EV71 Virus-Like Particle Purification via Chromatography
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Solution Overview
Problem
Current methods for producing EV71 virus-like particle vaccines face challenges such as low antigenic purity, significant clinical side effects, and complex purification processes, particularly in large-scale production, due to the use of prokaryotic and eukaryotic expression systems, which affect the safety and efficacy of the vaccine.
Innovation Solution
A method involving high-density fermentation of engineered Hansenula yeast with intermittent glycerin addition and continuous methanol induction, followed by cell disruption, precipitation, ultrafiltration, ion exchange chromatography, and hydroxyapatite chromatography to purify recombinant EV71 virus-like particles, resulting in a highly safe and effective vaccine.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If inactivated vaccine is used for EV71 prevention, then vaccine development progress is achieved, but antigenic purity is low and clinical side effects are significant
Solution Approach 1:
The patent extracts only the capsid protein antigens (VP1, VP2, VP3, VP4) from the complete virus, eliminating viral nucleic acid and other harmful components. This extraction approach produces virus-like particles that retain immunogenicity while removing pathogenic elements, thereby improving safety and antigenic purity simultaneously
Solution Approach 2:
The patent creates virus-like particles that copy the structural characteristics of the EV71 virus capsid without containing viral genetic material. These synthetic copies mimic the native virus structure to induce immune response while being inherently safer, resolving the contradiction between vaccine effectiveness and safety
2Reliability
If prokaryotic or eukaryotic expression systems are used for VLP production, then vaccine efficacy is improved, but purification process becomes complex for large-scale production
Solution Approach 1:
The patent segments the purification process into distinct stages: cell disruption, differential centrifugation, density gradient centrifugation, and chromatography. Each stage targets specific contaminants, systematically simplifying the overall purification process while maintaining high VLP purity and yield for large-scale production
Solution Approach 2:
The patent utilizes changes in physical parameters (density, size, charge) during sequential purification steps to selectively separate VLPs from contaminants. By optimizing these parameter-based separation techniques, the process achieves high purification efficiency with manageable complexity for industrial scale-up
3Reliability
If complete virus inactivation is performed, then vaccine safety is improved, but antigen targets are destroyed and immunogenicity is affected
Solution Approach 1:
The patent extracts only the essential capsid protein components needed for immunogenicity while removing the viral genome and other non-essential elements. This selective extraction preserves antigenic targets without requiring complete virus inactivation, maintaining immunogenicity while ensuring safety
Solution Approach 2:
The patent uses recombinant expression systems as intermediaries to produce capsid proteins that self-assemble into virus-like particles. This intermediary approach allows production of immunogenic structures without handling complete infectious viruses, eliminating the need for harsh inactivation processes that could damage antigen targets
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
The method achieves high recovery rates, simple operation, and large-scale industrial production feasibility, producing a vaccine with enhanced immunogenicity and safety, free from adverse toxic effects and RNA carcinogenesis risks.
Implementation Method 1
the yeast cells were disrupted by high-pressure homogenization
Implementation Method 2
the supernatant was concentrated and purified by ultrafiltration
Implementation Method 3
the concentrate was purified by ion exchange chromatography
Implementation Method 4
the eluate was purified by hydroxyapatite chromatography
Data Source
AI summary
The present invention provides a method for purifying an EV71 virus-like particle and a method for preparing a vaccine thereof. The virus-like particle is obtained by performing high density fermentation cultivation on recombinantly engineered bacteria; inducing expression of the EV71 virus-like particle protein expression using methanol; collecting the bacteria by centrifugation and performing high-pressure homogenization for disruption; performing precipitation on the supernatant with ammonium sulfate; and purifying by redissolution, ultrafiltration, ion exchange chromatography, molecular sieve chromatography, hydroxyapatite chromatography, etc.