Detergent-Free OMV Extraction via EDTA Chelation
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Solution Overview
Problem
Current methods for preparing outer membrane vesicles (OMV) from Gram-negative bacteria for vaccines face challenges such as low yield, low purity, and toxicity due to the use of detergent extraction, which affects the native structure and immunogenicity, particularly for serogroup B meningitis vaccines.
Innovation Solution
A detergent-free process involving cultivating Gram-negative bacteria to stationary growth phase, then incubating them in a medium with a metal chelating agent like EDTA at a pH higher than 8.0 to extract OMV, followed by sterilization and removal of bacteria, allowing for high yield and purity OMV production suitable for industrial scale.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If detergent extraction is used to remove LPS and increase vesicle release, then the immune response is enhanced and vesicle release is increased, but the native vesicle structure is changed, lipoproteins contributing to immunogenicity are removed, and aggregation is promoted
Solution Approach 1:
The patent extracts only the necessary component (LPS) from the OMV preparation while preserving the native structure. This is achieved through controlled extraction methods that selectively remove excess LPS without removing other immunogenic components like lipoproteins, thereby maintaining the native vesicle structure while still enhancing immune response.
Solution Approach 2:
The patent applies different treatment conditions to different aspects of the OMV preparation. By controlling extraction conditions locally (selective LPS removal without affecting other components), the method preserves the native structure and immunogenic lipoproteins while removing excess toxic LPS, thus achieving local quality optimization.
2Object-affected harmful factors
If detergent extraction is used to remove LPS, then LPS toxicity is decreased, but immunogenic lipoproteins are removed and cross-protection is not elicited
Solution Approach 1:
The patent converts the harmful effect of LPS into a beneficial outcome by using controlled extraction to remove only the toxic excess LPS while preserving the immunogenic lipoproteins. This selective removal transforms the harmful LPS presence into a benefit where toxicity is reduced but immunogenicity is maintained, enabling cross-protection.
Solution Approach 2:
The patent changes the concentration parameter of LPS selectively through controlled extraction. By adjusting extraction conditions to achieve optimal LPS concentration (removing excess toxic LPS while retaining sufficient immunogenic LPS and lipoproteins), the method reduces toxicity while maintaining cross-protective immunity.
3Stability of the object's composition
If detergent-free purification is used to retain all LPS, then native vesicle structure is preserved, but the vaccine becomes inherently toxic for parenteral immunization
Solution Approach 1:
The patent extracts excess LPS from the OMV preparation through controlled extraction methods that preserve the native vesicle structure. This selective extraction removes the toxic excess LPS while maintaining the intact native structure, making the vaccine suitable for parenteral immunization.
Solution Approach 2:
The patent changes the LPS concentration parameter through controlled extraction to achieve an optimal balance. By reducing LPS concentration from the high levels in detergent-free preparations to optimal levels, the method eliminates toxicity while preserving the native structure and immunogenicity.
4Productivity
If OMV yield is increased for feasible process development, then the sOMV process becomes viable, but the process complexity increases due to additional purification steps
Solution Approach 1:
The patent combines multiple functions into a single integrated process step. The extraction method simultaneously achieves OMV release, LPS concentration optimization, and purification in one step, thereby increasing yield without adding process complexity.
Solution Approach 2:
The patent creates a universal extraction process that serves multiple purposes: releasing OMV, optimizing LPS concentration, and purifying the preparation. This multi-functional approach increases productivity while avoiding additional purification steps, making the process suitable for industrial scale-up.
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 process achieves high yield and purity OMV production with reduced toxicity, maintaining native structure and immunogenicity, enabling effective vaccine development without the need for adjuvants, thus addressing the limitations of existing detergent-based methods.
Implementation Method 1
incubating them in a medium with a metal chelating agent like EDTA at a pH higher than 8.0 to extract OMV
Data Source
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AI summary
The present invention relates to the fields of medical microbiology and vaccines. In particular the invention relates to a process for detergent-free preparation of outer membrane vesicles (OMV) of Gram negative bacteria for use in vaccines, to OMV obtainable by said process, and to a pharmaceutical composition comprising such OMV. The present invention further relates to the use of OMV of the present invention as a medicament in particular for use in a method for eliciting an immune response.