Cynthia A. Challener is a contributing editor to Pharmaceutical Technology.

Cynthia A. Challener

Oligonucleotide drug substances generally are unstable at room temperature, and some are also degraded in the presence of certain enzymes. That is why, in part, they are formulated using special delivery solutions such as lipid nanoparticles (LNPs). (These delivery technologies also facilitate entry of nucleotide APIs into target cells.) Degradation can occur via loss of terminal nucleotides or certain functional groups, oxidation, and other mechanisms. Oligonucleotide–oligonucleotide interactions that can occur at higher concentrations are an added concern. These instability issues must be managed throughout the entire manufacturing process.

The challenges are particularly great for messenger RNA (mRNA), which can be degraded by enzymes found commonly in the environment, temperature, and due to the overall shear sensitivity of the
long-chained molecule.

The Real Message Behind Commercial mRNA Products

Managing mRNA Instability During Formulation, Manufacturing, and Shipment

Pharmaceutical Technology’s The Real Message Behind Commercial mRNAProducts eBook

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Cynthia A. Challener, PhD, is a contributing editor to 

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Unique solutions are required to protect inherently unstable messenger RNA.

Managing mRNA Instability

Many drug molecules, particularly biologics and vaccines, are unstable at room temperature when formulated in aqueous solutions and must be frozen or lyophilized (freeze-dried) to allow their storage and shipment. Freezing and freeze-drying involve harsh conditions that can degrade the active ingredients. Cryoprotectants and lyoprotectants are excipients used in drug product formulations to ensure that safe and efficacious products are obtained following these processes. As such, the quality of those excipients can have a direct impact on the final stability and performance of frozen and lyophilized drugs and vaccines, as explained independently by two industry experts.

Excipients used as cryoprotectants and lyoprotectants serve mainly to protect the drug substance in a formulation from degradation or disassociation during the freezing or freeze-drying process, according to Christy Eatmon, global subject matter expert, sterile drug products, in the pharma services business of Thermo Fisher Scientific. Lyoprotectants also provide a bulking effect so that the resulting dried cakes can be readily reconstituted.

Most cryoprotectants are monosaccharides (sugars), polysaccharides, or polyols, observes Virginie Le Brun, associate director of formulation development for Lonza Drug Product Services. There are limited options, she adds, because only certain excipients are suitable for use in drugs for human use, particular
 parenteral applications.

The selection of the best excipient or combination of materials and their quantity(ies) is dependent on the molecule type, according to Eatmon. She recommends the use of a design-of-experiment (DoE) approach to identify which materials work best with the target drug product.

Take a structured approach to development

Ideally, formulation compositions for drug products that must be frozen or lyophilized should be kept simple. “Whenever possible, just one excipient with sufficient ability to protect, and if necessary act as a bulking agent, should be selected,” Le Brun says. However, she adds that there might be situations where a combination is needed to achieve the most stable formulation conditions.

During formulation development, it is important, observes Heiko Nalenz, associate director of process development for Lonza Drug Product Services, to take a structured approach to development, addressing process challenges during early formulation selection. “Excipient selection should be based on the intended storage conditions and/or compatibility with the freeze-drying process. For this purpose, the thermally induced transitions of the complete product matrix (e.g., glass-transition temperature and collapse temperature) should be characterized,” he explains.

Eatmon agrees, once again underscoring the value of performing selective DoE studies to determine which excipients are most successful for the formulation and product under evaluation. “Characterization is important to understand and develop key parameters of the lyophilization cycle. For example, with a small molecule, it is important to understand whether the molecule is crystalline or amorphous. With that information in hand, the eutectic point or glass-transition temperature should be determined to enable excipient selection,” she comments.In the case of large molecules or more complex formulations, such as dispersions, Eatmon notes that the need for a combination of excipients is more likely.

Quality and purity can have multiple impacts

As with APIs, the quality and purity of lyo/cryoprotectants used in formulations that are frozen or lyophilized is important. “Impurities in materials may cause slight differences in critical attributes, which can lead to incomplete freezing or poor sublimation during the drying process,” Eatmon says. They may also negatively affect the product’s stability in the frozen or freeze-dried state, according to Nalenz. For crystallizing excipients, he points to the potential of varying impurity profiles to increase or decrease their propensity to crystallize, which might impact the quality of the drug component if it ends up in the final product in a non-desirable state.

The biggest potential impact of poor-quality cryo/lyo excipients is, according to Eatmon, for the resulting frozen or lyophilized product to fail to meet acceptance criteria, resulting in rejection of the material. “Robust quality systems should prevent the use of non-compliant materials, but if the quality issue is unexpected, timelines may be delayed while a replacement is sourced. Plans should be in place in advance to efficiently manage this type of scenario,” Eatmon emphasizes.

In addition, variability in the properties of excipients can also create challenges. “Even excipients in compliance with compendial quality specifications may have some variability in their properties or impurity profiles that may impact cryo/lyoprotective properties,” Nalenz remarks. This variability for many cryo/lyoprotectants can be attributed to the fact that they come from plant sources (e.g., starch), according to Le Brun.

