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

Cynthia A. Challener

Viral vectors must be free of contaminants, such as adventitious viruses introduced via raw materials and helper viruses introduced purposefully to stimulate expression of the desired viral product. Viral clearance used with incoming raw materials and, where possible, viral-vector products combined with testing is essential. Because conventional viral clearance methods used for protein biologics may degrade or destroy viral vectors, assessment of the potential risk for contamination and the implementation of prevention and control strategies are essential.

The most common source of viral contamination is usually traced back to animal-derived raw materials, such as certain cell-culture media (e.g., serum) and trypsin, according to Artur Padzik, adeno-associated virus (AAV) production manager at Biovian, a contract development and manufacturing organization (CDMO). Non-animal-derived raw materials can be an issue as well, particularly glucose if a chemically defined medium is used. A risk-based approach to process development combined with extensive testing of raw materials (and often the application of virus removal steps at raw material entry points), in-process materials, and final products is essential to ensuring the safety of viral vectors.

Similar to other biologics but challenging

The requirements for adventitious agent testing for viral vectors overall are consistent with those performed for other biological therapeutic products. As with other biologics, too, selection of appropriate testing methods is guided by the need to mitigate the risk of viral contamination taking into account the materials and processes used.

“Viral vectors are biologics, and as with all biologics, the concern around adventitious agents must be addressed with a thorough risk assessment that [considers] control and probability of contamination. The principals from the three-pronged complementary core strategy (material selection, testing, and clearance assessment) to prevent and minimize adventitious agents can and should be applied to viral vector manufacturing processes and products,” says Audrey Chang, executive director, Rightsource and Biologics at Charles River.

Direct adoption of strategies used for recombinant proteins and monoclonal antibodies may not be possible, however, Chang adds, due to biological differences between viral vectors and large protein molecules. Indeed, it may be necessary to modify standard assays or use alternative methods because viral vectors are viruses themselves and could interfere with virus detection methods, notes Leyla Diaz, a technical consultant in the Technical and Scientific Solutions unit of MilliporeSigma.

Specifically, adventitious agent detection can be challenging if cell-based assays are being used in combination with live-virus vaccines, replication-competent gene therapy vectors, or vectors exhibiting the cytopathic effect (CPE), Padzik observes. This issue arises largely because human cell lines are used for cell-based assays, and human cells are susceptible to human viruses. Lytic viruses such as adenoviral products, for example, will infect human and primate-based cells and can cause invalid assays. Robert Schrock, director of bioassay services at Lonza Houston, adds that even if adenovirus is only conditionally replicating and does not replicate, cellular toxicity may occur.

In these cases, neutralization of interfering and abundant product viruses is required, says Padzik. There are three approaches that can be taken, according to Schrock. One is to block antibodies specific to the virus product to “soak up” the product as much as possible without interfering with the detection of other adventitious viruses. Alternatively, the sample can be diluted so as to avoid overwhelming the detection cells. Often these two approaches are used in combination. The third option is to pull the test sample from an uninfected parallel satellite culture that is split off the bioreactor just prior to transduction with the virus bank. “For oncolytic vectors, FDA guidance mentions the use of #3 as acceptable. The first two have limitations, such as with test #2, where the value of the test can rapidly decline due to sample dilution,” Schrock comments.

In general, the key is to select the appropriate test method for a given product. For example, Sophia Nguyen, director and head of quality control at Lonza Houston, points to the selection of cell lines for infectivity assays as being important. “Viruses differ in their ability to infect host cell types.” For the endpoint assay used to determine whether a host cell is infected with a virus, a monolayer culture is preferable to a semi-adherent or suspension culture. With the former, CPE and hemadsorption (HAd) can be visualized by microscopy, making the analysis relevant both to viral detection and subsequent viral identification.

The plethora of potentially contaminating sources arising from the complexity of the viral-vector production process favors a holistic scheme for adventitious agent testing, according to Padzik. In this approach, both general and species-specific assays are used.

