How is 4basebio’s Synthetic DNA made?
The Future is Synthetic
For decades, scientists have relied on plasmid DNA (pDNA) manufacturing to produce genetic material for research and therapeutic development. Many modern genetic therapies, including mRNA therapeutics and vaccines, DNA vaccines, and viral vectors, require pDNA as a critical starting material or use it directly as the active pharmaceutical ingredient (API). However, pDNA production is associated with several limitations, including slow manufacturing processes, high costs, and susceptibility to contamination. Consequently, many companies are choosing to move to synthetic, enzymatic approaches to DNA manufacture. In addition to being cost and time efficient at scale, enzymatic approaches are suitable for complex and unstable sequences and cell-free processes avoid bacterial contaminants.
4basebio’s Synthetic DNA Manufacturing Process
4basebio’s synthetic DNA manufacturing process uses Trueprime® technology, which is an enzymatic, high-yielding DNA amplification process. 4basebio’s experienced molecular biology team works with you to design a strategy to convert your DNA sequence into a circular DNA master template for the enzymatic amplification. Starting material can be in the format of pDNA, synthetic DNA blocks or PCR fragments. Crucially, any bacterial sequences are removed during master template generation and are never amplified during the manufacturing process. The master template then feeds into the manufacturing process:
Amplification – the master template is used to amplify the sequence via rolling circle amplification (RCA) using proprietary, high-fidelity Trueprime® enzymes, currently operating at yields of 1 g/L.
Processing – This step produces the final DNA format, processing the DNA ends to add single-stranded hairpins or stabilised, open-ends that confer unique application-specific benefits.
Purification and Buffer Exchange – The DNA undergoes optimised purification steps to remove enzymes used during the process and to select for sequence specific impurities, ensuring a high-quality and homogenous final product.
Final Synthetic DNA Product
How can synthetic DNA support your genetic therapy manufacturing?
1. Enhanced Safety: Elimination of Endotoxins
Plasmid DNA production in bacterial systems introduces unwanted contaminants, including bacterial endotoxins and genomic DNA, as well as extraneous sequences, such as antibiotic resistance genes. These impurities pose both safety and regulatory challenges, particularly in therapeutic applications. Synthetic DNA, on the other hand, is cell-free and the bacterial backbone is absent during amplification steps, mitigating contamination risks and ensuring a higher standard of safety for clinical use.
2. Costs at Scale
Large scale production of plasmid DNA requires considerable plant footprint, due to the bioreactors used in the manufacturing process. In contrast, synthetic DNA operates with reduced process volumes and waste streams, which requires a smaller footprint and less capital demand. Additionally, due to the absence of the bacterial backbone, synthetic DNA provides a greater copy number per mass than the equivalent pDNA sequence, meaning dose can be titrated down.
3. Sequence Complexity: Overcoming Constraints
Certain DNA sequences, including regions of high GC-content, repetitive elements, and toxic genes, present challenges in bacterial systems. Plasmid DNA production is limited by these constraints, whereas enzymatic DNA synthesis allows for the design and manufacture of almost any DNA sequence, regardless of complexity. The enzymes used in 4basebio’s process, produce DNA at yields exceeding 1g/L, with high fidelity (error rate ~ 3.75 x 10-9).
4. Accelerated Production Timelines
Plasmid DNA production is a long process that involves bacterial growth, master cell bank generation, and extensive purification to extract pDNA from cellular contaminants. Enzymatic DNA synthesis reduces these timelines to a matter of weeks, enabling GMP-grade DNA for gene and cell therapies, vaccines and therapeutic applications. This is particularly crucial in rapidly evolving fields like pandemic response as well as personalised therapies, where speed can save lives.
4basebio’s unique application-specific constructs
One of the main advantages of 4basebio’s platform, is the carefully designed synthetic DNA templates based on the end application. Each construct has specific benefits that accelerate development of advanced therapies.
| 4basebio Construct | Benefits |
|---|---|
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hpDNA is suitable for viral and non-viral vector applications. No bacterial sequences present, which allows no risk of reverse packaging. |
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opDNA® is ideal for mRNA production due to its 3' open end, which feeds directly into IVT reactions. |
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oeDNA® is ideally suited for gene editing by homology directed repair. Suitable for sequences from 140 bp up to 20 kb. |
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ssDNA is suitable for gene editing by homology directed repair. 4basebio can manufacture long ssDNA sequences, with batches up to 1 milligram. |
The Future of DNA Manufacturing
While plasmid DNA has historically played a critical role in genetic research and therapeutics, the introduction of synthetic DNA solutions marks a paradigm shift. With improved safety, reduced costs, sequence flexibility, and accelerated production timelines, enzymatically produced DNA is redefining manufacturing. With varied applications from viral vector production to personalised mRNA therapeutics, this technology is driving the future of genetic medicines toward greater efficiency, safety, and accessibility.