Fungal DNase Treatment for Residual DNA Reduction
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Solution Overview
Problem
Existing methods fail to effectively reduce residual DNA in microbial fermentation products, particularly recombinant DNA, which poses environmental and health concerns due to its release into the fermentation broth during protein production.
Innovation Solution
A method involving a fungal DNase or its variant is applied at various stages of the fermentation process, including before, during, or after flocculation/precipitation and membrane filtration, to degrade residual DNA to single nucleotides or oligonucleotides less than 150 bp.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If host cells are disrupted/lysed to produce intracellular protein products, then the protein yield is improved, but residual DNA in the fermentation broth increases
Solution Approach 1:
The patent applies DNase enzyme treatment at multiple stages before the final product is obtained - specifically during fermentation broth processing and/or during purification steps. This preliminary degradation of DNA prevents the accumulation of harmful residual DNA while maintaining the protein production process, resolving the contradiction between achieving high protein yield through cell lysis and minimizing residual DNA contamination.
2Ease of manufacture
If conventional purification methods are used, then the processing is simple, but residual DNA cannot be effectively reduced to acceptable levels
Solution Approach 1:
The patent introduces a biochemical parameter change by adding DNase enzyme to the purification process. This enzymatic treatment fundamentally changes the DNA degradation parameter, enabling effective reduction of residual DNA to below 10 ng/g (and in some cases below 1 ng/g) without significantly complicating the overall manufacturing process. The enzyme treatment can be integrated into existing purification workflows, maintaining ease of manufacture while achieving the required manufacturing precision for DNA reduction.
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 significantly reduces residual DNA to undetectable or trace levels, enhancing the purity of the protein product and ensuring compliance with regulatory standards.
Implementation Method 1
a fungal DNase or a variant thereof is added to the fermentation broth before, in, or after step (a), to the fermentation broth supernatant after step (b), or to the fermentation product after step (c)
Implementation Method 2
subjecting the fermentation broth supernatant to a membrane filtration step to provide a fermentation product, where the membrane has a size exclusion limit of less than 100 kDa or less than 1 μm
Data Source
AI summary
The invention provides a method for reducing the amount of DNA in a microbial fermentation product by adding a fungal DNase from Aspergillus oryzae to the resulting fermentation product during the recovery process.