseria meningitidis selected from serotype A and serotype X has a recovery of 60-80%, while maintaining desired polysaccharide characteristics associated with the O-acetyl level. Additional refinements include optional treatment with a cationic detergent selected from cetyltrimethylammonium salt, tetrabutylammonium salt, myristyltrimethylammonium salt, hexadimethrine bromide, or combinations thereof, and then collection of a pellet, dissolving the pellet in alcohol, and performing salt-based precipitation.
Claims Coverage
One independent claim is provided, describing a detergent-based, non-enzymatic isolation workflow for polysaccharide from Neisseria meningitidis (serotype A and serotype X) with a defined recovery range and a defined potassium-salt detergent removal mechanism. The inventive features cover sodium dodecyl sulfate treatment, EDTA/sodium acetate addition, alcohol precipitation with supernatant retention, potassium-salt detergent precipitation, and subsequent concentration/diafiltration, with optional cationic detergent refinements in dependent claims.
Sodium dodecyl sulfate treatment at a defined concentration
Adding a detergent comprising sodium dodecyl sulfate to a crude polysaccharide solution comprising Neisseria meningitidis selected from serotype A and serotype X, wherein the final concentration of sodium dodecyl sulfate in the solution is 0.1 to less than 1%.
EDTA and sodium acetate addition
Adding EDTA and sodium acetate to the solution.
Alcohol precipitation with supernatant retention
Subjecting the solution to alcohol precipitation, followed by centrifugation and retention of supernatant.
Detergent removal by potassium-salt precipitation
Removing the detergent present in the supernatant by precipitation of detergent using a potassium salt selected from the group consisting of potassium chloride, potassium acetate, potassium sulfate, potassium carbonate, potassium bicarbonate, potassium phosphate, potassium hydrogen phosphate, potassium dihydrogen phosphate, potassium nitrate, and combinations thereof.
Concentrating and diafiltrating the polysaccharide
Concentrating and diafiltrating the polysaccharide.
Purified polysaccharide recovery range
Wherein the obtained purified polysaccharide from Neisseria meningitidis selected from serotype A and serotype X has a recovery of 60-80%.
Optional cationic-detergent pellet formation and salt-based precipitation
The method further comprises mixing the polysaccharide solution with a cationic detergent selected from cetyltrimethylammonium salt, tetrabutylammonium salt, myristyltrimethylammonium salt, hexadimethrine bromide, or combinations thereof, followed by centrifugation to collect a pellet, dissolving the pellet in alcohol, and performing salt-based precipitation.
Overall, the claim set centers on a defined sodium dodecyl sulfate level combined with EDTA and sodium acetate, alcohol precipitation with supernatant retention, and detergent removal via precipitation using specified potassium salts, followed by concentration and diafiltration, yielding a 60-80% recovery purified polysaccharide. Dependent claims further narrow optional use of specific cationic detergents followed by pellet handling and salt-based precipitation.
Stated Advantages
Provides a purified polysaccharide recovery of 60-80%.
Allows impurity reduction and maintenance of desired O-acetyl levels while purifying polysaccharide from Neisseria meningitidis (serotype A and serotype X).
Documented Applications
Producing purified polysaccharide fractions characterized by specified purity and impurity thresholds, including endotoxin impurity and serotype-specific impurity limits, for subsequent conjugate/immunogenic compositions as described in the document.
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