Suppliers can help avoid such issues by deeply characterizing the cryo/lyo excipients they produce and obtaining knowledge of how variability might impact their products’ cryo-protective and other properties, observes Nalenz. Drug manufacturers, meanwhile, can for lyophilized products perform lyo cycle tests using different lots of lyoprotectants to ensure that batch-to-batch variability is accounted for in the final formulation, according to Eatmon. She also recommends that conservative measures be taken when developing lyophilization cycles to account for some variability in excipients, upstream drug substances, and drug-product manufacturing processes.

Excipient sourcing and characterization crucial

Appropriate sourcing of any type of excipient is essential to ensuring security of supply and the use of high-quality, compliant materials. For instance, Eatmon stresses the importance of sourcing materials that are intended for pharmaceutical use rather than nutraceutical- or food-grade materials. She also indicates that excipients classified as ultra-pure or low bioburden are preferable for use in injectable products, which comprises most frozen and freeze-dried products. In addition, given the potential for variability in excipient batches, Le Brun emphasizes the importance for formulators of understanding that variability and its impact on the properties of the drug product before selecting a specific excipient supplier.

While common cryo/lyoprotectants and bulking agents are well understood, used in a variety of commercial products, have multiple high-quality sources, and comply with a variety of compendia, there are limited
numbers of them suitable for parenteral applications, according to Eatmon. Other than the challenges related to impurities and variability, Le Brun comments that the limited options can represent a significant hurdle, particularly for newer modalities.

It is hoped that the FDA’s Pilot Program for the Review of Innovation and Modernization of Excipients (referred to as PRIME), which was launched in September 2021 (1), will lead to a regulatory pathway for approval of novel excipients and innovation in the excipients space, including new cryo- and lyoprotectants.

FDA. Center for Drug Evaluation and Research Office of New Drugs Novel Excipient Review Pilot Program. 

Cynthia A. Challener, PhD, is a contributing edtior to 

When referring to this article, please cite it as Challener, C.A. Quality Challenges Associated with Cryo/Lyoprotectants. 

Impurities and batch-to-batch variability present the biggest challenges.

Quality Challenges Associated with Cryo/Lyoprotectants

For messenger RNA (mRNA) therapeutics and vaccines formulated as lipid nanoparticles (LNPs), the excipients receiving the most attention are the encapsulating lipids. While these compounds are crucial to the stability and delivery of mRNA-LNPs, the non-lipid excipients also play crucial roles in the function of final drug products. They impact the morphology and stability of these delivery systems, which in turn affects

performance and the overall usefulness of mRNA drugs developed using nanoparticle technology.

Non-lipid excipients used to formulate mRNA-LNP products include solvents, salts, and sugars/cryoprotectants, according to David Jung, senior manager RD&I, nucleic acids at Evonik. Overall, these excipients serve the function of controlling the pH during the initial mixing and formation of the particle and ensuring stability upon storage of the material, he notes.

More specifically, rapid mixing is the predominant technique for lipid nanoparticle–mRNA formulations: an ethanol phase (lipid components) and an aqueous phase (mRNA molecules, usually in acetate or citrate buffers) are mixed under specific conditions (pH and flow rate), explains Luca Bruno, segment marketing manager, MilliporeSigma, the Life Science business of Merck KGaA, Darmstadt, Germany. Rapid mixing, he adds, allows scalability and reproducibility of mRNA-LNP formulation.

Organic solvents and residual components are then removed by ultrafiltration. The mRNA-LNP drug substance is then formulated using a more physiological buffer, typically tris(hydroxymethyl)aminomethane- or phosphate-based together with salts such as sodium chloride or potassium chloride to adjust the ionic strength, Bruno observes.

Storage conditions (as aqueous solutions, frozen, or lyophilized) must also be carefully considered in the context of mRNA-LNP clinical translation, according to Bruno, because they can affect the long-term stability of mRNA-LNP formulations. “Sugar stabilizers (sucrose in particular, trehalose or mannitol as alternative options) are commonly used for this application,” he says.

Choosing the right non-lipid excipients important

In addition to assuring long-term stability and in vivo performance, non-lipid excipients for mRNA-LNPs also play an important role in controlling manufacturing processes, Jung comments. “The selection of these excipients must therefore be done carefully. Typically, they are initially chosen based on their chemical and physical properties (i.e., pKa and lyophilization characteristics, freezing point/state) because these attributes will determine compatibility with the drug substance. Because these choices may also impact the morphology of mRNA-LNPs, which can affect overall efficacy and stability, modifications may be required as determined [by] experimental testing,” he says.

The susceptibility of mRNA to degradation requires that formulation buffers be free of any ribonuclease contamination. Testing of excipients for the absence of nuclease activity is therefore the preferred approach, Bruno stresses. Other product-quality attributes should also be determined, including endotoxin content and bioburden, as they play an important role in controlling the risk of contamination by these materials.

Beyond the product-quality attributes, Bruno emphasizes the need to consider supplier related aspects for any excipients used in mRNA-LNPs, including non-lipid excipients. “Supply robustness and availability of supporting documentation are both essential if mRNA-LNP developers are to navigate regulatory challenges and ensure smooth manufacturing scale up and progression through clinical development to commercialization,” he adds.

Since the approval of the COVID-19 mRNA vaccines and demonstration of their effectiveness, interest in mRNA-LNP therapeutics and vaccines has risen dramatically from all perspectives. In particular, the rise of the mRNA technology due to the COVID-19 pandemic and the manufacturing scale needed to produce vaccines shifted the attention to mRNA manufacturing needs, according to Bruno.

