ARW's ATMP Manufacturing Must-Reads (ECLIPSE EDITION!)

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

Day-in and day-out, I write, read, listen to, and watch as much content as I can about CGT and RNA therapy manufacturing, in particular, and/or other ATMP industry-related topics that you should at least be aware of in the manufacturing facility. Once a month, I compile the articles and industry updates I think are most worthy of your time into an unconventional roundup article.

You may or may not have heard that there was an eclipse on April 8. As I’m based in Erie, PA, I was lucky enough to be living in “the path of totality.” Naturally, this also meant I was feeling particularly inspired to make this round-up “eclipse”- themed — and lucky for you, there are tons of songs about eclipses (both celestial and otherwise), some of the lyrics from which also “cross” over into ATMP CMC. Don your protective eye-gear: these articles/resources are bright.

Ain’t no sunshine when she’s [the materials are] gone, and [they’re] always gone too long anytime [they] go away. \

~ Bill Withers
[mRNA SUPPLY CHAIN]

• There ain’t no drug substances or drug products either.
• Fortunately, the outlook for materials in the mRNA supply chain remains bright — albeit with plenty of room to grow. I recently hosted the inaugural Advancing RNA LIVE event, featuring two mRNA-LNP supply chain experts who shared their perspectives on material supply considerations for linear and next-gen modalities of mRNA; navigating the patent landscape for materials; and how they’re defining “phase-appropriate” with their teams/partners.
  • The discussion can be watched here in full, and I’ve also broken it out into smaller segments for more selective watching.

Losin’ everything is like the sun going down on me. ~ Elton John
[CRYOPRESERVATION]

• Good news, Elton — drugs last longer when they’re not in the sun.
  • #ShadowsSaveLives.
• For those of us who hate the sun and prefer living in the cold darkness (just me?), there have been a couple of good resources penned on ATMP cryopreservation trends, challenges, and advancements:
  • PDA recently announced that its 02-2021 Cryopreservation of Cells for Use in Gene Therapies, Cell Therapies, and Regenerative Medicine Manufacturing garnered “complete recognition” from the FDA under its Voluntary Standards Recognition Program. You can purchase the standard here.
  • This is an incredibly thorough and thoughtful interview outlining the past, present, and future of cryopreservation in the cell therapy space. Not only do we continue to face scientific complications with current approaches, I also appreciated the emphasis on standardization of thawing processes at clinics, as well as the packaging considerations and constraints many of us will face as we scale and commercialize our products.
  • This may be from March 2023, but as the mRNA-LNP (or just LNP delivery) space continues to grow, this publication investigating the impact of cryopreservation on the transfection efficiency, stability, and safety of mRNA-LNPs is worth a gander.

I saw the crescent while you saw the whole of the moon. ~ The Waterboys
[THE WHOLE OF THE ATMP “SPACE”]

• Most of the time, we get stuck staring at the same sliver of the ATMP universe/functional area within which we work. If you’re going a bit stir-crazy, I’d like to offer a few ways to see the rest of the (lunar-like) ATMP landscape.
  • On May 16-17, ARM will be hosting an in-person workshop in Rockville, MD with presentations and panel discussions on CGT advanced manufacturing approaches. Automation, decentralized manufacturing, AI/ML approaches, and data digitization are just some of the topics on the table for discussion. You can register here.
  • Nobel winner Drew Weissman and several Penn colleagues just published an article in The Lancet reviewing the research and/or clinical progress we’ve made thus far with infectious disease mRNA vaccines, cancer vaccines, protein replacement therapeutics, and targeted (ex-hepatic) therapeutic products. As he concludes, “higher standards will need to be met” as the RNA space transitions from vaccines to therapeutics. (Institutional access needed to read in full.)

It’s no secret, the stars are falling from the sky…Look, I gotta go, yeah, I’m running out of change; there’s a lot of things if I could I’d rearrange. ~ U2
[GENE EDITING]

• Yo, Bono — ever heard of CRISPR?
• If ever a paper could cause a bar fight, it might be this one comparing LNP-delivered mRNA Cas9 to LNP-delivered Cas9 RNP (in mice). The conclusion? mRNA Cas9 (accompanied by gRNA and HDR template) was “the bigger star” in terms of particle size, editing efficiency, and biodistribution in ex- and in vivo editing.
• Last month, Caribou and Precision BioSciences inked a licensing deal around a circular RNA-encoded CRISPR-cas12a prime editor. Should you be curious about this specific editing technology, I’d refer you to this interview in CRISPR Medicine News which outlines one company’s approach using this technology in plant and mammalian cells.

