Plasmid Production: 3 Key Takeaways For mRNA Manufacturing

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

There’s the well-known cliché statement: “Curiosity killed the cat.”

As both an ardent cat lover and an incredibly curious person, I am thrilled to report that no cats have been harmed in the process of planning any of my Advancing RNA Live panel discussions. And that’s saying something since my unchecked curiosity often translates into overly ambitious question sets and discussion plans.  

My latest Live panel discussion on analytical and manufacturing technology innovations for mRNA production was no exception. I sat down with three experts — Life Edit Therapeutics’ April Sena; Tune Therapeutics’ Tyler Goodwin; and University of Sheffield’s Adithya Nair. Together we planned to — and did — tackle the current state of mRNA-LNP production, starting with plasmid production moving into mRNA drug substance and drug product production.

The panel discussion in its entirety is available to stream (or rewatch) on demand here on Advancing RNA. But for those of you who prefer reading to watching (you rare breed, you), I thought I would provide a few high-level takeaways from the discussion — starting with the underdiscussed but critical darling of the mRNA space: The plasmid.

The past decade of plasmid production has led to increasingly controlled production — albeit with some caveats.

Gone are the days of increasing scale to improve plasmid yield. In upstream processing, for example, our increasing knowledge of our cells and the integration of in-process analytics has enabled us to focus more closely on overall cell health and, in turn, to improve the quality and yield of the plasmids those cells can produce. As Goodwin explained, thanks to the integration of real-time monitoring, we can ascertain changes in lactate levels and pH, as well as make informed decisions about when we need to apply more glycerol or glucose to ensure our cells are well fed and capable of producing the desired yields and quality of plasmids.

However, despite our advancements upstream, Goodwin admitted that variations in plasmid size and GC content can still challenge our downstream processes. In turn, the onus is on the mRNA therapeutics company to understand the plasmid’s impact on their process and the quality of mRNA it produces.  

“There are a lot of options in terms of chromatography,” he explained. “So, you need to understand what your product requires. What type of ion exchange or HIC chromatography steps do you need to clean up the plasmid to achieve the appropriate plasmid quality and maintain a good yield?”  

Likewise, it’s exceptionally important for mRNA makers to pay attention to GMP master cell banks for plasmid production. As Sena went on to explain, encoding the poly-A tail into the plasmid itself can certainly be beneficial from a production standpoint, seeing as it eliminates a step from our IVT process. However, encoding the poly-A tail into the plasmid also demands additional due-diligence on behalf of the mRNA company and the plasmid manufacturer.

“If you’re working with a manufacturer who understands how to screen the clones going into your master cell bank, you can make sure that your poly-A tail is the length you need it to be and that it hasn’t been truncated,” she added.  

We still have a lot of open-ended questions about plasmid quality for mRNA production.

“We’ve taken a lot of what the AAV gene therapy space has needed in terms of plasmid quality and copy and pasted those requirements over,” Goodwin explained. “But I’m not sure this is the best approach. There may be different critical quality attributes we should be focusing on instead for higher quality RNA production.”

I’ve heard similar statements from members of the industry for the past few years. In fact, the gaps in our knowledge about plasmid DNA quality for IVT production were discussed at length in this fantastic article from one of Advancing RNA’s expert contributors, as well.

If one thing was clear from this recent Advancing RNA Live, we still have more questions than answers around which plasmid DNA quality attributes are directly correlated to higher (or lower) quality mRNA. For example, as Nair pointed out, while some literature reveals a direct correlation between supercoiled percentage and the quality of IVT-produced RNA, there is also literature demonstrating a much less conclusive relationship.

The panelists were also aligned around the increasing importance of working with NGS to better understand plasmid quality — especially when trying to determine whether a plasmid has been nicked. On the one hand, identifying a nick can help pinpoint and ameliorate the cause of nicking in the production process. But as our constructs become bigger, so too, do our plasmids, which opens them up to greater opportunities for nicking. “This can be a huge problem when you’re making larger RNA constructs,” Sena added.   

Finding the “right” plasmid can still be “a lot” for mRNA makers.

As Goodwin walked through the improvements he’s observed in fermentation processes, he continued to reiterate the importance of understanding the quality and yield of plasmid your product will ultimately need/demand. However, as the previous takeaway revealed, it’s early days, and we’re still trying to figure out what it means to “know our plasmids and our products”— and to know them well.

In turn, I appreciated Goodwin’s reminder that, despite advancements in fermentation processes, plasmid production remains “an art” and is “absolutely the most complex unit operation compared to RNA and LNP.”  Not only are you reliant on cells in general, you’re faced with choices about the strain you ultimately want to use, as well as how you/a partner need to properly “take care” of those cells to ensure you arrive at the yield/quality of plasmid you’ll ultimately need for your product. 

“There’s a lot of optionality there, which is good,” Goodwin admitted, “But it can also be a lot for anyone who is at the beginning of developing their own mRNA products.”

If you want the best of both worlds, you can also watch the speakers discuss this topic during this brief installment from the panel discussion.