“The development of non-lipid excipients has primarily focused on the quality attributes based on the specificity of the technology,” Bruno notes. For instance, the need for endonuclease-free materials required establishment of additional product-release testing methods and services to support drug manufacturers.

There has also been a significant amount of effort placed on assessing non-lipid excipients that may improve the long-term stability of mRNA-LNP formulations, observes Jung. He points specifically to the use of different cryoprotectants to improve refrigerated storage stability. Lyophilization is also being explored as a means for eliminating cold-chain requirements, with several clinical-stage studies underway to assess the impact of lyophilization on the stability and in vivo efficacy of traditional mRNA-LNPs.

Separate efforts are also underway to create novel delivery mechanisms for RNAs that are not dependent upon LNPs, Jung adds.

A key focus in the mRNA field is on lipid excipients and that will continue to be true for some time, contends Aditi Mehta, head of mRNA process and delivery, MilliporeSigma, the Life Science business of Merck KGaA, Darmstadt, Germany. “Despite the success of the COVID-19 vaccines, we still have not reached the full potential of mRNA therapeutics, and there are several hurdles. Insufficient targeting of organs beyond the liver or vaccinations, an acute immune response against LNP administration, stability issues, and the need of extreme low temperature for storage are key bottlenecks in the development of LNP medicines,” she says.

These issues, Mehta observes, can be addressed through improvements in lipid and LNP design, (e.g., optimization of linker chemistry, addition of degradable bonds, more optimal synthetic routes, better LNP components, etc.). “Strategies such as design of novel ionizable lipids based on large libraries produced with combinatorial synthesis have been aiding the understanding of structure-function properties and enabling the design of unique lipids with improved properties (targeting, stability, tolerability),” she states.

Data continues to be published on new lipid libraries that demonstrate delivery to different tissues/cells (1), Jung agrees. Targeting of mRNA-LNPs to specific tissues or cells can be achieved through either passive or active means. A majority of studies investigating ways to target LNPs have focused on passive targeting through modification of the LNP size, surface, and composition. Substitution or addition of an extra moiety (polymeric or lipidic) has also been shown to be beneficial for achieving passive targeting toward specific organs, according to Mehta.

“Active targeting approaches leverage conjugation of lipid components with small molecules, antibodies, proteins, or aptamers,” Mehta comments. She points specifically to flexible platforms and highlights the ASSET platform developed in the lab of Prof. Dan Peer at Tel Aviv University (2). “This type of approach enables customized targeting towards any cell receptor and, therefore, any tissue or organ,” she says.

Mehta does caution, however, that while there has been a tremendous push (and success) in this area over the past few years, there is still a long way to go and many challenges to overcome.

While lipids have predominated the mRNA delivery space, they are not the only materials that can provide effective delivery of these sensitive and highly charged drug substances. Hybrid lipid–polymer delivery systems, for instance, have been developed to harness and combine the advantages of both lipid and polymer nanoparticles, according to Mehta.

“Polymers possess an inherent high degree of chemical variety that is not feasible with lipids, increasing tremendously the possibilities of chemical functions, such as promoting endosomal escape, controlled released of RNA, targeting, and finally stability,” she explains. Lipids, meanwhile, show great potential for enhancing bioavailability, improving pharmacokinetic profiles, and increasing biocompatibility.

Strategies investigated to formulate hybrid systems include lipopolyplexes, hybrid LNPs, and others. As an example, Mehta notes that incorporation of biodegradable polymers such as poly(beta amino esters) in LNP formulations leads to improved and specific lung delivery, highlighting the potential of hybrid lipid-polymer nanoparticles (3).

Evonik has a research agreement with Stanford University to develop and commercialize the charge-altering releasable transporter (CARTs) technology initially developed by the Waymouth lab. These polymeric materials can effectively encapsulate mRNA and then rapidly degrade through controlled self-immolative reaction to release the mRNA, according to Jung.

“The first generation of CARTs showed mRNA delivery efficacy in a range of cell types in both in vitro and in vivo models,” Jung notes. He adds that subsequent generations of CARTs incorporating lipid side chains on the CART backbone structure (4) showed an improved delivery efficiency.Further generations of CARTs that are still under development, says Jung, have shown specific targeting capabilities in preliminary testing (5,6).

Kularatne, R.N.; Crist, R.M.; Stern, S.T. The Future of Tissue-Targeted Lipid Nanoparticle-Mediated Nucleic Acid Delivery. 

Kedmi, R.; Veiga, N; Ramishetti, S. Goldsmith, M.; Rosenblum, R. et al. A Modular Platform for Targeted RNAi Therapeutics. 

https://doi.org/10.1038/s41565-017-0043-5. https://www.nature.com/articles/s41565-017-0043-5

Yan Cao; Zongxing He; Qimingxing Chen; Xiaoyan He; Lili Su, et al. Helper-Polymer Based Five-Element Nanoparticles (FNPs) for Lung-Specific mRNA Delivery with Long-Term Stability after Lyophilization. 

https://doi.org/10.1021/acs.nanolett.2c01784. https://pubs.acs.org/doi/10.1021/acs.nanolett.2c01784

McKinlay, C.J.; Vargas, J.R.; Blake, T.R.; and Waymouth, R.M. Charge-altering Releasable Transporters (CARTs) for the Delivery and Release of mRNA in Living Animals, 

McKinlay, C.J.; Benner, N.L.; Haabeth, O.A; and Wender, P.A. Enhanced mRNA Delivery into Lymphocytes Enabled by Lipid-varied Libraries of Charge-altering Releasable Transporters, 

Haabeth, O. A. W.; Lohmeyer, J. J. K.; Sallets, A.; Blake, T.R.; Sagiv-Barfi, I. et al. An mRNA SARS-CoV-2 Vaccine Employing Charge-Altering Releasable Transporters with a TLR-9 Agonist Induces Neutralizing Antibodies and T Cell Memory. 

