The mRNA-based COVID-19 vaccines were made possible through the use of lipid nanoparticles (LNPs) as delivery vehicles to protect the mRNA from degradation and to aid intracellular delivery and endosomal escape. However, LNPs are not the only solution for the delivery of mRNA and other sensitive molecules used in gene therapies. Charge-altering releasable transporters (CARTs) are polymeric alternatives that are simple to produce and robust, with the ability to deliver many types of nucleic acids and potentially other negatively charged biomolecules. Evonik is collaborating with the Stanford University researchers that initially developed this technology to commercialize CARTs and offer drug developers this complementary formulation solution for mRNA- and other nucleic acid–based therapies.
Significant Therapeutic Potential, But Delivery Challenges Remain
The approval of the mRNA–LNP COVID-19 vaccines established confirmation of the utility of these molecules as prophylactic vaccines in humans. A key advantage of mRNA in drug development is that the molecules are fairly similar irrespective of their target. As a result, mRNA can encode a variety of proteins implicated in cancer or infectious disease, among others, but the physicochemical properties of the respective mRNAs are similar from a manufacturing perspective and dictate how they must be produced and formulated for delivery and adsorption within the body. There are some nuances, of course, but in general it is possible to take a platform approach to manufacturing and formulation.
Production of mRNA drug substances promises to be easier than for traditional biologics, such as recombinant proteins and monoclonal antibodies (mAbs), which are expressed in specialized cell lines and then must be purified through a series of complex unit operations. mRNA is manufactured in vitro via enzymatic reactions that are more controlled, leading to simpler purification operations. Once a process is established, it can be applied to the production of many different mRNA drug substances. There is no need to develop new cell lines for each new product, as is the case with mAbs and other proteins.
On the other hand, mRNA molecules require encapsulation, because mRNAs are inherently biologically transient and are readily degraded in the body, a problem that affects most proteins to a lesser extent. mRNA molecules must therefore be protected for long enough for delivery to the site of action, which is inside the cell. Another challenge that must be overcome is the high charge density of mRNA. Negatively charged molecules do not readily enter cells, and the large size of mRNA molecules makes transport into the cytoplasm even more difficult. In contrast, protein therapeutics, such as antibodies, typically target receptors on the surfaces of cells and do not have to enter cells in order to exert their therapeutic action.
The COVID-19 vaccines leveraged lipid nanoparticles (LNPs), but there are other options, including polymeric platforms, such as the new charge-altering releasable transporters (CARTs) Evonik develops in conjunction with researchers at Stanford University.
Lipid Nanoparticles as a Delivery Solution
LNPs comprise four different lipids, including an ionizable lipid that complexes the mRNA; a structural, stabilizing lipid, such as a phosphatidylcholine; polyethylene glycol (PEG)-modified lipids that prevent aggregation; and cholesterol. The LNP must be formulated to protect the mRNA, aid in its transport into the cell, and then release it into the cytoplasm. In some cases, LNPs also function as adjuvants, providing additional immunostimulation.
The required properties of the LNP formulation are dictated by the characteristics of the mRNA molecule (e.g., size, charge), and the delivery target for achieving a therapeutic effect can vary significantly from one drug substance to another. The complexity and need for specialized solutions can be a disadvantage. In addition, most established LNPs exhibit a strong tropism toward the liver, and a portion will reach the liver even when administered locally.
The chemical design space for ionizable lipids, which have the greatest impact on the functionality of LNPs, has been fairly thoroughly explored at this point. New ionizable lipids with unique structural motifs are introduced rarely. From an intellectual property perspective, there is consequently added risk to developing proprietary LNPs.
The LNPs consist of four lipids, three of which provide the structure of the nanoparticle (a PEGylated lipid, cholesterol and phospholipid distearoylphosphatidylcholine (DSPC)). The fourth is an ionizable lipid with a positive charge that becomes neutral under physiological conditions, reducing the toxicity of the LNPs.
Merging Lipid and Polymer Technologies
Evonik has over three decades of experience developing biodegradable polymers (RESOMER® brand) with pharmaceutical applications, particularly in parenteral drug delivery, including long-acting injectables. This expertise is complemented by considerable experience with lipid manufacturing, which has been leveraged for the production of lipids used in the Pfizer/BioNTech COVID-19 vaccine.
Recognizing the need for other solutions to complement LNPs that could effectively deliver mRNA to organs in the body other than the liver, Evonik began a discovery project to identify other excipients that could be used to deliver nucleic acids — not just mRNA, but also short interfering RNA (siRNA) and plasmid DNA. At the same time, the company was surveying the external landscape for potential new technologies and came across the CARTs being developed by a team of researchers, including Professor Robert Waymouth, Professor Paul Wender, and Professor Ronald Levy, at Stanford University.
