The success of the mRNA-based vaccines against SARS-CoV-2 merely hint at the healthcare implications of mRNA technology. More mRNA-based vaccines are in the works, as are mRNA-based therapeutics for various maladies. However, the medical boons promised by mRNA technology won’t be realized unless there are improvements in mRNA manufacturing.
The most obvious improvement has to do with scale. As mRNA technology proves itself, demand for mRNA-based vaccines and therapeutics will soar, necessitating increases in production. But it is unclear, exactly, how issues of scale should be addressed, particularly since mRNA technology is still evolving. Fortunately, the chemistry, manufacturing, and controls (CMC) for mRNA-based vaccines and therapeutics are evolving, too. The situation is well summarized by Ye Gu, PhD, co-founder and CTO of Crystal Bio in Cranbury, NJ. She says, “We’re still exploring, and nobody can say that they have the best way to do it.”
Applying phase-appropriate analytics
Crystal Bio provides advanced analytical services to support the development of biologics, including mRNA-based biologics, from discovery through clinical trials, and CMC is a crucial component across the life cycle of a therapy.
“Over the past two decades, modern analytical techniques have significantly reduced the CMC failures in biotherapeutics, particularly for antibodies,” Gu observes. “However, mRNA—as a relatively new and a rapidly emerging field—is still in the process of establishing standardized characterization and testing methods, so the analytics and quality controls for mRNA vaccines and the therapeutics still have gaps.”
To address these gaps, Crystal Bio offers phase-appropriate analytical strategies throughout the development process. According to Gu, such strategies provide “drug developers with cost-effective, regulatory-guided testing, ensuring that the analyses are tailored to each stage of development.”
As a product moves into clinical trials, for example, the U.S. Food and Drug Administration (FDA) “requires more detailed information about the quality attributes at the later stages,” Gu states. “At Phase I, they give you time to develop methods based on the gradually collected knowledge regarding the mRNA molecule or drug product.”
To analyze mRNA being manufactured, Crystal Bio uses a variety of tools. In some tests, for example, “you need to have a good separation, and then you have a good analyzer or detector,” Gu says. “These two techniques need to be coupled together, and you need to carefully select the right technique for this type of analysis, which requires a lot of method development, especially for challenging analytical projects.”
The separation can be performed with processes such as high-performance liquid chromatography or capillary electrophoresis. For in-depth characterization of mRNA, Crystal Bio utilizes high-resolution mass spectrometry following a suitable separation. This approach can provide high sensitivity and high accuracy in analyzing critical quality attributes of the mRNA, including the capping efficiency of the mRNA and the distribution of the poly(A) tail.
As Gu explains, “The effective and timely application of advanced technologies, such as high-resolution mass spectrometry, can help you understand the molecular characteristics and assess the suitability of the manufacturing platform early in development, and thereby mitigate the risk of CMC delays later on.”
Reducing risks
Beyond analyzing the efficiency of steps such as mRNA capping, companies can reduce risk by optimizing manufacturing processes. That’s just what TriLink BioTechnologies in San Diego, CA, does with the entire mRNA manufacturing workflow.
TriLink’s CleanCap technology enables streamlined mRNA manufacturing with more than 95% capping efficiency. Plus, the company’s years of mRNA experience help ensure that “important aspects of the mRNA—such as the poly(A) tail length and integrity—are where they need to be for product efficacy and safety,” states Kevin Lynch, TriLink’s vice president and general manager of GMP operations. “When I think of the requirements to remain compliant and to continuously improve with robust processes, I think of how process questions might be addressed from a quality-by-design perspective.
“How are you measuring or using in-process controls that define a successful process step? What contaminants are you removing? What functionality are you ensuring is integral with your product? These are the types of questions that need to be asked and answered with improved in-process control testing and analysis.”
The mRNA-based product, though, depends on several factors. For one thing, a manufacturer must properly screen “suppliers to make sure that you’re having the best materials introduced,” Lynch emphasizes. For example, the process requires critical enzymes and nucleoside triphosphates for the in vitro transcription reaction. Also, reaction durations must be determined, and cleanup tasks, such as the removal of double-stranded DNA and any residual enzymes, must be performed.
TriLink works out as many of these issues as possible before it gets started on a project. “We always take an approach with our customers internally to do feasibility runs, as they bridge the gap from process development and R&D into the clinical space,” Lynch says. “Then, when you get into that GMP space, you have to do your design of experiments with proven scale-down models to trust the data.”
“All along,” Lynch says, “we make sure we’re hitting the same in-process controls in the scale-down process that we expect to hit in the larger scale GMP process.” For example, when transmembrane flow filtration is performed, the removal of contaminants can be “pretty challenging,” Lynch points out. “You have to select the proper membrane sizes and surface areas as well as diafiltration buffers correctly, because you don’t want to lose your product as you concentrate and purify. This ensures the process is capable of gently and effectively leveraging molecular-weight cutoff points for product size and buffer formulations to exploit charge differences between products and contaminants.”
Upping throughput
In a healthcare crisis, manufacturing flexibility and speed can be crucial. So, Ginkgo Bioworks in Boston, MA, developed a modular high-throughput platform for manufacturing mRNA-based therapeutics and vaccines.
