Bionanoparticle Purification Using Salt-Active Endonuclease Chromatography
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Solution Overview
Problem
Current methods for purifying bionanoparticles, such as viruses and viral vectors, are inefficient and require multiple unit operations, leading to impurities that can induce unwanted immune responses and reduce infectivity, with high salt concentrations needed to solubilize chromatin for enzymatic digestion.
Innovation Solution
A method using a salt-active endonuclease at low salt concentrations to enzymatically digest chromatin, followed by capture chromatography, enhancing purification efficiency and increasing loading volume without particle breakthrough.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If high salt concentrations are used to solubilize chromatin for enzymatic digestion, then chromatin solubility and enzymatic efficiency are improved, but virus stability and infectivity are worsened due to the sensitive nature of enveloped viruses
Solution Approach 1:
The patent changes the salt concentration parameter from high (conventional) to low (innovative), achieving chromatin digestion at physiological salt concentrations (10-500 mM NaCl) that are compatible with virus stability. This parameter change resolves the contradiction by finding an optimal range that satisfies both chromatin solubility and virus protection requirements
Solution Approach 2:
The patent introduces a salt-tolerant endonuclease as an intermediary enzyme that can function effectively at low salt concentrations where conventional enzymes fail. This intermediary enables chromatin digestion under gentle conditions that preserve virus integrity, resolving the contradiction between enzymatic efficiency and virus stability
2Reliability
If multiple unit operations (ultracentrifugation, filtration, chromatography) are combined to achieve high purity, then purification effectiveness is improved, but processing time and loss of infectivity are worsened
Solution Approach 1:
The patent merges the chromatin digestion step with the chromatography purification step by performing digestion in the chromatography buffer before loading onto the column. This consolidation eliminates separate processing steps, reducing overall time while maintaining high purity through the chromatography step that captures both viruses and digestion products
Solution Approach 2:
The patent performs chromatin digestion as a preliminary action before chromatography, using salt-tolerant endonuclease to break down chromatin into smaller fragments that are then retained on the chromatography column while viruses are captured. This preliminary digestion simplifies subsequent purification steps and reduces overall processing time
3Object-affected harmful factors
If conventional endonucleases are used at low salt concentrations, then virus stability is improved, but chromatin solubility and enzymatic activity are worsened
Solution Approach 1:
The patent changes the salt concentration parameter from low (conventional for virus stability) to a low-but-sufficient range (10-500 mM NaCl) that enables salt-tolerant endonuclease activity. This parameter optimization resolves the contradiction by finding a salt concentration range that provides both virus stability and adequate enzymatic function
Solution Approach 2:
The patent introduces salt-tolerant endonuclease as a specialized intermediary enzyme that bridges the gap between low salt conditions (good for virus stability) and chromatin digestion requirements. This enzyme variant functions effectively at low salt concentrations where conventional enzymes fail, resolving the contradiction between virus stability and enzymatic activity
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 a significant increase in loading volume for chromatographic purification, reducing impurities like chromatin and dsDNA, thereby improving yield and maintaining high infectivity levels.
Implementation Method 1
enzymatic treatment using a salt-active endonuclease to digest double-stranded DNA (dsDNA) or dsDNA in the form of chromatin
Implementation Method 2
digesting extraneous genetic material
Implementation Method 3
purify the bionanoparticles from the culture fraction by capture chromatography
Data Source
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AI summary
A method for purifying bionanoparticles from a host cell culture fraction, comprising the steps of: (a) adding a non-specific salt-active endonuclease to the host cell culture fraction which comprises a conductivity between 11.9 to 35.1 mS/cm, thereby obtaining an enzymatically treated cell culture fraction; and (b) purifying the bionanoparticles from said enzymatically treated cell culture fraction by capture chromatography, whereby the bionanoparticles are adsorbed, and further eluted, thereby obtaining a purified bionanoparticle composition, wherein the endonuclease has an optimum enzymatic activity at a sodium chloride concentration within the range of 150 mM to 1 M.