Linear Closed DNA Preparation for High-Purity GMP Production
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Solution Overview
Problem
Large-scale industrial production of linear closed DNA (LcDNA) under GMP conditions faces challenges such as oversized and non-universal circular plasmid backbones, instability in large-scale digestion, insufficient purity, and potential introduction of organic reagents, leading to mutations and lower fidelity.
Innovation Solution
A plasmid design with editable regions of tandem prokaryotic telomerase target sequences and restriction endonuclease digestion sites, combined with a method involving prokaryotic telomerase digestion, exonuclease treatment, and chromatography for purification, to produce a linear closed DNA.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If conventional plasmid vectors are used for large-scale LcDNA production, then the plasmid can carry gene of interest and replication elements, but the plasmid backbone becomes oversized and non-universal, causing instability in large-scale digestion and insufficient purity
Solution Approach 1:
The patent extracts and removes the problematic plasmid backbone elements (prokaryotic replicon, resistance gene, CpG sequence) that cause immune response and production issues. Only the essential gene of interest and TeIN recognition sites are retained, creating a streamlined LcDNA structure that eliminates the source of digestion instability and purity problems while maintaining the ability to carry therapeutic genes at large scale
Solution Approach 2:
The patent segments the plasmid structure into removable backbone elements and essential functional elements (gene of interest + TeIN sites). This segmentation allows the essential parts to be retained for LcDNA production while the problematic backbone segments are extracted and discarded, resolving the contradiction between production scale and purity
2Ease of manufacture
If organic reagents such as Triton, isopropanol, and ethanol are used in LcDNA production, then the production process can proceed, but residual organic reagents are introduced with high residual risk
Solution Approach 1:
The patent replaces persistent organic reagents with water-based or enzyme-based alternatives that are biodegradable and leave no harmful residues. The production process uses disposable-like approach where reagents are completely degraded or removed, ensuring no residual risk in the final LcDNA product while maintaining manufacturing feasibility
Solution Approach 2:
The patent converts the potentially harmful organic reagent step into a beneficial enzyme-based process. Instead of using organic solvents that leave residues, the patent employs biological enzymes that naturally degrade without harmful byproducts, turning a harmful manufacturing step into a safe and even advantageous process feature
3Productivity
If polymerase Phi29 is used for in vitro amplification, then LcDNA can be amplified, but the fidelity is lower compared to in vivo replication in E. coli, resulting in unnecessary mutations
Solution Approach 1:
The patent introduces E. coli as an intermediary host system for DNA replication. Instead of direct in vitro amplification with low-fidelity polymerase, the LcDNA is replicated within living E. coli cells that provide high-fidelity in vivo replication machinery. The E. coli acts as a mediator that ensures accurate DNA copying while still enabling production scalability
Solution Approach 2:
The patent replaces the mechanical/enzymatic in vitro amplification system (polymerase Phi29) with a biological in vivo replication system (E. coli cellular machinery). This substitution leverages the natural high-fidelity replication mechanisms of living cells, achieving both productivity and reliability that neither system can achieve alone
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 ensures high fidelity and purity of LcDNA production, maintaining a small size and improving yield, addressing the challenges of large-scale production.
Implementation Method 1
digesting a recombinant plasmid comprising a gene of interest with a prokaryotic telomerase to separate the gene of interest from a heterogeneous nucleic acid in the recombinant plasmid
Implementation Method 2
using at least two restriction endonucleases for digestion such that the ends of the heterogeneous nucleic acid are exposed, and removing the heterogeneous nucleic acid by using an exonuclease
Implementation Method 3
separating and purifying the linear closed DNA containing the gene of interest
Data Source
AI summary
Provided are a method for preparing a linear closed DNA and a plasmid for use in the method. The plasmid comprises two editable regions of prokaryotic telomerase target sequences which are connected in tandem in the same direction, and both ends of the editing regions are independently provided with one or more restriction endonuclease digestion sites. The method can yield high-purity LcDNA and can be better applied to clinical research and commercial applications.