Cyclic Dinucleotide Fermentation and Nanofiltration at Commercial Scale
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Solution Overview
Problem
Current methods for producing cyclic dinucleotides (CDNs) are inefficient, time-consuming, and costly, and do not allow for large-scale production, with chemical synthesis being environmentally unsound and enzymatic synthesis requiring costly precursors and low bacterial cell densities.
Innovation Solution
A method involving fermentation of recombinant E. coli cells in a culture medium, followed by purification using nanofiltration without affinity chromatography, to produce and purify CDNs at a commercial scale, utilizing a codon-optimized mcGAS gene and specific E. coli strains for high cell density and efficient CDN production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of time
If chemical synthesis is used to produce cyclic dinucleotides, then production time is reduced, but environmental soundness deteriorates and production cost increases
Solution Approach 1:
The patent replaces chemical synthesis methods with biological synthesis using recombinant E. coli cells expressing cGAS enzyme. This substitution eliminates the need for harsh chemical reactions, toxic reagents, and complex purification steps associated with chemical synthesis, thereby improving environmental soundness while maintaining production efficiency through controlled fermentation processes
Solution Approach 2:
The recombinant E. coli cells are engineered to self-produce cyclic dinucleotides through the cGAS enzyme catalyzing GTP to CDN conversion. The cells utilize their own metabolic machinery and intracellular GTP pools to synthesize the target molecule, eliminating the need for external chemical reagents and simplifying the production process to a straightforward fermentation and filtration sequence
2Manufacturing precision
If enzymatic synthesis using dinucleotide cyclases is used, then production specificity is improved, but production cost increases due to costly GTP precursor requirements
Solution Approach 1:
The patent introduces recombinant E. coli cells as an intermediary system that expresses cGAS enzyme. These engineered cells serve as a biological factory that converts inexpensive GTP (a natural bacterial metabolite) into cyclic dinucleotides through enzymatic catalysis. This intermediary approach replaces direct chemical enzymatic synthesis with a biological expression system, eliminating the need to purchase expensive purified GTP precursors while maintaining high production specificity
Solution Approach 2:
The patent changes the production parameters by shifting from a chemical synthesis approach requiring expensive GTP precursors to a biological fermentation approach where GTP is endogenously produced by the bacterial cells. By optimizing fermentation conditions (temperature, pH, nutrient composition) and expressing cGAS at controlled levels, the system achieves cost-effective production while maintaining high specificity for the target cyclic dinucleotide
3Quantity of substance
If conventional bacterial cell culture methods are used, then CDN production is achieved, but scalability deteriorates due to low cell density limitations
Solution Approach 1:
The patent changes the fundamental parameter of cell density by transitioning from conventional low-density batch culture to high-density fed-batch fermentation. The engineered E. coli cells are optimized for rapid growth and high-density cultivation, allowing large-scale production in industrial fermenters. By controlling nutrient feed rates, oxygen transfer, and pH to maintain high cell densities throughout the fermentation process, the system achieves scalable production capable of meeting commercial demand
4Manufacturing precision
If affinity chromatography is used for CDN purification, then purification efficiency is improved, but device complexity and production cost increase
Solution Approach 1:
The patent extracts and removes the complex affinity chromatography step from the purification process. Instead of using affinity columns that require specific ligands, flow rates, and extensive optimization, the method employs simple filtration and concentration techniques to isolate cyclic dinucleotides from the fermentation broth. This extraction of the complex purification step maintains high purification efficiency while dramatically reducing device complexity and operational difficulty
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 high-purity CDN production with minimal endotoxin and host cell protein contamination, enabling scalable and cost-effective commercial production of CDNs such as 2′,3′-cGAMP and 3′,3′-cGAMP.
Implementation Method 1
incubating CDN-producing recombinant E. coli cells in a culture medium in a fermenter to produce a desired amount of CDN
Implementation Method 2
purifying the isolated CDN through nanofiltration
Data Source
AI summary
The present disclosure provides a method of producing cyclic dinucleotides (CDNs) on a commercial scale. Also provided are pharmaceutical compositions comprising a purified CDN preparation and use thereof to stimulate the immune system in a subject.