CRM 197 Purification Using Redox Control
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Solution Overview
Problem
Current methods for purifying CRM 197 from bacterial production organisms face challenges in maintaining the proper disulfide bridges, leading to protein degradation and low yields, which results in high production costs and supply shortages for conjugate vaccines.
Innovation Solution
The use of oxidized disulfide compounds, such as glutathione disulfide, during purification to maintain the redox state of CRM 197, preventing cleavage and degradation, and employing novel purification schemes involving chromatography steps like hydrophobic interaction, anion exchange, and hydroxyapatite chromatography to recover intact CRM 197 with high purity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional purification methods are used without redox control, then the purification process is simpler, but CRM 197 degrades due to disulfide bridge disruption
Solution Approach 1:
The patent introduces oxidized disulfide compounds (such as glutathione disulfide, cystine, or cystamine) as intermediary substances that mediate between the purification process and CRM 197's disulfide bridges. These compounds maintain the redox state of CRM 197 throughout purification steps, preventing degradation while enabling standard chromatographic procedures to proceed effectively.
Solution Approach 2:
The patent modifies the chemical environment parameters by controlling redox conditions through the addition of oxidized disulfide compounds. This parameter change (maintaining oxidative redox state) prevents disulfide bridge reduction and subsequent protein degradation, allowing the purification process to proceed with standard techniques without requiring specialized equipment.
2Productivity
If recombinant bacteria are used to produce CRM 197, then production cost decreases, but yield remains low due to protein degradation
Solution Approach 1:
The patent applies preliminary protective action by adding oxidized disulfide compounds to the purification stream before CRM 197 degradation can occur. This preliminary redox control prevents proteolytic degradation during the purification process, ensuring that the majority of CRM 197 produced by recombinant bacteria is recovered in intact form, thereby increasing yield.
3Manufacturing precision
If multiple purification steps are performed, then purity increases, but CRM 197 degradation increases due to extended exposure to harmful conditions
Solution Approach 1:
The patent ensures continuity of protective action by maintaining oxidized disulfide compounds throughout all purification steps. This continuous redox control ensures that CRM 197 is protected from degradation during each stage (chromatography, filtration, concentration), allowing multiple purification steps to be performed sequentially while maintaining both high purity and high recovery.
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 increases the yield of intact CRM 197, reduces degradation, and improves its stability, enabling higher purity and reduced contamination compared to traditional methods, thus addressing the supply and cost issues in vaccine production.
Implementation Method 1
The use of oxidized disulfide compounds, such as glutathione disulfide, during purification to maintain the redox state of CRM 197, preventing cleavage and degradation
Implementation Method 2
employing novel purification schemes involving chromatography steps like hydrophobic interaction, anion exchange, and hydroxyapatite chromatography
Implementation Method 3
employing novel purification schemes involving chromatography steps like hydrophobic interaction, anion exchange, and hydroxyapatite chromatography
Implementation Method 4
employing novel purification schemes involving chromatography steps like hydrophobic interaction, anion exchange, and hydroxyapatite chromatography
Data Source
AI summary
The present invention relates to the use of redox agents for purification of the CRM 197 variant of diphtheria toxin. The invention further relates to multi-step purification of CRM 197 from bacterial fermentates.