 2021 7 (7), 1191-1204 DOI:10.1021/acscentsci.1c00361

Cynthia A. Challener, PhD has been a freelance technical writer for over 20 years and contributes regularly to 


Vol. 47, No. 3
March 2023
Pages: 20–22, 32

When referring to this article, please cite it as Challener, C. Excipients Impact Stability in mRNA-LNP Formulations. 

Lipids aren’t the only important ingredients influencing stability and in vivo performance.

Excipients Impact Stability in mRNA-LNP Formulations

There is no doubt that chimeric antigen receptor (CAR) T-cell therapies have had a tremendous positive impact on patients that have received them; however, autologous cell therapies produced in centralized manufacturing facilities have inherent limitations that inhibit widespread availability and access. Allogeneic, or off-the-shelf versions, are one potential solution, but developers of these therapies must overcome different challenges, such as graft-versus-host disease, the potential for introduction of chromosomal abnormalities, ensuring successful gene editing of all donor cells, and problems with T-cell exhaustion (1).

Another option is to produce autologous therapies at the point of care (POC) (2). Companies such as Miltenyi Biotech, Lonza, SQZ Biotech, Orgenesis, and Ori Biotech have developed or are commercializing closed, automated cell- and gene-therapy production platforms with small footprints that can be installed at hospitals and institutions for good manufacturing practice (GMP) production of CAR T-cell therapies onsite. There are various university hospitals around the world exploring the potential of these systems. Orgenesis and aCGT Vector are establishing networks of POC sites leveraging their own production technologies.

A third approach being explored by several companies eliminates the need for gene editing of immune cells outside the body altogether. These solutions involve in vivo generation of CAR cells. This approach also has its challenges, but offers the potential to combine some of the benefits of both autologous and allogeneic cell therapies.

Autologous cell therapies today require collection of immune cells from the patient that are then shipped to a central manufacturing facility. Most patients are extremely ill and have endured multiple previous therapies, leading to variability in the starting material and the potential for manufacturing failures. Production slots are also limited, often creating delays that such ill patients cannot generally afford. Facilities that offer cell therapy are limited as well, and as a result, patients must travel to them, adding to the burden placed on them and their families. Furthermore, the cost of gene-modified cell therapies is high.

An IQVIA survey (3), notes Gregory Frost, chairman and CEO of EXUMA Biotech, revealed that the patient’s health status, cost to patient, and geographic distance for the patient to travel were the top three reasons why community oncologists do not refer patients for CAR-T therapy.

Allogenic cell therapies offer compelling benefits relative to autologous CAR T-cell treatments for certain indications. As off-the-shelf products, they are available immediately, eliminating the logistics complexities that add time and cost to autologous therapies. They also do not suffer from the variability associated with patient-derived cells.

Avoiding the natural immune response to foreign CAR-T cells has, however, proven much more challenging than past industry hurdles such as the advancement from mouse antibodies to human antibodies, according to Frost. “Although many different strategies are being explored, the only clinically validated solutions to this challenge to date has been sustained immunosuppressive treatment regimens to eliminate the body’s T and NK cell function, thereby increasing the risk of opportunistic infections without transplant-ward-level care,” he says. Generally as a result, the time that must be spent in the specialized care setting is often extended and introduces additional burdens for patients and their families that do not live near one of these specialized facilities.

 CAR therapies have the potential to offer the benefits of both autologous and allogeneic therapies. “The most apparent advantage that 

 CAR therapy could provide over autologous or allogeneic cellular therapy is to capture the durability benefits of autologous cell therapy with the reduced cost and complexity of an off-the-shelf product,” contends Frost.

If other safety parameters can be maintained clinically, Frost explains a CAR therapy that delivers a cost-of-goods-per-patient-treated on par with biologics may change the efficacy bar from 30–50% durable complete responses to a 30+% improvement in progression-free survival or overall survival, a particularly relevant goal for indications in solid tumors. “Moreover,” he adds, “lymphodepletion may not be necessary for 

 therapies, thereby potentially expanding the setting of care beyond tertiary care centers with intensive-care units and extending CAR therapy into indications not previously addressed (e.g., autoimmune disease) due to an unfavorable benefit-to-risk ratio.” Furthermore, as a true off-the-shelf option

 CAR therapy products are unburdened by the complex logistics for cryopreservation of a cellular drug product.

 CAR therapy “may address the significant concerns associated with autologous and allogenic gene-modified cell therapies and thus function to expand patient access to CAR therapy by making it safer, potentially more efficacious, less financially toxic, and available in settings more convenient to patients and their families,” Frost comments.