Adventitious virus detection assays, explains Nguyen, mostly fall into three testing categories: cell-culture-based infectivity assays, targeted nucleic acid-based amplification, and antibody production tests. Both in vitro cell-culture detection methods and in vivo assays are employed. No universal method is available to detect all adventitious agents, and therefore various complementary methods must be used. “There is a clear tendency, however, to move away from assays that require animal models toward assays with higher sensitivity, specificity, and rapid turnover, such as polymerase chain reaction (PCR),” Padzik remarks.

In vitro cell-based detection methods are typically broad-spectrum and intended to detect viruses that are able to replicate in cell lines—a human diploid cell line, a non-human primate cell line, and a cell line of the same species as the one used to produce the vector, Diaz observes. In vivo, animal-based detection methods are also broad-spectrum and intended to detect viruses that replicate in animal models—typically mice and embryonated eggs, but also other animal species based on specific risks associated with the product being tested.

With these non-specific 14- and 28-day adventitious agent tests, CPE or haemagglutination and HAd values serve as the readouts. The obvious limitation, notes Padzik, is the difficulty in detecting adventitious viruses that do not cause CPE. The limits of detection for these assays can also be an issue. Padzik says a partial remedy, but with limited sensitivity, is the use of transmission electron microscopy (TEM) to detect, for example, retroviruses. XC plaque, S+L focus assays, co-cultivation assays, or fluorescent product enhanced reverse transcriptase (FPERT) testing are also commonly used to detect retroviruses.

Molecular methods such as PCR tests are used to detect specific viruses that are either not detectable by other methods or that are emerging as particular biosafety concerns, according to Diaz. Known contaminating entities, including porcine and bovine viruses, minute virus of mice, vesivirus, rhabdovirus, polyomavirus SV40, and arboviruses for various animal and insect cells are addressed with PCR-based specific assays, Padzik says. PCR-based methods that can detect virus families rather than specific viruses are also being employed. They are also used to detect mycoplasma, spiroplasma, mycobacterium species, and mice DNA sequences, according to Nguyen.

Nguyen stresses that each method exhibits a different level of sensitivity, and the testing requirements and sample preparation protocols differ as well. It is imperative, therefore, to select the appropriate test for each vector product.

Adventitious agent testing of raw materials should be proportional to the risk posed by the raw materials, and the testing methods chosen based on such an assessment, states Horst Ruppach, executive director, scientific and portfolio global biologics for Charles River.

The most common methods of adventitious agent testing for raw materials are cell-culture-based assays using specific indicator cell lines, with Vero cells preferable because they can detect viruses from all sources, according to Nguyen. The added risk for introducing animal viruses posed by animal-derived components requires the use of sensitive cell-based assays that can detect replicating infectious viruses, Ruppach adds.

PCR-based methods are also used depending on the raw material and turnaround requirements. It is important to recognize, though, Ruppach stresses, that while using molecular methods can be advantageous due to their speed, they do not discern between a nucleic acid positive signal and an actively replicating virus.

Viral vectors produced to modify cells for the production of ex vivo cell therapies have some additional raw material concerns with respect to adventitious agents. Human serum albumin (HSA), a commonly used excipient for cell therapies, is a high-risk material with respect to virus and prion transmission, but there is no practical assay for direct prion testing, according to Ruppach. “Here, special emphasis must be placed on HSA donor screening and the manufacturing process in order to mitigate the risk of virus and prion transmission,” he says.

In addition, Ruppach notes that for gene-modified cell therapies, the viral vector is considered a critical raw material, and the same three-pronged complementary core strategy involving material selection, testing, and clearance assessment should be applied to mitigate the introduction of adventitious agents into the final cell therapy product.

Allogeneic, off-the-shelf cell therapies in which the cells used to produce ex vivo-modified cell products are derived from multiple human donors are worth a special mention, according to Diaz. They require a carefully derived risk-mitigation strategy, because each donor contributes a separate, individual virus risk to the product. “Upfront donor screening plays a large role in the development of a strategy that can mitigate the risk of virus contamination of these cell-based therapeutic products,” she states. In addition to normally performed adventitious agent product testing, it may be necessary to also perform additional virus testing to address potential risks associated with a specific set of donors or even a specific geography.

While traditional in vivo and in vitro methods for adventitious agent testing of viral vectors are sufficient to ensure the production of safe and effective products, they do suffer from some limitations, including sensitivity, the time required to perform the assays, throughput, and the detection of unknown contaminants, according to Padzik.

