The Cell-Free Revolution: Breaking The Plasmid DNA Bottleneck

By Anna Rose Welch, Editorial & Community Director, Advancing RNA

It was during my first mRNA-centric conference in 2022 that the concept of cell-free manufacturing was raised during a panel of CEOs. Pfizer had previously announced its partnership around synthetic DNA with Codex in 2021, and 2023 brought with it Moderna’s acquisition of the Japanese firm OriCiro, which specialized in synthetic DNA production.

Since then, it seems as though there’s a new biotech and/or existing supplier announcing the availability of synthetic DNA every other day. One such biotech is Ribbon Bio, which just announced the launch of its first commercial product. I was excited to be given the opportunity to sit down with Jodi Barrientos, CEO, Ribbon Bio, to learn more about the current state of the synthetic DNA space. In the following Q&A, Barrientos and I discuss some of the synthetic DNA industry’s biggest goals and the challenges that stand in the way of bringing synthetic plasmids more commonly into the mRNA development paradigm.

Anna Rose Welch: We talk a lot about the merits of a completely cell free mRNA manufacturing process. On the one hand, it frees us from the challenges of dealing with endotoxin contamination inherent with e-coli-based plasmid production. But what else is driving the synthetic DNA “movement” today?

Jodi Barrientos: The speed at which we can manufacture mRNA products is going to be critical moving forward. We know we want to go from blood draw to an injection in the arm in as quickly as nine days; there's a patient life underneath this workflow. So, the criticality of being able to reduce the end-to-end process time is what we're thinking about. How do we remove the DNA bottleneck so that we can get the next steps underway? While I think cell-free providers are doing a great job right now — it takes days to produce synthetic plasmid — we can do better. I think the goal for cell-free companies is to get highly accurate and highly pure DNA production down to 48 hours.

Welch: Where are you finding that the technology for making synthetic DNA and the science are still struggling to match up today?  

Barrientos: Though we don’t have to worry about the bacteria that’s inherent to cell-based processes, we do have some different challenges that we’re facing in the synthetic production world. For one, we're relying on techniques like PCR, and PCR is an old technique. We are at the mercy of the amplification efficiency to make sure that we get high accuracy. Likewise, as technology gets more advanced and we think about things such as gap mining and larger RNA molecules like saRNA, we’re often relying on a basic polymerase that’s been used on the “read” side to now “write.”

Welch: How are we as a field working to overcome the limitations posed by PCR amplification & enzyme inefficiencies?

Barrientos: There are several strategies. The first is improved amplification methods. Approaches like LAMP and RCA have legs and are making their impacts felt and I’m glad to see companies succeed in commercializing them.

But then there is what we do at Ribbon – cell-free DNA synthesis. This is a huge step forward that negates many of the challenges around enzyme inefficiencies because you no longer need to work around them. Moreover, this does away with many of the challenges of amplification. Amplification amplifies the target sequences, but it also amplifies the errors – if you have fewer errors, amplification is no longer an issue.

Welch: Pharma is a risk-averse industry, and many of the conversations I’ve had around making the transition to cell-free synthetic DNA have come back to the need for regulatory experience. From where you sit, how have you seen regulators responding to/evaluating cell-free synthetic DNA compared to plasmid derived DNA?

Barrientos: The honest answer is that regulators aren't evaluating cell-free DNA as a fundamentally different category; they're asking the same questions they always ask, just through a cleaner lens: “What's in it? How consistent is it? Can you control it?”

And that's where cell-free has a compelling story. The traditional concerns with bacterially-derived plasmid DNA like endotoxin contamination, unmethylated CpG motifs, and antibiotic resistance markers are exactly what regulators have been increasingly vocal about wanting minimized or eliminated. Cell-free manufacturing removes those from the conversation almost entirely.

What regulators do want to see is comparable and, in some cases, superior performance. Sequence accuracy, batch-to-batch consistency, defined critical quality attributes. The good news is that cell-free processes are inherently more controllable than fermentation. You're not managing a living organism. That consistency is something you can demonstrate and document in a way that regulators respond well to.

The risk-aversion we hear about in these conversations is less about the technology itself and more about change control anxiety — Companies mid-program who'd have to justify a switch. For new programs, starting with cell-free means it's simply your process from day one. There's no comparability burden because there's nothing to compare against. So, from where I sit, the regulatory path isn't harder; in many ways it's more straightforward, because the product is cleaner to begin with.

Welch: How do you see the synthetic DNA space evolving in the next 5 years, and in what ways do you anticipate it will impact the manufacturing paradigm for mRNA?

Barrientos: I think in the next five years, we're going to finally be able to say that DNA isn't the bottleneck for getting things done, which opens a whole new world of answers to the question of “what’s next?” DNA is empowering so many cool things in the market, whether it's in agriculture, biotech, biofuels, or even just in the research community. DNA is at the beginning of everything. There’s real value in not having to think about DNA as an impediment to starting your process. You don't want your first step to be a hard step. If we’re able to streamline the transition between DNA and mRNA drug substance production, I think we'll see that the downstream applications start to flourish. We’ll start to see therapeutics get to market faster. We’ll start to see new innovations that we never even knew were possible because we’ve made DNA production more accessible.