I think it’s kind of interesting, the way things get to be, the way that people work with their machines, Serenity’s a long time coming to me ~ “Eclipse” by John Denver
[CAR-T MANUFACTURING]

• Once you realize that Serenity was also the name of a spaceship in the tv show Firefly, this song about John Denver’s life goals takes on a whole new world of meaning.
• ISCT recently published “CAR-T Cell Expansion Platforms Yield Distinct T-Cell Differentiation Rates,” which reveals the absolutely critical role your T-cell manufacturing platform (i.e., CliniMACS Prodigy, Xuri W25 rocking platform, G-Rex gas-permeable bioreactor, static bag culture) plays in cell expansion/differentiation.
• This ASGCT article gives us an inside look at the results of a partnership between GSK and Miltenyi to automate CAR-T cell production — a process now termed the “T-Cell Transduction-Large Scale” process — and how the partners tech transferred this process across three different manufacturing sites.

It’s a total eclipse of the sun, can’t come to grips with the total eclipse, one slip of the lips and you’re done ~ Klaus Nomi
[VIRAL VECTOR DEVELOPMENT]

• Despite what these lyrics suggest, reading these articles full of insightful “lip slippage” will not kill you.
  • BioProcess Online: “Stable Cell Lines: Stepping Stones for Continuous Viral Vector Manufacturing”
  • Biotechnology Progress: “High-yield recombinant adeno-associated viral vector production by multivariate optimization of bioprocess and transfection conditions”
    • If staring into the sun has made focusing on a whole article hard/impossible, check out Ben McLeod’s quick summary of key takeaways in his recent LinkedIn post.
  • Nature Scientific Reports [Jan. 2023]: “Machine learning and metabolic modelling assisted implementation of a novel process analytical technology in cell and gene therapy manufacturing” [LVV vectors]

So we cross that line into the crypt, total eclipse, suffer under my apocalypse ~ Metallica
[IVT]

• I’d like to propose “suffer under my apocalypse” as the RNA industry’s next battle cry against dsRNA.
• If for some reason that doesn’t magically make the dsRNA cower and eliminate itself in fear, this publication finds that urea supplementation may be a more practical solution for reducing dsRNA and truncated RNA transcripts.

Every now and then, I get a little bit lonely, and you’re never coming around... ~ Bonnie Tyler, Total Eclipse of the Heart
[PACKAGING EFFICIENCY]

• Hello, emptiness, my old friend.
• In addition to whiskey and/or ice cream, some of our peers are developing a few more fruitful solutions for dealing with “emptiness” in both AAVs and mRNA-LNPs.
  • My colleague Tyler Menichiello wrote this fantastic article following an interview with the CTO of Ultragenyx about the company’s efforts to limit the upstream production of empty AAV capsids via a producer cell line.
  • Likewise, this piece in Nature details the use of a multi-laser cylindrical illumination confocal spectroscopy (CICS) technique to identify unencapsulated mRNAs, empty LNPs, and full mRNA-LNPs, as well as to identify mRNA distribution patterns when packaged in LNPs.

In the dark of the sun, we will stand together, yeah we will stand as one in the dark of the sun.

~ Tom Petty & The Heartbreakers
[LNP ENCAPSULATION]

• ^^What we desperately hope our mRNA & LNPs will say to each other as we force them to mingle.
• For those looking to play matchmakers between their mRNA and LNPs, you can give these articles a spin:
  • Chemical Engineering Science: “Encapsulation of mRNA in LNPs by Micromixing”
  • Methods in Molecular Biology [Book chapter]: “Encapsulating IVT-transcribed Circular RNA into LNPs via Microfluidic Mixing” ($$ to access)
• Or maybe we get annoyed by how cute mRNA-LNPs are together and just choose to break them up instead. In that case, I give you this article on LNP deformulation methods/mRNA extraction from LNPs by the heartbreakers over at Sanofi.