“Delivery times of adventitious virus test results have always been a serious challenge regardless of the method used, with some of them taking four to five weeks, and up to a few months with full analysis and documentation,” Padzik observes. For some gene therapy products that have a short shelf life, Diaz notes that these lengthy periods can be prohibitive. This concern is particularly relevant for autologous gene-modified cell therapies that must be administered to patients shortly after production. Molecular methods are being used to fill this gap, in particular PCR-based methods are being fine-tuned or developed to fill this need.

“The growing scope of required assays is creating continuous pressure, especially on early-stage clinical projects where results from the first human clinical studies are pivotal in securing the funding for full clinical medicine development,” Padzik adds.

“A partial solution could be a new pan-microbial microarray comprising 29,455 sixty-mer oligonucleotide probes for screening on a single chip vertebrate viruses, bacteria, fungi, and parasites,” Padzik comments. He cautions, though, that PCR-based methods are not able to help with the recognition of undefined adventitious agents and they suffer from noticeable instances of false positive cases.

Sample volume is another concern, particularly for viral vectors used in gene-therapy products. “Depending on the scale of vector production, adventitious virus testing can take up a significant amount of volume that could be otherwise used for patient dosing,” Diaz explains. This issue is of particular concern in early stages of product development before a production scale-up strategy has been implemented.

Nguyen reiterates that the different methods used for adventitious agent testing have different sensitivities for the detection of different contaminants and limitations. “These methods are intended to complement each other to provide a greater understanding of the detection of adventitious agents. In addition, the methods also have specific pharmacopeial and regulatory requirements/guidelines, such as sampling size. As always, these requirements must be considered when establishing a testing strategy,” she contends.

Tried-and-true test methods such as in vivo and in vitro virus assays and mycoplasma culture and microbiology culture assays have been used to demonstrate the biosafety of biologics for decades and can be applied in viral-vector manufacturing for certain steps such as cell-line and viral-seed-stock characterization, Ruppach emphasizes. During the past few years, adds Nguyen, understanding and methodologies have evolved significantly for adventitious agent testing, and Lonza believes this trend will continue to accelerate.

Given the limitations of existing methods and the advances being made in molecular-based methods, Ruppach expects the eventually the industry will switch to molecular test methods and rapid alternatives such mycoplasma PCR and rapid microbiological testing.

NGS-based assays are attractive for a number of reasons. The biopharma industry is focusing on using the full potential of NGS for adventitious testing requirements due to its low turnaround time in particular, according to Nguyen. The other main reason NGS is attractive is its ability to detect unknown contaminants, says Padzik. NGS can also be used to detect low-level known virus DNA/RNA sequences, according to Schrock. Identification will, of course, still require a comprehensive library of known and potential viral genomic sequences, and verification will be necessary by comparing results to those obtained using established methods.

In addition to these technical benefits, rapid NGS development has the potential to reduce costs and increase accessibility. “These advantages are driving significant interest and the urgency within the industry to validate this technique so that it can be swiftly implemented and applied for routine use,” Padzik remarks. The Advanced Virus Detection Technology Interest Group (AVDTIG) is an industry-based organization facilitating the exchange of experiences with the development of NGS-based assays to accelerate the introduction of practical solutions.

For Ruppach, one of the main challenges to effective viral-vector adventitious agent testing today is the resistance to new analytical methods. “There are several barriers to the adoption of new techniques such as NGS, including fear of regulatory delays, the risk associated with changing platforms, the resources (financial, personnel, time) needed, the tendency within the biopharma industry to be more focused on the short-term and less on the long-term, and the hesitancy to share information and desire to protect company trade secrets,” he explains.

“While all are valid reasons, it is essential to develop and share data that will support progress in the NGS field,” Ruppach says. He points specifically to the need for reference standards that will allow sharing of data obtained during development, comparability studies, validation and re-validation, technical transfer and assay trending, and ultimately the design of appropriate bridging studies that will enable translation to commercial application.