“We have the capabilities to synthesize and characterize thousands of different sequences in an arrayed format, and hundreds of thousands of different sequences in a pooled format, to create large datasets that are robust enough to train highly accurate AI models,” says Stephanie Khan, director, business development, Ginkgo Bioworks. “One model reached 93% accuracy in predicting stability-assay performance.” She adds that Ginkgo Bioworks can “perform characterization across dimensions like half-life mRNA stability, polysomal profiling, immune profiling, and proteomics.”
Still, optimizing the manufacturing of mRNA-based vaccines faces some challenges. The top challenges that Khan points out are shelf-life stability, cost of reagents, impurities (such as double-stranded RNA), and analytics. “If we can overcome these challenges, mRNA-based vaccines could be manufactured at lower cost, and the biggest advantage would be that efficacy would increase,” Khan suggests. “The industry could really significantly improve its rates of therapeutic success.”
As one example, sequence design and optimization “would significantly increase shelf-life stability and lower impurities, which is an area Ginkgo Bioworks excels in,” Khan remarks. To maintain this excellence, the company invested in automation processes and development teams that focus solely on mRNA purification and analytics.
Nonetheless, further investment and continued advances are required to further improve the manufacturing of mRNA-based vaccines and therapeutics. “There is so much in the mRNA field left to explore, and so many various ways Ginkgo Bioworks would be able to provide support to pharmaceutical companies to optimize their therapeutic drugs and accelerate their development timelines, that it was challenging to narrow our scope of work when first developing our platform,” Khan explains. “We were able to resolve this challenge by relying heavily on market research, data, and applying our learnings from our other successful platforms and work we accomplished in the circular RNA field.”
Intensifying mRNA manufacturing
Other companies have also developed platforms to improve mRNA manufacturing. For example, Quantoom Biosciences, headquartered in Belgium, developed its Ntensify™ technology.
“The Ntensify solution brings a novel approach that provides turnkey solutions for mRNA production and purification, achieved through advanced design of experiments and after four years of optimization,” says Lionel Malbec, PhD, product manager, Quantoom. “It enables high-yield, low-contaminant mRNA to be produced while simultaneously reducing the overall costs.”
Plus, the Ntensify solution works with any mRNA construct, and it is processed through one-pot in vitro transcription with co-transcriptional capping and a redesigned single-step purification to minimize mRNA losses and increase available drug substance mRNA. “It can scale seamlessly from milligram scale (R&D level) to kilogram scale (commercial production) using the same process design principles,” Malbec notes. “[This system] increases the accessibility of mRNA bioprocessing.”
Developing the Ntensify solution, though, required that several technical hurdles be cleared. “The biggest challenges in developing our platform involved optimizing the RNA production process for high quality, high yield, and cost efficiency, while ensuring it could be RNA construct–agnostic,” Malbec says. “Additionally, we needed to integrate this optimized process into a compact, automated system that could eliminate scale-up needs, minimize operator errors, and reduce both the physical and financial footprints, particularly for deployment in resource-scarce areas such as low- and middle-income countries.”
This platform is designed for ease of use. In one piece of equipment, Quantoom’s automated system can perform in vitro transcription and downstream processing, which “reduces the need for operator intervention and eliminates scale-up needs,” Malbec asserts. “This approach minimized errors and ensured a reliable, streamlined production process independently of production needs.”
Tomorrow’s mRNA
One of the companies working to bring about fundamental advances in mRNA-based therapies is Parexel, a global clinical research organization headquartered in Durham, NC. Rajiv Gangurde, PhD, the company’s vice president, technical operations, cell and gene therapy, offers this observation: “Currently available mRNA vaccines simply deliver the message that gets translated into the corresponding protein by the same cellular machinery that translates one’s own mRNA. Beyond translation into protein, the message is not useful and degrades in the cell.”
Gangurde is based in Cambridge, MA, where several biotechnology companies are developing mRNA-based therapies. He notes that Paraxel helps them “navigate interactions with regulatory agencies at various stages of development, from early pre-IND stage to licensure.”
Some of the next generation of RNA-based therapies and vaccines could use self-amplifying RNA (saRNA). “Such RNA carries the message that encodes the desired protein as well as viral genes that code for replicase, an enzyme that can replicate the mRNA,” Gangurde points out.
Gangurde emphasizes that getting tomorrow’s RNA-based treatments to patients will take teamwork: “Parexel helps companies developing mRNA-based drugs on multiple levels. For example, we consult on regulatory strategy, and we plan and execute clinical trials.”
In terms of CMC, Gangurde points out that mRNA-based therapies face the same challenges as any other new modality. “As any modality develops, the stringency on CMC increases—as it should—and drug developers need to pay more attention to manufacturing considerations balancing safety and efficacy,” Gangurde explains. “Specific to mRNA-based therapies, CMC activities need to focus on developing platform technologies for optimizing mRNA cargos and tissue tropism for lipid carriers.”
Tomorrow’s manufacturing of various forms of RNA will face even more challenges as the range of applications expands. “RNA-based therapies are rapidly moving beyond vaccines,” Gangurde notes. “Newer forms of mRNA—for example, circular RNA—are being developed as therapies for autoimmune diseases, infectious diseases, and cancer.”