It is also worth noting that in addition to the reduced cost and complexity of therapy and the potential to expand to a wider array of care settings

 CAR/T-cell receptor (TCR) therapies reduce the complexity of cell processing validation, according to Frost. “While the product is the process,” he observes, “the 

 approach does not require the associated costs and time to develop an optimized and validated manufacturing cell process, which can introduce new challenges in comparability assessment with subsequent process changes.”

Frost also points out that enhancements and minor modifications to the transduction program encoded in the vector can easily be made, allowing for iterative evaluation preclinically and potentially clinically. “The versatility of the technology is anticipated to extend the technology broadly into academia and small and large biotechnology companies, which could result in the accelerated discovery and development of transformative therapies at substantially reduced costs,” he concludes.

 gene-modified cell therapies face their own set of technical challenges. A key issue, according to Frost, is the inability to perform preparative lymphodepletion of the patient prior to the infusion of the engineered cells. This procedure removes other immune cells that would otherwise compete with the CAR cells for growth factors and other nutrients, reducing their chances to survive, expand, and exhibit antitumor function. “Lymphodepletion is not a viable option for 

 CAR therapy since it would kill off the very cells targeted for in vivo engineering. As such, any 

 approach will need a means for ensuring robust proliferation and survival in an environment that is rife with competition for the same nutrients,” Frost says.

It is also essential that the vehicle delivering the genetic material for reengineering of the target immune cells is highly selective for the right cells. “Whether the vehicle is a viral vector or lipid nanoparticle (LNP), it must target delivery to the specific cell type to be re-engineered (e.g., T cell, natural killer cell) in order to prevent any unintended gene modification,” comments Frost.

Viral vectors and lipid nanoparticles represent two different approaches being explored for in vivo engineering. Nonviral approaches typically involve LNPs encapsulating nucleic acids, while viral approaches are generally based on the same retroviral vectors used in current 

Non-viral approaches, notes Frost, have the possible advantage of a more defined regulatory pathway based on the recent approvals of the COVID-19 vaccines. Non-integrating approaches may, however, not provide a sustained clinical response given that CAR transcripts become diluted (and eventually lost) as the cells divide, he observes.

Such therapies would likely require persistent retreatment in the absence of a clinical response, especially for the treatment of solid tumors, which require a prolonged T-cell response. “Integrating vectors, such as lentiviral (LV) vectors, may offer the benefit of stably
integrating into the host-cell genome to allow for a sustained response with a single dose,” says Frost. As such, he anticipates further investigations into the tropism of viral approaches to reduce the risks of adverse events associated with non-target cell integration.

The level of investment in in vivo gene-modified cell therapies suggests that this new approach truly does have real potential. Several startups have been established in the past few years with strong financial backing.

 CAR/TCR therapy platform, GCAR, comprises a lentivector with a CD3- targeting/activation element on its surface to target T cells and provide them with their initial proliferation stimulus. The genetic payload within the LV encodes for either a CAR or TCR, as well as EXUMA’s intracellular “DRIVER” signaling domain. Once integrated and expressed by T cells, DRIVER signaling and initial CD3 activation causes robust in vivo proliferation with persistence and antitumor activity in a lymphoreplete environment occurring from continued antigen engagement, according to Frost.

“Our proprietary LV design elements address the challenges faced by 

 CAR therapy, as the DRIVER allows for proliferation and persistence in the midst of competing cells, and the CD3-targeting moiety directs the lentivector to T cells,” Frost states. In preclinical animal models, EXUMA has confirmed enhanced tropism to T cells, circulating CAR-positive cells, and a potent, dose-dependent elimination of target cells upon 

Umoja Biopharma has developed its VivoVec surface-engineered LV vector-based off-the-shelf viral-vector particles for the generation of CAR-T cells 

 (4). The particles include rapamycin-activated cytokine receptors (RACRs) designed to selectively provide costimulatory molecules to CAR-T cells in the presence of rapamycin. Preclinical study results indicate the particle design demonstrates enhanced T-cell binding and activation both in vitro and 

, as well as generation of large numbers of CAR-T cells, thus enabling enhanced antitumor activity at lower doses.

Vector BioPharma’s Shielded, Retargeted Adenovirus (SHREAD) platform uses engineered adenoviral vectors combined with exogenous, high-avidity adapter proteins (in the form of virus-like particles) to achieve delivery to defined biomarkers on specific cells or tissues of DNA encoding various genes and regulators, allowing local production of potent drugs with reduced risk of systemic toxicities (5,6). The company was established in August 2022 to commercialize technology developed in the laboratory of Andreas Plückthun, PhD at the University of Zürich (7).

Ensoma, launched in February 2021, is also leveraging adenoviral vectors (8). Ensoma’s Engenious vectors are devoid of any viral genome, thus minimizing the chance of an immune response and freeing up to 35 kilobases of DNA packaging capacity to deliver a diverse range of genome modification technologies. The vectors can be used in vivo to engineer hematopoietic stem cells as well as erythroid, lymphoid (e.g., T cells, B cells), and myeloid (e.g., macrophages, microglia) cell types with precision. The company has entered into a strategic collaboration with Takeda Pharmaceutical Company (9).

Capstan Therapeutics, which was co-founded by Carl June, one of the original innovators in the autologous CAR T-cell-therapy space, and launched in September 2022 (10), is using mRNA technology to generate CAR-T cells 

. In one example, they injected CD-5 targeted mRNA-LNPs to reduce fibrosis and restore cardiac function in a mouse model of heart disease (11).

