By Anna Rose Welch, Director, Cell & Gene Collaborative
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One of my favorite sayings when I was a kid was an old Irish proverb: “The older the fiddle, the sweeter the tune.”
If I’m being honest, I liked it mostly for a selfish reason: I was the extremely proud 12-year-old owner of a 107-year-old violin. My violin, which was made in France in 1894, remains one of my most prized possessions even though — believe it or not — a centennial is not exactly “old” in the violin world. Some of the most valuable violins were made between 1644-1737 by an Italian luthier named Antonio Stradivari who is revered as the father of the modern violin. Seeing as only 600 (of 1200) “Strads” are making music or being displayed in museums today, they are highly coveted and exorbitantly priced (some to the “tune” of $16 million dollars).
It wasn’t until recently reading a headline about what makes a Stradivarius violin “sound like a Stradivarius” that my brain forged a connection between the art and science of violin making and the mRNA therapeutics industry’s ongoing quest to establish high quality processes and procedures.
Though the goals of violin-making and pharmaceutical manufacturing ultimately diverge when it comes to mass-production, violin craftmanship serves as a fantastic metaphor for the work the mRNA therapeutics space is tackling today. In addition to navigating a limited supply chain of critical raw materials, companies are also challenged to carefully optimize the IVT reaction for making their mRNA drug substance.
Managing supply chain and working in process development may not look anything like the work a luthier does to hew an instrument from an ancient spruce tree — but I’d argue they require a similar mindset and level of dedication.
Mass production of an mRNA therapeutic requiring thousands of IVT reactions may be the end goal for many mRNA companies. However, in these early days of the industry, there’s a lot of value in taking the time to source and assess the quality of each raw material to understand how it can/will impact your mRNA drug substance. Like a master luthier working on a violin that will, one day, be considered a rare commodity, those of us in supply chain and process development should be approaching that all-important IVT reaction as if it, too, will produce a rare commodity that will carry on our legacy for hundreds of years.
The Raw Materials & “CQAs” Behind A Violin’s Sound
According to a German fairytale, the first violin was created out of a box, a few hairs from a fairy queen, and a rod. At the urging of the fairy queen, a love-sick boy put these materials together and played them. As he played, the voice of the fairy queen laughing and crying came forth, creating the first violin and winning the heart of a princess (or, rather, the permission of her father, the king).
The reality behind how and from what a violin is created is almost as poetic. Picture venturing up a mountain in Central or Eastern Europe in the winter. You have one goal: to chop down an old growth tree — ideally a Spruce and/or Maple, both of which will be essential for the face (Spruce), ribs (Maple), and back (Maple) of a future violin. For the wood to be a prime candidate for violin making, it must be growing at a high altitude. The colder temperature strongly influences the grain or the size of the rings in the wood — the narrower and tighter grains/rings being the most optimum for tone wood. It’s also important that the wood be dormant when it’s harvested — hence your winter journey up a mountain.
That wood is then cut into cylinders, sealed, and left to oxidize and dry in an environmentally controlled space for at least 10 years. Some wood ages for as long as 40-50 years before a violin maker will touch it. (And because I know the engineering minds among us are thinking it: yes, there are artificial ways — namely kiln drying — that can be used to speed up the aging process. But like human cells, wood cells are delicate; they don’t respond well to kiln drying and, in turn, lose their acoustic properties.)
Given that the sound of a violin is dictated in large part by the type, grain, and age of the wood from which it is carved, we can certainly classify the wood as a critical raw material. However, recent research argues that a violin’s superior tone can and should be attributed to much more than just the biological properties of the wood itself. Rather, the chemicals with which that wood is treated play a large role in shaping a string instrument’s acoustic profile.
As this recent article shares, one scientist at Texas A&M University has finally identified several chemicals he has long asserted were a predominant reason for the “unmatched” tone of a Stradivarius’ and/or the other violins made by master Italian luthiers during the same time period. Borax, copper, zinc, alum, and lime water were all used to preserve the wood to keep it safe from worm infestations. However, as this was long prior to the day and age of patents and process documentation, the exact recipes, ratios, and methods were perfected over time and kept secret to each violin maker — all the way to the grave.
Current Challenges In Navigating The mRNA Raw Material Supply Chain
In many ways, mRNA companies today are working much like these violin masters during the Italian violin “golden age”— albeit with patents and more thorough documentation. They’re relying upon techniques and building processes in-house based on individual knowledge and experiences, all of which vary widely. This lack of standardization makes it infinitely more difficult to determine how the processes and materials influence the structure, stability, and purity of mRNA.
There are two parts to the challenge of developing high quality mRNA — the first, of course, being navigating a currently immature supply chain. As the highly specific quest for violin wood outlined above should indicate, not all raw materials (for violins or therapeutics) are created equal.
Finding high quality (particularly GMP) raw materials is exceptionally challenging in the mRNA space today. Though the mRNA-centric supplier network is growing larger by the day, there is still plenty of room for this network to expand and further enable dual-source supply. Even in cases where suppliers do exist and have the available supplies, companies are challenged to suss out whether the GMP touted on a raw material’s label is the true, regulatory definition of GMP or just the “skilled marketer’s” definition of GMP.
The as-yet undefined regulatory landscape for MRNA therapeutics adds additional uncertainty around regulatory purity/quality expectations, particularly for the raw materials used in the IVT process. In many cases, the question plaguing the industry about their raw materials today is: “How pure is pure enough?” In lieu of clear regulatory guidelines outlining these quality expectations, the industry simply has to make a well-informed decision between sourcing, for example, the “purest” plasmid or to strive for greater homogeneity in the plasmid supply.