The Stanford team was looking for a provider partner like Evonik rather than a pharmaceutical company to avoid limiting the potential use of the technology. Working with an excipient supplier / contract development and manufacturing organization (CDMO) like Evonik that provides solutions and services to biopharmaceutical companies enables the use of the technology in many different applications by many different drug developers.
The Molecular Makeup of CARTs
CARTs are oligomeric molecules of 10–20 repeating monomer units with a molecular weight of approximately 3–5 kilodaltons. Polycarbonate and poly(α-amino acid) segments make up the backbone, both of which contain hydrolyzable groups and thus are degradable in aqueous environments and potentially biodegradable. The poly(α-amino acid) block is ionizable and hydrophilic, whereas the polycarbonate block is functionalized with lipid side chains and is therefore hydrophobic. Together, they form an amphiphilic molecule with a lipid-like head and tail structure.
The ionizable hydrophilic block binds to the negatively charged mRNA. The multiple charged oligomers can bind to nucleic acids efficiently and without the need for PEGylated lipids to impart solubility. Cellular uptake — rather than being driven by apolipoprotein binding, as is the case with LNPs — is mediated by interactions between the negatively charged cellular membrane and the positively charged ionizable block. Once inside the cell, physiological pH conditions result in deprotonation of the ionizable block. The lack of a charge leads to disassembly of the particle and release of the mRNA. The polymer then breaks down into smaller molecules that can be metabolized or excreted.
Advantages of Polymer Technology
CARTs can offer several advantages over LNPs, most notably that polymer-based formulations enable the design of delivery vehicles that target different organs beyond the liver, such as the spleen. CARTs have the potential to provide targeting opportunities that are complementary to LNPs.
CARTs are also based only on one oligomer structure. The design of the oligomer can be fairly complex, but it is the only excipient that must be mixed with the mRNA, overall making it a simpler system than LNPs from the perspective of manufacturing. Fewer inputs also mean that less analytical testing is required. Importantly, physical mixing is all that is required to complex the CART with the mRNA. Initial evaluations indicate that CART particles are more robust than LNPs, where production of formulated mRNA products make it as much of an art as a science. This robustness facilitates the development of reproducible production processes and thus transfer to the clinic.
CARTs also present an advantage from an IP perspective, as they represent a completely new area of chemistry with a wide-open design space. Evonik will be licensing the technology from Stanford, which holds the patents.
Collaboration is Essential
The challenge with CARTs is synergizing together the various types of expertise required to develop a solution that can serve as a platform technology for the delivery of nucleic acids. A multidisciplinary approach is needed to effectively design molecules with the right balance of lipidic and polymeric properties. With expertise in both polymers and lipids, Evonik is well positioned to partner with the Stanford researchers to advance CART technology. The knowledge of monomer synthesis, polymerization, nanoparticle formation, final product formulation, and bioanalytical testing at Evonik has all been applied to the further development of CARTs.
Groups at our polymer production facility in Birmingham, Alabama, are working closely with the lipid experts at our site in Vancouver, Canada, educating each other on the complementary aspects of CART technology that are unfamiliar to each group. For instance, the polymer group has not previously worked with nucleic acids as APIs, while the lipid group has not previously worked with polymers. The key to success has been engaging the teams, building on the close collaborations that already existed between the sites, and further cementing them. Indeed, the CART project has confirmed for many at Evonik that our company is truly interdisciplinary in nature.
Initial evaluations indicate that CART particles are more robust than LNPs, where production of formulated mRNA products make it as much of an art as a science. This robustness facilitates the development of reproducible production processes and thus transfer to the clinic.
Getting to GMP
One of the important aspects of the work at Evonik is developing a scalable manufacturing process that will be suitable for production of CARTs under GMP conditions. The process used by the Stanford group leverages laboratory-scale academic techniques. That synthetic route has to be modified so that it can be readily scaled to produce anywhere from grams to hundreds of grams and potentially kilograms of material in a manner that is compliant with GMP requirements.
Much progress has been made in this area, and, once the manufacturing process has been finalized, the first CART product will be launched. Extensive stability studies are also underway in advance of a product launch to establish a body of work regarding the storage and handling of CART particles. Part of this effort involves discussing the technology with potential customers, sharing the information that is available in the public domain, and outlining the pros and cons relative to other available delivery solutions on the market.
Early Safety Data are Promising
One of the advantages of the CART technology is that it is being commercialized based upon an extensive foundation of work completed at Stanford University. Some of that work includes peer-reviewed, published articles reporting results of toxicity studies. The published data suggest that CARTs are highly biocompatible systems with respect to potential toxicity.