Interius BioTherapeutics is commercializing technology developed by Saar Gill at the University of Pennsylvania (12). The company is initially focused on treating hematologic malignancies using an in vivo CAR treatment, but hopes to expand to applications beyond immuno-oncology that address diseases not amenable to current gene-therapy modalities. It has provided minimal information about the specific technology involved.

gene-modified cell therapies at the preclinical development stage. SG295 is an 

 CAR T with CD8-targeted fusogen delivery of a CD19-targeted CAR for treatment of patients with B cell malignancies and for which the company expects to file an investigational new drug application (IND) in 2023 (13). SG418 is a hematopoietic stem cell-targeted fusosome with the ability to deliver gene-editing material 

 to repair genetic abnormalities such as those that cause sickle cell disease and beta-thalassemia. Sana hopes to demonstrate preclinical proof of concept in 2023.

Gene therapy provides a potential regulatory roadmap

gene-modified cell therapies constitute a novel drug class, they are related to direct gene therapies, several of which have received regulatory approval in the United States, European Union, and other countries around the world. These gene-therapy approvals, according to Frost, provide a helpful roadmap for regulatory evaluation of 

 CAR therapies. “While each product and platform has its own unique considerations that must be addressed for a specific indication, the FDA has also published numerous guidance documents regarding gene therapy that are helpful to sponsors developing 

As is the case with recent regulatory reviews of retroviral gene vectors in cells other than T lymphocytes, Frost expects that regulators will expect developers of 

 therapies based on retroviral vectors to demonstrate that those vectors are indeed transducing the target cell type (e.g., T cells) and not other cell types, such as hematopoietic stem cells or gametes. In this context, he notes that significant consideration must be given to the tropism of the delivery vehicle, whether viral or non-viral.

Clinical candidates anticipated in the next few years

Given the growing number of companies developing 

 CAR therapies using many different approaches, Frost anticipates candidates entering clinical trials within the next several years. The data gathered during those trials will serve to better refine the science. “With the advances that will be achieved and the growing body of evidence that will be generated as a result of these studies, it can be expected that the overall industry will become more comfortable with 

 gene therapy, ultimately facilitating the progression of other products to the clinic and eventually the market. The widespread use and breadth of enhancements to monoclonal antibodies, which were first discovered in 1975 and first approved in 1986, is suggestive of the potential for in vivo CAR therapy. With today’s technology and know-how, the innovation cycle for this technology is expected to be dramatically accelerated,” he concludes.

Preparing for the Allogeneic Cell Therapy Tipping Point

Alvaro, D. and Challener, C.A. Bedside Cell Therapies: Point-of-Care Manufacturing Is Slowly Becoming a Reality. Pharma’s Almanac, Nov. 15, 2022.

Saez-Ibañez, A. R.; Upadhaya, S.; Partridge, T.; Shah, M.; Correa, D.; and Campbell, J.Landscape of Cancer Cell Therapies: Trends and Real-world Data. Nature Reviews Drug Discovery, 2022. 21, 631-632, Supplementary Fig. 9,. doi: https://doi.org/10.1038/d41573-022-00095-1.

Umoja Biopharma. Umoja Biopharma Presents New Preclinical Data on its Integrated In Vivo CAR T and Engineered Induced Pluripotent Stem Cell Platform Technologies at the Society for Immunotherapy of Cancer (SITC) 37th Annual Meeting. Press Release, Nov. 10, 2022.

Freitag, P. C. et al. Modular Adapters Utilizing Binders of Different Molecular Types Expand Cell-Targeting Options for Adenovirus Gene Delivery. Bioconjug Chem. 2022 Sep 21;33(9):1595-1601. doi: 10.1021/acs.bioconjchem.2c00346.

Smith, P. C.et al. The SHREAD Gene Therapy Platform for Paracrine Delivery Improves Tumor Localization and Intratumoral Effects of a Clinical Antibody. Proc Natl Acad Sci U S A. 2021 May 25;118(21):e2017925118. doi: 10.1073/pnas.2017925118.

Vector BioPharma AG. Versant Ventures Launches Vector BioPharma AG. Press Release. August 10, 2022.

Ensoma. Ensoma Launches to Pioneer Next-Generation 

 Approach to Deliver First “Off-the-shelf” Genomic Medicines. Press Release, Feb. 11, 2021.

Ensoma. Ensoma Announces Strategic Collaboration with Takeda to Accelerate Next-Generation 

 Gene Therapies. Press Release, February 2021.

Capstan Therapeutics. Capstan Therapeutics Launches with $165 Million to Deliver on the Clinical Promise of Precise 

 Cell Engineering. Press Release, Sept. 14, 2022.

Rurik, J.G. et al., CAR T Cells Produced 

 to Treat Cardiac Injury. Science. 2022. 375 (6576), pp. 91-96, Jan. 6, 2022. DOI: 10.1126/science.abm0594

Interius BioTherapeutics. Interius BioTherapeutics Raises $76M Series A Financing to Advance Breakthrough Cell and Gene Therapy Platform. Press Release, May 18, 2021.

Sana Biotechnology. Sana Biotechnology Confirms Key Program Timelines and Announces Portfolio Prioritization. Press Release, Nov. 29, 2022.

