Continuous DNA Plasmid Purification via Venturi Mixing
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Solution Overview
Problem
Current methods for purifying polynucleotide sequences, such as plasmid DNA, face challenges including contamination, degradation due to mechanical stress and high operational costs, especially when using alkaline solutions or batch-wise methods, which require handling at low temperatures and can lead to inefficient recovery of high-purity DNA.
Innovation Solution
A method and apparatus for continuous production and purification of DNA plasmids that involves alkaline lysis, neutralization with an acetic acid/acetate composition, and precipitation using specific salts, performed at room temperature without the need for chromatographic steps, utilizing peristaltic pumps and Venturi effect to minimize mechanical stress and contamination.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If alkaline lysis solution is used to disintegrate cells, then cell disintegration efficiency is improved, but DNA plasmid degradation increases
Solution Approach 1:
The patent applies preliminary action by performing rapid neutralization immediately after alkaline lysis. The neutralization solution is added in a controlled manner right after cell disintegration, preventing prolonged exposure to alkaline conditions that would degrade DNA. This timing strategy allows efficient cell breaking while protecting plasmid integrity.
Solution Approach 2:
The patent employs the skipping principle by minimizing the contact time between alkaline lysis solution and cellular contents. The rapid mixing and immediate neutralization rush through the potentially harmful alkaline phase, reducing degradation while maintaining disintegration efficiency. The continuous flow system ensures quick transition through the lysis stage.
2Manufacturing precision
If batch-wise purification methods are used, then purification effectiveness is improved, but contamination risk and operational complexity increase
Solution Approach 1:
The patent transforms the batch-wise purification process into a continuous flow system. Cell suspension, lysis solution, and neutralization solution are continuously mixed and processed through the column, eliminating the need for repeated manual operations. This continuous action maintains purification effectiveness while reducing contamination risk and operational complexity.
Solution Approach 2:
The patent applies self-service by designing a system where the continuous flow automatically performs mixing, neutralization, and purification functions. The fluid dynamics and column design enable self-regulating flow patterns that maintain effective purification without requiring complex external control mechanisms or frequent manual interventions.
3Manufacturing precision
If concentrated salt solutions are used for precipitation, then contaminant removal is improved, but mechanical stress on DNA increases
Solution Approach 1:
The patent uses hydraulic principles by leveraging the flow dynamics of concentrated salt solutions through the continuous flow system. The controlled fluid flow and pressure gradients enable effective salt precipitation of contaminants while distributing mechanical stress uniformly throughout the solution, preventing localized damage to DNA molecules.
Solution Approach 2:
The patent applies parameter changes by carefully controlling the concentration, flow rate, and contact time of salt solutions. These parameter optimizations allow effective contaminant precipitation while minimizing mechanical stress on DNA. The continuous flow system maintains parameters within optimal ranges throughout the process.
4Manufacturing precision
If chromatographic steps are included for high-purity DNA recovery, then DNA purity is improved, but process cost and complexity increase
Solution Approach 1:
The patent merges multiple purification functions into a single continuous flow column system. The column performs cell debris removal, contaminant precipitation, and plasmid purification simultaneously in one integrated unit, eliminating the need for separate chromatographic steps while achieving high DNA purity.
Solution Approach 2:
The patent applies universality by designing a multi-functional continuous flow column that handles multiple purification tasks. The same column structure and flow system perform lysis, neutralization, precipitation, and purification functions, replacing multiple specialized chromatographic devices with a single universal unit.
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 enables efficient, rapid, and cost-effective production of high-purity DNA plasmids with reduced contamination and degradation, achieving pharmaceutical-grade quality without the need for chromatographic steps or low-temperature handling.
Implementation Method 1
performing continuously in the passageway a precipitation of DNA Plasmid contaminants by adding a solution containing one or more salts to the first mixture of step b) to form a second mixture by the use of means to provoke Venturi effect
Data Source
Figure 1
AI summary
The present invention relates to an apparatus and a method for obtaining a (poly) nucleotide sequence of interest and comprising the steps of cultivating hosts cells to produce a nucleotide sequence of interest and harvesting these cells, introducing these cells in a passageway and disintegrating them in a continuous process, in this continuous process, performing in the passageway a precipitation of contaminants by a mixing of these disintegrated cells with a solution containing one or more salt(s) and obtaining a mixture and allowing a precipitate to separate from the solution of this mixture, preferably to float and/or to sediment from the solution of this mixture for 1-48 hours and pumping out a soluble material from this solution, while excluding recovering the precipitate.