RNA Bacteriophage VLP Oligonucleotide Packaging Efficiency
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Solution Overview
Problem
Current processes for producing recombinant virus-like particles of RNA bacteriophages packaged with oligonucleotides are not optimized for efficiency, scalability, and purity, particularly in the reassembly process, where the use of aggregated oligonucleotides with specific particle sizes can significantly enhance efficiency and purity but are not adequately addressed in prior art.
Innovation Solution
A process involving the self-assembly of coat protein in the presence of aggregated oligonucleotides, where oligonucleotides with a relative peak start time of 50 to 110% are achieved through controlled aggregation and disaggregation steps, optimizing the size exclusion HPLC conditions and temperature ramps to enhance the packaging efficiency and purity to at least 99%.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional reassembly processes are used to produce virus-like particles packaged with oligonucleotides, then production can proceed with standard methods, but the efficiency and purity of the assembled product are insufficient
Solution Approach 1:
The patent applies parameter changes by optimizing the aggregation state of oligonucleotides to a specific relative peak start time range (50-110%, preferably 80-95%) and controlling particle size distribution during the reassembly process. These parameter optimizations dramatically improve both packaging efficiency and product purity, achieving at least 99% purity while maintaining high efficiency.
2Productivity
If oligonucleotide aggregation is increased to improve packaging efficiency, then more oligonucleotides are packaged into virus-like particles, but the particle size distribution becomes heterogeneous and purity decreases
Solution Approach 1:
The patent precisely controls the aggregation parameter of oligonucleotides by adjusting the relative peak start time to fall within 50-110% (preferably 80-95%) of the capsid standard in size exclusion HPLC. This optimized aggregation state ensures efficient packaging while maintaining homogeneous particle size distribution and high purity.
Solution Approach 2:
The patent uses size exclusion HPLC with capsid standard as a reference to monitor and control the aggregation state of oligonucleotides during the process. This feedback mechanism allows real-time adjustment of aggregation parameters to maintain optimal particle size distribution and purity.
3Productivity
If the reassembly process is scaled up for large-scale production, then manufacturing capacity increases, but maintaining high purity and efficiency becomes more difficult
Solution Approach 1:
The patent performs preliminary optimization of oligonucleotide aggregation state before the actual reassembly process. By pre-adjusting the aggregation to the optimal relative peak start time range (50-110%, preferably 80-95%), the process ensures that scaling up production will maintain both high efficiency and purity without requiring complex adjustments during large-scale operations.
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 dramatically improves the efficiency and purity of the packaging process, achieving a protein yield and oligonucleotide yield of at least 75% with a product that is at least 99% pure, facilitating the production of high-quality virus-like particles packaged with oligonucleotides.
Implementation Method 1
Oligonucleotides comprising at least one poly G stretch are capable of aggregation. The aggregation state of an oligonucleotide can be characterized by the relative peak start time in size exclusion HPLC using the capsid of said RNA bacteriophage as standard.
Implementation Method 2
adjusting the temperature of solution II to temperature III, wherein said temperature III is 50 to 99° C.; and (c) incubating said oligonucleotide in solution II at temperature III, wherein said incubating is performed until said oligonucleotide comprises a relative peak start time of 50 to 110%
Implementation Method 3
the self assembly of the coat protein is performed in the presence of aggregated oligonucleotide. During said process said virus-like particle is formed by self assembly of coat protein of said RNA bacteriophage in the presence of an oligonucleotide.
Data Source
AI summary
The invention provides processes for the producing compositions comprising (i) a virus-like particle, wherein said virus-like particle is a virus-like particle of an RNA bacteriophage, and (ii) an oligonucleotide, wherein said oligonucleotide is packaged into said virus-like particle. The invention further provides processes for producing nucleotide compositions comprising oligonucleotides suitable to be used in the processes mentioned before. The invention further provides nucleotide compositions obtainable by the processes of the invention and uses thereof. The invention further provides compositions comprising (i) a virus-like particle, wherein said virus-like particle is a virus-like particle of an RNA bacteriophage, and (ii) an oligonucleotide, wherein said oligonucleotide is packaged into said virus-like particle, wherein said compositions are obtainable by the processes of the invention and wherein said compositions preferably comprises a purity of at least 98%, most preferably of at least 99%.