Method for separation of protein and other impurities from microbial capsular polysaccharides
Inventors
Matur, Ramesh Venkat • Kandimalla, Vivek Babu • Mantena, Narender Dev • Datla, Mahima • Reddy, Muthyala Venkateswara • Charan, Kantam
Assignees
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Publication Number
US-10836839-B2
Publication Date
2020-11-17
Expiration Date
Abstract
The invention relates to a method for the removal of protein and other impurities from microbial capsular polysaccharides. More particularly, the present invention relates to isolation of microbial capsular polysaccharides in pure form after removal of protein and other impurities.
Core Innovation
The invention provides a method for isolating a polysaccharide in a substantially pure form from bacteria selected from Neisseria meningitidis, Streptococcus pneumoniae, Haemophilus influenzae type b, and Salmonella typhi. The method contacts a solution of lysed cells comprising the polysaccharide, proteins, nucleic acids, cell wall components and other impurities with silicone dioxide (SiO2), and the polysaccharide is separated from the SiO2 by filtration or by centrifugation.
The method isolates the polysaccharide without using chromatography. In the described approach, polysaccharide solutions from lysed microbial cells are exposed to SiO2 to enrich polysaccharides while reducing protein and other impurities, including nucleic acids and cell-wall polysaccharides, with substantial protein impurity reduction shown across multiple Streptococcus pneumoniae serotypes.
The document further specifies embodiments by defining properties of SiO2 and conditions of exposure or addition, including SiO2 particle size ranges, SiO2 amount, and contact or addition temperature and time windows. Depyrogenated or pyrogenated SiO2, including Aerosil® and Aeroperl®, is also described, together with reported yield and relative purity metrics.
Claims Coverage
The partial content includes two independent claims. Across them, the coverage centers on isolating substantially pure polysaccharides from specified bacteria by contacting lysed-cell solutions with silicone dioxide (SiO2) and separating the polysaccharide from SiO2 by filtration or centrifugation, expressly without chromatography. Additional dependent features refine SiO2 material parameters and contact or addition conditions, and narrow the bacterial starting material to Streptococcus pneumoniae with specified serotypes.
Silicone dioxide contacting and separation without chromatography
A method for isolating a polysaccharide in a substantially pure form from bacteria selected from Neisseria meningitidis, Streptococcus pneumoniae, Haemophilus influenzae type b, and Salmonella typhi by exposing or contacting a solution of lysed cells comprising the polysaccharide, proteins, nucleic acids, cell wall components and other impurities with silicone dioxide (SiO2), and separating the polysaccharide from the SiO2 by filtration or by centrifugation, without using chromatography.
Adding an SiO2 suspension to a lysed-cell solution and separating by filtration or centrifugation without chromatography
A method of isolating a polysaccharide in a substantially pure form from bacteria selected from Neisseria meningitidis, Streptococcus pneumoniae, Haemophilus influenzae type b, and Salmonella typhi comprising preparing a solution of lysed cells comprising the polysaccharide, proteins, nucleic acids, cell wall components and other impurities and a suspension of silicone dioxide (SiO2) in water or a buffer; adding the suspension of SiO2 to the solution of lysed cells; and isolating the polysaccharide from the SiO2 in a substantially pure form, wherein the polysaccharide is isolated from the SiO2 by filtration or by centrifugation and without using chromatography.
Both independent claims cover polysaccharide isolation from the listed bacterial species using SiO2 contact or addition followed by filtration or centrifugation separation, with the distinguishing limitation that chromatography is not used. Dependent claims further specify SiO2 particle size and amount ranges and define temperature and period ranges for SiO2 exposure or addition, and narrow embodiments to Streptococcus pneumoniae with explicitly listed capsular serotypes.
Stated Advantages
Substantially pure polysaccharide isolation without using chromatography.
Reduction of protein impurities while enriching polysaccharides.
Relative purity and yield are reported.
Documented Applications
Preparation of immunogenic or conjugate compositions using a carrier, including CRM197, and related toxoid components described as immunogenic composition components.
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