Cynthia A. Challener, PhD has been a freelance technical writer for over 20 years, leveraging her education from Stanford University (BS) and University of Chicago (PhD) and 10+ years of industry experience. She currently focuses on pharma/biopharma topics, writing technical articles, white papers, blogs, and other content for a variety of clients in addition to contributing regularly to 

Key challenges posed to autologous and allogeneic treatments could be resolved by in-vivo CAR-T gene therapies.


Advances in In Vivo CAR T-cell Therapies

Drug delivery to the human eye is a rapidly growing area in the pharmaceutical industry, but it is also one of the most challenging. The inherent structure and physiology of the eye make it difficult for drugs to reach the site of action and persist over time. Despite these constraints, ocular therapies continue to garner significant interest due to growing patient need, according to Ashley Rezak, Global Market Manager for Topical Drug Delivery at Lubrizol Life Science Health (LLS Health). Drug product developers are actively seeking options for overcoming these barriers and effectively delivering ophthalmic drugs.

Ocular injectable products in particular are experiencing strong growth with the development of gene and other advanced therapies. Even so, topicals (mostly liquids including solutions and suspensions, but also semi-solids) remain the most prominent dosage form for ophthalmic commercialized products and those in development, says Dr. Robert Lee, President of LLS Health’s CDMO Division.

Topicals are appealing due to their ease of administration, non-invasive nature, and ability to be administered at the site of action, all of which make them attractive to treat eye diseases such as conjunctivitis, glaucoma, and dry eye, among many others. The big challenge is delivery, according to Gregg J. Berdy, MD, voluntary associate professor of clinical ophthalmology in the Department of Ophthalmology at Washington University School of Medicine and a physician with Ophthalmology Associates of St. Louis, Missouri.

Formulations must be stable and homogeneous and designed such that when a drop leaves the medication container and hits the surface of the eye, it has the appropriate concentration of API and can reach the intended target within the eye—the surface, the anterior segment tissues (cornea, aqueous humor, iris, ciliary body, and lens), or the posterior segment tissues (vitreous humor, retina, and choroid)—providing the correct dosage.

Patient centricity an important driver in topical development

One of the challenges with topical ophthalmic drugs is ensuring patient compliance. Topical drugs are easy for patients to apply themselves, but there is no way to ensure that patients do so at the proper frequency, according to Berdy.

“Studies have shown that patients don’t generally take medicines more than twice per day, so it can be difficult to get them to apply ophthalmic drugs as often as needed in some cases. In addition, refill rates for many basic drugs are well below 50%. There is no way to guarantee medication adherence, which requires that patients buy/order, pickup, and then take their prescriptions. Whether the reason may be price, pain, or forgetfulness, compliance is a big challenge with self-administered treatments,” Berdy explains.

For this reason, Berdy notes that physicians often prefer intraocular injectable drugs that can be administered in the office. With these medications, proper dosing is assured. In many cases, he adds that the injectable treatment schedule may be every three months to one year, and patients don’t need to worry about refilling prescriptions on a monthly basis for a prolonged period. Furthermore, there is no worry of accidental harm to the ocular surface while patients apply topical solutions.

Given the benefits and concerns associated with topicals, it is not surprising that much research has focused on developing topical ophthalmic drug products that are easier to use, more comfortable, and specifically reduce the frequency of administration. Such efforts include developing sustained/extended release and increasing the bioavailability of the active drug substances in formulations.

Natural barriers create delivery challenges

Frequent dosing requirements for topical ophthalmic drugs can be attributed to the fact that typically only approximately 5% of the of the administered drug substance in a standard eye drop is absorbed at
the site of action, according to Rezak. There are several reasons why this issue exists. Primarily, she observes, it is due to the tricky path a drug must take to effectively penetrate the target tissue.

In addition, the average eye drop is 40–50 μL, whereas the tear film capacity of the eye is only 7 μL, and the eye can only hold about 20–25 μL, according to Berdy. This situation results in a large waste of product and presents a significant delivery limitation, Rezak notes. Furthermore, the little liquid that does remain is subjected to innate solution drainage and blinking, making it difficult to keep a topical drug product in place long enough to be absorbed.

Given that there are significant protective barriers and mechanisms on the ocular surface and achieving relevant drug concentrations in various parts of the eye is so challenging, it is crucial to understand the pharmacokinetics of any topical ophthalmic drug (1). Part of this knowledge must include the ability to correlate data generated in animal models—often rabbits because of anatomical and physiological similarities—to humans (2).

As a result, some suggest that successful topical ophthalmic drugs for the treatment of diseases of the anterior and posterior segments of the eye must be highly potent and administered only to the eye, avoiding delivery of the topical dose to systemic circulation (1).

“Without incorporating differentiated ingredients or technologies that help overcome these challenges, frequent administration of the drug product is required to maintain an effective drug level,” says Rezak. “In addition to decreased patient compliance due to the added inconvenience of more frequent administrations, toxicity and overdosing can become an issue, as can the increased cost if dealing with an expensive API,” she continues.

Early products focus on outer eye conditions

Most topical ophthalmic drugs are formulated as liquids delivered as drops to the eye. These drugs generally treat issues involving the eye surface or anterior segment tissues, including both acute problems (conjunctivitis, keratitis, iritis) and chronic conditions (dry eye syndrome, glaucoma).

