Streptococcus Pneumoniae Polysaccharide Purification via pH Adjustment
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Solution Overview
Problem
Current methods struggle to effectively reduce residual protein levels in pneumococcal polysaccharide preparations to meet specifications, as excess protein remains solubilized in the lysate after purification, posing a risk of adverse events and requiring improved upstream processing.
Innovation Solution
A process involving the growth of Streptococcus pneumoniae in a soy-based medium, followed by controlled pH adjustment to less than 5.5 after cell lysis, which precipitates soluble proteins, allowing for their removal through centrifugation and filtration, thereby preserving capsular polysaccharides and reducing protein content.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If conventional purification processing is used to remove protein from polysaccharide, then some protein is removed, but residual protein content remains above specification
Solution Approach 1:
The patent applies preliminary action by adjusting the pH to 6.6 before purification to precipitate deoxycholate and cell membrane complexes, and then lowering the pH to less than 5.5 to precipitate additional soluble proteins. This pre-precipitation step removes proteins before the main purification process, enabling the final product to meet protein content specifications that conventional purification alone cannot achieve.
Solution Approach 2:
The patent utilizes parameter changes by adjusting the pH of the lysate broth through controlled addition of acid. The pH is first adjusted to 6.6 to precipitate detergent and membrane complexes, then further lowered to less than 5.5 to precipitate soluble proteins. This systematic pH modification changes the solubility characteristics of different components, enabling selective precipitation and effective protein removal while preserving polysaccharide in solution.
2Manufacturing precision
If pH is lowered to precipitate proteins, then soluble protein is removed, but capsular polysaccharide must be preserved in solution
Solution Approach 1:
The patent exploits differential parameter responses of proteins and polysaccharides to pH changes. Proteins precipitate at pH less than 5.5 due to isoelectric point effects, while capsular polysaccharides remain soluble in this acidic range. This selective parameter-based separation enables effective protein removal without significant polysaccharide loss, maintaining high recovery yields.
Solution Approach 2:
The patent replaces mechanical separation methods with chemical precipitation controlled by pH adjustment. Instead of relying solely on mechanical filtration or centrifugation to remove proteins, the process uses controlled chemical precipitation through acidification, which selectively precipitates proteins while leaving polysaccharides in solution, followed by simple filtration or centrifugation to remove the precipitated protein material.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach significantly reduces soluble protein levels, resulting in a cleaner product for purification, achieving higher polysaccharide recoveries and yields, and meeting protein specification without significant modification to the purification process.
Implementation Method 1
lowering the pH of the cellular lysate to less than 5.5 to precipitate out the detergent and most of the soluble proteins
Implementation Method 2
agitating the cellular lysate for a time sufficient to assure complete lysis and polysaccharide release
Implementation Method 3
processing the solution and precipitate by centrifugation and/or filtration, whereby the capsular polysaccharide in solution is preserved and the soluble protein is effectively reduced
Data Source
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AI summary
A process for reducing the protein content and preserving the capsular polysaccharide content in a complex cellular Streptococcus pneumoniae lysate broth prior to purification is described. Utilizing pH reduction after cellular lysis has resulted in a purified polysaccharide that consistently meets the protein specification, and higher recovery yields of polysaccharide during the purification process.