Experiments involving repeated injections will be needed to establish the immunogenicity of CARTs, but their biodegradability is expected to limit the potential for an immune response. In addition, for each drug product developed with clients, it will be necessary to perform GLP tox and other testing as for any drug candidate. However, the body of literature developed by the Stanford group offers positive answers to the basic questions that provide reasons for optimism.
Wide Applications
While current efforts at Evonik are focused on the development of CARTs for the delivery of mRNA, the technology is expected to have applicability for other nucleic acids, such as siRNA and other types of RNA, as well as plasmid DNA. In fact, any biological molecules with strong negative charges could be compatible. There is therefore also potential to use CARTs with proteins that have the correct type of negative charges.
It may also be possible to use combinations of these different types of molecules, depending on the particular application, such as a protein and a nucleic acid. In addition, the use of CARTs in conjunction with other delivery technologies — microparticles for depot injections, capsules for oral delivery of biologics, and so on — could open up new applications. There is significant opportunity for further development.
Anticipating Regulatory Responses
The challenge with any novel excipient is that currently there is no separate approval pathway, and thus the excipient must be used with an API and submitted as part of a new drug application (NDA). It is then approved for that type of use only.
Evonik is following FDA’s pilot excipient approval program with interest, which, if implemented as a new regulatory pathway, would broaden the applicability of excipients approved using this approach. Until that happens, however, CARTs will get approved when used in a drug product that receives approval. With the biodegradability and biocompatibility of CARTs, the expectation is that there will not be any significant regulatory hurdles.
In addition, once the first product leveraging CART technology has received approval, subsequent products can leverage previously generated knowledge, as the backbone of each CART is essentially the same. Only the side chains and functional groups attached to the backbone change to accommodate the different properties of each biomolecule being encapsulated, so there may be different combinations of monomers in different ratios and overall different molecular weights, but the biodegradable and biocompatible properties will be the same.
Ultimately, Evonik will offer a reasonable library of CARTs that target different organs, have different cellular and other bioactivities, and can accommodate different types of biomolecules. The idea is to have sufficient flexibility to provide solutions for a range of nucleic acids — and potentially other molecules — intended for delivery to different organs and cell types.
Part of a Broad Excipient Portfolio
CARTs cross the boundaries of existing polymeric and lipid-based systems offered by Evonik. On the lipid side, Evonik produces custom ionizable lipids, along with the vegetable-derived cholesterol PhytoChol® and collagen-based products. On the polymer side, we offer the RESOMER® range of standard and custom biocompatible and biodegradable poly(D,L-lactide) and poly(D,L-lactide-co-glycolide) polymers with acid and ester end group chemistries and specialized co-polymers. All of these excipients are produced at high quality and backed by a secure supply chain. In addition to these excipients, we also offer drug formulation and manufacturing services and can support projects from the proof-of-concept stage through development and onto clinical and commercial production.
The new CART technology will be included in Evonik’s extensive service portfolio as a complementary solution for the delivery of nucleic acids. The intention is to include CARTs as part of the formulation development services offered by Evonik for APIs. For example, when no formulation exists but some data are available, Evonik can develop a certain number of trial formulations in a “blinded” fashion that the client can then test. If one of the trial formulations appears promising and the client wishes to go forward, contractual agreements are put in place, the specific formulation solution is revealed, and formal and more comprehensive formulation and process development proceed.
This approach has been well received in the marketplace, because it takes the guesswork out of the hands of the drug developer and places it in the hands of Evonik’s experienced formulators, who can make highly educated choices. Clients then can generate some initial data and thus make informed decisions about which formulation approach will be optimal for their specific applications.
Formulation Services Offered in the Second Half of 2022
The goal is to begin offering the new formulation services using CARTs in the second half of 2022. Clients will send their APIs to Evonik to be formulated using our experience and expertise to ensure the best chance of success. Clients will then be able to test the formulations with respect to bioactivity, behavior using in vivo models, and so on.
Although the launch will include just one product initially, Evonik will eventually offer a family of CART molecules through continued collaboration with the Stanford researchers, who have developed a library of CARTs and are continuously expanding their portfolio. Once the first product is launched, Evonik will add additional CARTs based on the available data, with the focus on molecules that provide the best solutions for the range of APIs and applications that may be forthcoming from clients in the future.
Ultimately, Evonik will offer a reasonable library of CARTs that target different organs, have different cellular and other bioactivities, and can accommodate different types of biomolecules. The idea is to have sufficient flexibility to provide solutions for a range of nucleic acids — and potentially other molecules — intended for delivery to different organs and cell types.