Immunogenic Protein Refolding for Malaria Vaccine Stability
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Solution Overview
Problem
Current methods fail to produce a stable, immunogenic protein with multiple cysteines that maintains purity and functionality, leading to low yields and instability, making it difficult to develop effective vaccines for malaria, particularly due to challenges in disulfide bond formation and co-purification of host proteins during refolding.
Innovation Solution
A process involving a detergent-dextrin mixture, a co-solvent, and a redox pair in the refolding buffer, along with specific chromatography steps, is used to achieve high purity (>98%) and stability up to 2 years, with a yield increase from 5% to 20%, and the use of lyophilization with sucrose enhances the stability of the protein composition.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If recombinant protein with multiple cysteines is expressed in E. coli at high levels, then productivity increases, but the protein aggregates into inclusion bodies and loses functionality
Solution Approach 1:
The patent applies preliminary action by performing refolding under controlled conditions before final purification. The inclusion bodies are solubilized and refolded in a specific buffer system containing redox agents (GSH/GSSG) and additives (arginine, detergents) that promote correct disulfide bond formation. This preliminary refolding step ensures the protein regains its native structure and functionality before being purified to high purity, thus resolving the contradiction between high yield and functional integrity.
2Manufacturing precision
If inclusion bodies are solubilized using strong denaturants, then the protein can be purified, but the complexity of the process increases and yield decreases
Solution Approach 1:
The patent employs parameter changes by systematically optimizing the refolding buffer composition and conditions. The buffer contains specific concentrations of redox agents (GSH 5-20 mM, GSSG 0.5-5 mM), arginine (0.1-1.0 M), and detergents (0.01-0.5% v/v). The pH is maintained at 7.0-8.5, and refolding is performed at 4-25°C for 2-24 hours. These optimized parameters enable efficient refolding and purification with reduced process complexity and improved yield while achieving >90% purity.
3Reliability
If the number of cysteines in the protein increases, then immunogenicity may improve, but the number of possible disulfide bond combinations increases exponentially making correct folding difficult
Solution Approach 1:
The patent uses intermediary substances to facilitate correct disulfide bond formation in proteins with multiple cysteines. The refolding buffer contains redox agents (GSH and GSSG) that act as intermediaries to promote oxidative folding. Additionally, arginine and small amounts of detergents serve as intermediaries to prevent aggregation and promote proper folding. These intermediaries enable the protein to navigate the complex energy landscape of multiple possible disulfide configurations and reach the native functional state, thus achieving high immunogenicity with accurate folding.
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 process results in a stable immunogenic protein with enhanced shelf life and immunogenicity, achieving high antibody titers and partial protection against malaria, with the protein remaining functional and stable for up to 3 years, overcoming previous limitations in yield and stability.
Implementation Method 1
a refolding buffer comprising a detergent-dextrin mixture, a co-solvent and a redox pair
Implementation Method 2
the use of lyophilization with sucrose enhances the stability of the protein composition
Data Source
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AI summary
The invention describes a stable immunogenic protein having multiple cysteines molecules wherein the protein is having stability up to two years and purity more than 98% particularly rPvRII and/or rPfF2. It also discloses a method for producing said immunogenic protein comprising the following steps: culturing the host E.coli cells containing a desired recombinant gene construct comprising a codon optimized gene sequence of rPvRII and/or rPfF2 to produce cells in high density; inducing expression rPvRII and/or rPfF2 as inclusion bodies; harvesting the cells and isolating the said inclusion bodies; separating rPvRII and/or rPfF2 from inclusion bodies by repeated sequential washing and solubilizing with chaotrophic agents comprising guanidine hydrochloride and / or urea; purifying the protein by subjecting to metal-chelate affinity chromatography; re-folding of the purified rPvRII and/or rPfF2 obtained in step e) with a redox system to recover a high yield of the soluble protein, followed by further purifying the desired protein by removing impurities by subjecting to chromatography. Further the invention discloses formulation comprising rPvRII or rPfF2, preferably being lyophilized using polysaccharides preferably sucrose, lactose, and pharmaceutically acceptable adjuvants such as aluminum hydroxide, aluminum phosphate, CpG nucleotides, non-CpG nucleotides, Montanide ISA-720, MF-59, Mono- phosphoryl Lipid-A (MPL-A) and QS-21.