Eye drops have also been formulated to treat surface abrasions and infections and to prevent swelling of the retina following surgery, according to Berdy. The most common use of eye drops today, he observes, is for the treatment of glaucoma. These treatments take many forms, but all must reach the cornea and penetrate into the anterior chamber to be effective.

The first topical treatment for presbyopia (age-related hardening of the lens) was approved in 2021 (3), and several other candidates are in development. These drugs function by modulating the size of
the pupil or changing the thickness of the optical lens. There are also several new treatments for dry eye disease currently advancing through the clinic (4).

Bioadhesion one way to widen topical applications

Despite the challenges to treating diseases at the back of the eye with topical drugs, there are several candidates in development for the treatment of ocular posterior segment diseases including age-related macular degeneration, diabetic retinopathy, diabetic macular edema, retinal vein occlusion, and optic neuropathy glaucoma disease (1).

“The back of the eye is regarded as one of the most difficult areas to effectively treat. Compared to the anterior portion, the posterior segment generally cannot be accessed via a topical route due to the natural impermeability of the eye exterior and the distance a drug would have to travel to reach the site of action, which is usually the retina. However, the industry is making great strides in the area of exploring topicals to treat both anterior and posterior diseases of the eye,” Lee contends.

The best way to enhance drug delivery of ocular topicals, Lee continues, is by prolonging a drug’s residence time on the surface of the eye. “For eye drops, the key is thus muco- or bioadhesion,” he states.

Bioadhesion is commonly defined as the adherence between two surfaces, at least one of which is biological. In the case of topical ophthalmic delivery, the surface of the liquid drug product and the front of the eye are the relevant interfaces, Lee explains. Bioadhesion can be introduced in a formulation via a variety of ingredients and delivery systems.

There are some drug product developers working on advanced delivery systems to instill bioadhesive properties, such as those utilizing solid-lipid nanoparticles, micelles, and liposomes, according to Lee. “Some of these approaches are very promising, and it is exciting to see so much innovation in the ophthalmic space,” he says.

Both small-molecule drugs and biologics can feasibly be delivered using these systems, Lee adds. He does note, however, that the specific active ingredient must be evaluated in the given system to assess suitability, and biologics are often much more challenging to deliver topically due to their large molecule size.

With respect to successful bioadhesive solutions, Rezak stresses that one of the most effective and well-established methods to impart muco- or bioadhesion to a formulation is through strategic excipient selection. “There are many polymers a formulator could select for establishing bioadhesion in a formulation, such as carbomers, carboxymethylcellulose, polycarbophil, and sodium alginate, to name a few,” she comments.

LLS Health provides muco- and bio-adhesive excipients designed to increase the effectiveness of ophthalmic topical therapies. They have unique functionalities that impact the mucoadhesive product profile and come in multiple grades so that profiles can be fine-tuned for the individual needs of different products, according to Rezak. “LLS Health has conducted studies (5) that show formulations containing its Carbopol polymers and Noveon polycarbophil excipients display significantly longer product retention at the target site than those containing alternative polymers,” she says.

In addition to the use of excipients to improve bioadhesion, other innovative approaches to improving the performance of topical ophthalmic drugs are being investigated. Two primary approaches include decreasing the particle size to allow greater penetration through the mucous barrier in the eye and the use of hydrophilic coatings to increase API solubility and bioavailability, particularly for hydrophobic drug substances, according to Berdy.

Some specific examples include nanospheres, nanocapsules, nanomicelles, nanovesicles, dendrimers, in situ gels, and microneedles (2). In situ gels are applied as solutions but once in the eye form a gel in the target tissue in response to a specific stimulus (pH, temperature, or solvent change, ultraviolet irradiation, the presence of specific ions or molecules, etc.) The application of physical or electrical forces (iontophoresis and sonophoresis) to enhance API penetration are also under investigation. Extracellular vesicles, biological particles including exosomes, ectosomes, microvesicles, microparticles, oncosomes, and apoptotic bodies, are also being explored as delivery vehicles in topical ophthalmic drug formulations.

“Every major company in the ophthalmic drug sector is developing topical formulations aimed at a wide range of diseases from those more traditionally treated with drops to disorders of the posterior of the eye. They are exploring both novel compounds and new delivery technologies that will enable both greater efficacy and improved ease of use and comfort for patients,” Berdy concludes.

del Amo, E. M. Topical Ophthalmic Administration: Can a Drug Instilled onto the Ocular Surface Exert an Effect at the Back of the Eye? 

8 Sept. 2022. https://doi.org/10.3389/fddev.2022.954771.

Lixiang Wang, L; Zhou, M. B.; and Zhang, H.The Emerging Role of Topical Ocular Drugs to Target the Posterior Eye. 

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Lubrizol Life Science. Mucoadhesive Polymers in Pharmaceutical Formulations. Technical Brief. 2021.

 
Vol. 47, No. 2 
February 2023 
Pages: 18–19

When referring to this article, please cite it as Challener, C.A. Aiming for Improved Efficacy and Patient Compliance for Topical Ophthalmics. 

Topical eye treatments can be beneficial for patients with a growing array of eye diseases, but only if taken appropriately.

Aiming for Improved Efficacy and Patient Compliance for Topical Ophthalmics


Vol. 35, No. 2
February 2023
Pages: 11–14

When referring to this article, please cite it as Challener, C.A. Technology for 

In vivo CAR-T gene therapies could resolve challenges faced by autologous and allogeneic treatments.