AAV Purification Using Positively Charged Surface and DNase
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Solution Overview
Problem
Current methods for purifying adeno-associated virus (AAV) capsids are inadequate in reducing contaminating DNA to very low levels, particularly due to strong binding of DNA to AAV capsids, which complicates the removal process and results in filter clogging and limited concentration factors during tangential flow filtration.
Innovation Solution
The method involves exposing the cell culture harvest to a positively charged surface to bind and remove contaminating DNA, followed by tangential flow filtration and DNase treatment to achieve significant DNA reduction and facilitate concentration, allowing for higher concentration factors and improved filterability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If simple DNase enzyme treatment is used to reduce contaminating DNA, then the process is simple and easy to operate, but the DNA reduction is modest and does not achieve very low contaminating DNA levels
Solution Approach 1:
The DNA removal process is divided into multiple sequential steps: (1) pre-treatment with positively charged surface to bind contaminating DNA, (2) tangential flow filtration to separate bound DNA from AAV capsids, (3) DNase treatment to digest residual DNA, and (4) additional filtration to remove enzyme and fragments. This segmentation allows each step to optimize for its specific function, achieving comprehensive DNA reduction while maintaining operational feasibility.
Solution Approach 2:
The method applies preliminary action by first treating the AAV harvest with a positively charged surface to bind and remove contaminating DNA before performing DNase treatment. This pre-step reduces the DNA burden on subsequent filtration and enzymatic treatment, making the overall process more effective and efficient.
2Manufacturing precision
If DNase treatment is applied to cell harvest, then DNA reduction is achieved, but filter clogging occurs and concentration factors are limited to 2 or less
Solution Approach 1:
The method performs preliminary DNA binding to positively charged surfaces and removal via tangential flow filtration before DNase treatment. This pre-step clears most contaminating DNA from the harvest, reducing the load on subsequent filtration steps and enabling higher concentration factors (10-20 or more) without filter clogging.
Solution Approach 2:
The method extracts and removes contaminating DNA through binding to positively charged surfaces and filtration before the DNase treatment step. This extraction of DNA contaminants early in the process prevents them from interfering with subsequent concentration and purification steps, thereby enabling higher productivity.
3Ease of manufacture
If standard chromatography methods are used for AAV purification, then the process is well-established, but contaminating DNA cannot be reduced to very low levels
Solution Approach 1:
The method introduces an intermediary step using positively charged surfaces that mediate the binding and removal of contaminating DNA. These surfaces act as an intermediate between the AAV harvest and the final purification steps, selectively capturing DNA contaminants while allowing AAV capsids to pass through, thereby achieving very low DNA contamination levels.
Solution Approach 2:
The method replaces or enhances standard mechanical filtration and chromatography steps with a biochemically-based approach using positively charged surfaces to bind DNA. This substitution leverages electrostatic interactions between the positive surfaces and electronegative DNA, providing superior DNA removal capability while maintaining process feasibility.
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 approach effectively reduces contaminating DNA to low levels, enhances filterability, and enables concentration factors of 10-20 or more, surpassing the limitations of existing DNase treatment methods, thereby improving the overall purification efficiency of AAV capsids.
Implementation Method 1
DNA is electronegative and binds strongly to positively charged surfaces
Implementation Method 2
Treatment with the positively charged surface(s) achieves an initial reduction of DNA and facilitates tangential flow filtration
Implementation Method 3
Diafiltering the first fraction by a first tangential flow filtration to obtain a second fraction
Data Source
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AI summary
A method for reducing a contaminating DNA content of a preparation containing AAV capsids and contaminating DNA, comprising the steps of a) Performing an extraction of DNA with a solid phase bearing positive charges at its surface said solid phase is contacted with the preparation at a pH of 7.0 ± 1.0, and a salt concentration of 10 mM to 200 mM yielding a first fraction, (b) Diafiltering the first fraction by a first tangential flow filtration to obtain a second fraction, (c) Treating the second fraction with DNase, (d) Diafiltering the DNase treated second fraction obtained by step c) by a second tangential flow, (e) filtration to a buffer with pH of 7.0 ± 1.0, and a salt concentration of 10 mM to 20 mM to yield a third fraction, and optionally (f) Concentrating the third fraction by tangential flow filtration before supplemental chromatography.