We also can’t overlook the comparability and optimization challenges posed by hopping from supplier to supplier — whether this switch be driven by a phase-appropriate approach to supply chain management/scale-up and/or a supplier shortage. I particularly appreciated the quote in this article that emphasized just how challenging it can be to optimize the IVT process and achieve a high yield of quality mRNA given the “different flavors” of vendors’ enzymes — specifically T7 polymerase. Not only is there less experience with these enzymes across the pharma industry, but they can be highly variable from supplier to supplier and/or contain residual DNA from the enzyme production process.
Adopting A “Rare Commodity” Mindset In Pursuit of mRNA Quality
However, herein lies the industry’s challenge and opportunity. Just as the Italian violin masters like Stradivari “fine-tuned” their wood-seasoning mixtures to improve the strength and acoustic properties of their instruments, the mRNA industry is facing the same challenging task. We’re attempting to learn firstly, how all the raw materials “play” together when combined in the IVT reaction. Secondly, we need to identify in what specific ways they leave their own signature on the preliminary mRNA drug substance that emerges from the IVT reaction and proceeds to downstream processing.
Any conference panel, webinar, or conversation will reiterate the importance of construct-specific optimization of the IVT reaction (particularly in the upstream). Broadly speaking, a company has to identify the best IVT process conditions for their batch or fed-batch IVT process. These parameters include but are not limited to reaction time; pH; temperature; feed volume/ timing; and raw material concentration (e.g., salt, DNA template, NTPs & NTP ratio; T7RNAP, etc.).
Of course, the capping strategy may also require a capping analog or additional considerations of the conditions and concentrations needed for efficient enzymatic capping. Each (data-driven!) decision will not only impact the stability and purity of the mRNA substance emerging from the reaction, but it will also impact the tools needed and the extent of purification required in downstream processing. Today, the industry is defined by its process diversity, all of which is dictated by the construct’s size as well as the quality/efficiency of the many different IVT reactions and capping strategies. In turn, the variability in upstream processing is nicely reflected in the current diversity of downstream processing strategies following the IVT reaction/DNAse l treatment (e.g., Affinity + Hydrophobic Interaction [HIC]; Ion Exchange [IEX] + HIC; Affinity only; Tangential flow filtration [TFF] only).
Much like the mRNA industry today, Stradivarius and his predecessors didn’t have clearly written instruction manuals to achieve the “golden violin standard”; rather, they employed “design-of-experiment”-like models that eventually evolved the shape/physical structure of their violins to embody the best acoustical properties. The individual nuances of a violin’s composition (e.g., the thickness and graduations of the front/back plates; the location of the sound post inside the violin, etc.,) all impact its acoustic properties, sound projection, tone, and resonance — a violin’s CQAs, if you will.
In the mRNA world, we’re in the midst of the same journey today, albeit with much higher stakes. Though we have a general understanding today of mRNA’s CQAs and impurities, we still have a long way to go to better understand the linkage of process and product and, in turn, the overall therapeutic/clinical profile of our product.
This is where I see the greatest opportunity for the mRNA industry. At the end of the day, we hope to be able to touch the lives of millions of patients. Based on my interactions with experts in this space so far, it’s clear that the scientific knowhow, excitement, and passion all exist to accomplish this goal. But as quickly as we, our patients, and the regulatory agencies may hope to get to that point, we have to embrace the same Stradivari-like level of curiosity and craftmanship to find the most ideal materials and process parameters/conditions from which we’ll chisel out “the golden standard” of mRNA.
*Editor’s Note: Putting together this piece required hours of additional research and fact-checking (and lots of editing) — for both the violin making & mRNA process development considerations. In addition to the links shared throughout the text above, I’d like to single-out a few additional resources I used to flesh out the scope of this article and/or fact-check my own understanding of the raw materials required for the IVT reaction and/or overall production of mRNA therapeutics.
- Whitley J., et.al. Development of mRNA manufacturing for vaccines and therapeutics: mRNA platform requirements and development of a scalable production process to support early phase clinical trials. Transl Res. 2022 Apr; 242:38-55.
- Boulais, A., et. al. The rise of mRNA: New era, new challenges. The Medicine Maker. 2021 Sept.
- Lowe, D. Myths of vaccine manufacturing. Science. 2021 Feb.
- Heilweil, R. The key ingredient that could hold back vaccine manufacturing. Vox. 2021 March.
- Silver, K. Shot of a lifetime: How Pfizer developed its own raw materials to ensure a steady supply for the COVID-19 vaccine. Pfizer.
- Phacilitate. Discussing key challenges with plasmid DNA: Top 3 takeaways from an interactive roundtable. 2021 May.
- Bozenhardt, H. & E. PIC/S annex update: What is your ATMP control strategy? Cell & Gene. 2021 June.
- Kibbey, M. Best practices to ensure quality of raw materials used to manufacturing therapeutic proteins. Bioprocess Online. 2021 Aug.
- Grooms, K. In Vitro Transcription: Common causes of reaction failure. Promega Connections. 2019 April.
- Bibel, B. T7 RNA Polymerase — discovery and uses (in vitro transcription & protein overexpression). The Bumbling Biochemist. 2020 Sept.
- Khan Academy. Overview of transcription.
- ThermoFisher Scientific. The basics: In Vitro Transcription.