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-11680111-B2
Publication Date
2023-06-20
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 preparing an immunogenic composition by preparing a purified capsular polysaccharide and conjugating it to a carrier protein selected from diphtheria toxoid, tetanus toxoid, and CRM197. The method begins with exposing or contacting a solution of lysed Streptococcus pneumoniae cells comprising the capsular polysaccharide, proteins, nucleic acids, cell wall components, and other impurities with silicone dioxide (SiO2). This exposure is used to address impurities associated with the lysed cell solution.
After exposure, the capsular polysaccharide is separated from the SiO2 by filtration or by centrifugation. The method isolates the capsular polysaccharide in substantially pure form without using chromatography. This approach is directed to removing protein and other impurities, including nucleic acids and cell wall components, while maintaining the capsular polysaccharide as a purified product suitable for subsequent conjugation.
The disclosed approach is applied to microbial capsular polysaccharides, notably Streptococcus pneumoniae capsular polysaccharides, and is supported by performance data showing reduced protein levels across multiple pneumococcal serotypes. Additional results are described for removal of cell wall polysaccharide (CWPS) and visualization by SDS-PAGE, reflecting impurity reduction after SiO2 exposure and separation.
Claims Coverage
The document includes one independent claim describing the SiO2-based non-chromatographic purification of capsular polysaccharide from lysed Streptococcus pneumoniae, followed by conjugation to a selected carrier protein. Four additional dependent claims specify selected Streptococcus pneumoniae serotypes and quantify SiO2 particle size, contact temperature/time, and SiO2 amount.
SiO2 non-chromatographic purification from lysed Streptococcus pneumoniae
Exposing or contacting a solution of lysed Streptococcus pneumoniae cells comprising the capsular polysaccharide, proteins, nucleic acids, cell wall components and other impurities with silicone dioxide (SiO2); and separating the capsular polysaccharide from the SiO2 by filtration or by centrifugation without using chromatography to isolate the capsular polysaccharide in substantially pure form.
Carrier protein conjugation to prepared capsular polysaccharide
Conjugating the purified capsular polysaccharide to a carrier protein selected from the group consisting of diphtheria toxoid, tetanus toxoid, and CRM197.
Specified pneumococcal serotypes
Carrying out the method using Streptococcus pneumoniae that includes one or more specified serotypes selected from a defined list.
SiO2 particle size range
Providing SiO2 particles with a size between 0.01 μm and 200 μm.
SiO2 exposure temperature and contact duration
Exposing or contacting the solution of lysed cells with SiO2 at 15°C to 60°C for a period of time ranging from 10 minutes to 16 hours.
SiO2 amount by weight/volume
Presenting SiO2 in an amount ranging from 0.5% to 20% by weight/volume (w/v).
Overall, the claim set centers on SiO2 exposure of a lysed Streptococcus pneumoniae solution followed by filtration or centrifugation separation without chromatography to obtain substantially pure capsular polysaccharide, which is then conjugated to diphtheria toxoid, tetanus toxoid, or CRM197; dependent claims further specify pneumococcal serotypes and define SiO2 particle size, exposure temperature/time, and SiO2 amount.
Stated Advantages
Capsular polysaccharide is isolated in substantially pure form without using chromatography.
Reduction of protein levels in pneumococcal serotypes after SiO2 exposure and separation.
Removal of cell wall polysaccharide (CWPS).
Documented Applications
Preparation of an immunogenic composition comprising a purified capsular polysaccharide conjugated to a carrier protein (diphtheria toxoid, tetanus toxoid, or CRM197).
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