mRNA-based therapies catapulted to the forefront of public consciousness in the form of vaccines against the SARS-CoV-2 virus. After that success, mRNA therapeutics are now being developed for an ever-growing number of indications and applications that include cancer, cystic fibrosis, and infectious diseases, as well as gene and stem cell therapies based upon either gene replacement or gene editing.
However, the purification bottleneck must be solved before these therapies can be scaled up and produced in adequate quantities for clinical trials or commercialization.
“Traditionally, small-scale tools and products have been used to purify mRNA, such as reverse-phase high-performance liquid chromatography (HPLC), precipitation, and in some cases, cellulose-based chromatography,” says Sirat Sikka, field application scientist at Thermo Fisher Scientific (Thermo Fisher). Those methods can be used to purify a few grams of mRNA and are adequate for bench work and some applications. Scale-up for clinical trials and commercialization, however, requires the ability to purify tens of grams or even tens of kilograms of mRNA.
“At Thermo Fisher, we understood the importance of mRNA and knew, even before the pandemic, that mRNA would be widely used,” Sikka recalls. Since then, scientific and trade journals alike have cited mRNA therapeutics and vaccines as disruptive advances that can change the future of medicine and ease of manufacturing, and the ability to target pathways that otherwise are undruggable. Industry analyst Research and Markets predicts the global segment for mRNA therapeutics will grow from $46.7 billion in 2021 to $101.3 billion by 2026. That’s a compound annual growth rate of 16.8%.
Relieving the bottleneck
To be ready for such rapid growth in mRNA development, Thermo Fisher began developing a new affinity chromatography resin to isolate and purify mRNA long before the technology became a “household word.” The team sought to develop a resin enabling improved recovery, increased purity, and enhanced reproducibility.
The POROS™ Oligo (dT)25 Affinity Resin—the resulting product—is a 50 µm poly(styrene-co-divinylbenzene) cross-linked porous bead functionalized with deoxythymidine (dT) strands that bind to mRNA via the poly-A tail (a chain of adenine nucleotides) that is on the three-prime end of all mRNA molecules.
One of the challenges is the size of mRNA. It is a large molecule —20 to 50 nm or greater in size that varies with construct length and solution composition—so there can be limitation to diffusion through the chromatography media and, therefore, hindrance to mass transfer, Sikka explains. Because the POROS™ beads have large throughpores the surface area available for interaction between the resin and mRNA molecule is increased leading to higher capacity. The large pores also result in a reduced mass transfer resistance, which helps to improve process efficiency and productivity.
“The POROS Oligo (dT)25 Affinity Resin minimizes the need to deal with organic solvents that are often used with HPLC systems,” Sikka continues. “Using organic solvents in large volumes becomes an issue for manufacturing.” She cites safety concerns regarding solvent disposal as well as the need to retrofit facilities to deal with them.
Instead of using toxic chemicals, after mRNA synthesis the column is loaded “with mRNA plus salt (for example, NaCl).” This neutralizes the negative charges on the RNA molecules so the poly-A tail can bind with the dT strands on the beads. Then, she says, “Elution can be performed using a low-conductivity buffer, or even water in some cases.” Impurities and salt ions are washed away. With the sodium removed, the negative charges on poly-dT and the poly-A tail repel each other, freeing the purified mRNA and generating a recovery, typically above 90%, depending on the elution buffer and mRNA construct size.
From bench to manufacturing
The POROS Oligo (dT)25 Affinity Resin is designed for scalable purification processes, so it is used to pack fast protein liquid chromatography (FPLC) columns. “The columns can be packed to multiple column size according to customers’ needs, based on their process development and optimization,” Sikka says. “We also have small-volume prepacked columns and Robocolumns. The 1 mL and 5 mL prepacked columns could be used with HPLC if needed, but that would only allow customers to purify very small sample volumes, may require re-plumbing and is usually not ideal for process development, so switching to FPLC is preferred. What customers mostly are looking for when they choose this resin is to scale-up purification, so they use it with FPLC systems.”
Sikka says this affinity resin is a good option for scientists interested in developing a platform process that can be implemented for a variety of mRNA constructs. One of the benefits of using mRNA is that the same construct backbone could potentially be used to express different proteins. As a result, scientists can potentially use a platform process for multiple mRNA programs.
While researchers may switch out the gene of interest, “they could still be working with mRNA of comparable sizes,” Sikka explains. “For example, depending on the protein they are trying to express, if the size range of all the constructs is between 4000 to 6000 bases, they could use this as the first capture step and develop a platform process.” Working with much larger mRNA, such as self-amplifying could require some additional development.
As a platform technology, the first purification step with POROS Oligo (dT)25 would remove digested DNA template, nucleotides, enzymes, and buffer components. This could be the only step in the process before concentration and buffer exchange. If needed, a second chromatography step can be developed with POROS™ hydrophobic interaction chromatography (HIC) or anion exchange chromatography (AEX) resins to remove double-stranded RNA and uncapped or residual incomplete RNA transcripts.
“Starting with this resin during the research and discovery phase lets scientists continue using the same purification resin all the way to commercial manufacturing,” she says. “This also eases the process of transitioning from one mRNA construct to another of similar size.”
As she elaborates, “Once used in a process, the resin is already in the system and accepted by the customer’s quality team.” Additionally, scientists needn’t redevelop their purification steps during each phase of scale-up, which minimizes the need to onboard a variety of chemicals and solutions or develop different buffer compositions, thus accelerating process development and reducing time to market. The resins are also reusable, which reduces the cost of goods.
“Importantly, POROS Oligo (dT)25 Affinity Resin beads are available for GMP production, and we provide the regulatory support package,” Sikka says.
Transitioning to a new bead
Switching to the POROS Oligo (dT)25 Affinity Resin is just a matter of ordering the prepacked columns if customers already use FPLC.
“A lot of our customers, however, are still at the research scale and are interested in scaling up,” Sikka points out. “They don’t necessarily have FPLC systems, and are trying to understand their options.”
In those instances, she recommends ordering loose POROS Oligo (dT)25
Affinity Resin, which can be used in spin columns or microfuge tubes in a batch mode. If that works well for their purposes, they may consider investing in an FPLC for further purification optimization and scale-up.
“Thermo Fisher is very focused on the modern day,” Sikka says, with solutions that address current and emerging purification challenges. Today, that means an intense focus on mRNA purification.
As interest in mRNA therapeutics continues to increase, the company’s R&D is focusing on understanding the complexity associated with purifying self-amplifying mRNA, removal of product related impurities such as double-stranded RNA and abortive transcripts, and use POROS Oligo (dT)25 Affinity Resin and other technologies to resolve existing and emerging challenges.