“Switching to rapid methods for adventitious agent testing must be done carefully, taking into consideration the fact that viral-vector manufacturing processes offer limited viral clearance steps,” states Ruppach. “Certainly, new alternatives such as NGS have applicability for viral assessment,” he adds. Padzik is hopeful as well. “The requests for using unbiased NGS are significantly increasing, especially in vaccine development. The current COVID-19 pandemic accelerated this transition even further,” he comments.

Ruppach also observes that a revision to International Council for Harmonisation (ICH) Q5A Quality of Biotechnological Products: Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin is currently in the works and expected to include updated guidance to reflect updated scientific knowledge and advances in analytical technologies for new modalities, including viral vectors. For Padzik, the changing regulatory landscape and pressure to reduce animal testing are important drivers for NGS adoption, along with the apparent increasing acceptance of NGS-based assays in vaccines and other products where contaminating virus detection by the classical test is difficult and vaccine release is time-sensitive. “It is possible to envision gradual supplementation and, ultimately, the replacement of in vivo and other nucleic-acid technologies by NGS,” he concludes.

Taking a holistic view is the best strategy

“For most virus-based vaccines and viral vectors, the removal of contaminating viruses is not feasible, so the prevention and detection of virus contamination become more critical in a virus risk mitigation strategy,” emphasizes Diaz.

Although the principles used for adventitious agent testing of monoclonal antibodies (mAbs) apply to viral vectors, it is certainly not a “plug-and-play situation”, according to Chang. She stresses that a holistic view of the product, manufacturing, and ultimate use of the viral vector must be taken into account when performing risk assessments and establishing risk-control strategies.

For instance, the batch size for viral vectors is different from that for mAbs, with only a small number of doses produced per batch. In addition, some aspects of viral-vector manufacturing, such as the level of empty capsid production and consequent impact to the patient, are not fully understood. “Furthermore,” says Chang, “it is not possible to introduce robust viral removal/inactivation steps that exist with antibody manufacturing. Therefore, closer characterization of the starting raw materials, including biological characterization that assesses adventitious agents thoroughly, is prudent.”

Diaz agrees that an effective adventitious agent testing strategy starts with an appropriate risk assessment that evaluates all possible sources of virus contamination, including cell banks, virus seeds, plasmids, media, and other substances used in production. The risk assessment should also include identification of any steps in the manufacturing process (upstream and downstream) where virus contamination could be introduced.

“Evaluating the manufacturing components and processes for their contribution to the virus contamination risk allows for a multi-pronged approach that includes appropriate sourcing of materials, the selection of relevant virus detection methods for the risks identified, and the potential removal of contaminants associated with specific viruses in downstream processes,” Diaz summarizes.

There are several key aspects of any strategy for adventitious agent testing, according to Nguyen. Appropriate testing regimens must be included that monitor the possible introduction of adventitious agents. These methods must be suitably sensitive or use indicator cell lines or animal models in which a specific viral entity is known to replicate. The potential for matrix interference must be avoided; spiking of the test sample matrix with a positive control virus enables assessment of potential interference and the limit of detection of the method. “As a general rule of thumb,” she says, “the assays should be sufficiently developed with the appropriate set of predetermined system suitability and acceptance criteria, including relevant positive and negative controls, and fully established before validation.”

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

Articles

Production of viral vectors requires a holistic view of the product, including its manufacturing process and its ultimate end use.

Testing for Adventitious Agents in Viral Vector Production

Electrochemistry offers many strategic advantages for increasing the sustainability and efficiency of small-molecule synthesis. It also has the potential to enable the creation of molecules not accessible using more traditional chemistry. As a consequence, interest in this technology is increasing in both academia and industry. Three researchers having a strong impact in this area through their independent work and collaborations with industry include Phil Baran of The Scripps Research Institute (1), Siegfried Waldvogel of Johannes Gutenberg University in Mainz, Germany (2), and Song Lin at Cornell University (vide infra).While its use in medicinal chemistry and drug discovery has been increasing, implementing these reactions at large scale under good manufacturing practice (GMP) conditions has been challenging.

The potential benefits are driving companies to develop chemical and engineering solutions that allow the use of electroorganic chemistry for the production of complex pharmaceutical intermediates at the kilogram scale (3).

Song Lin, associate professor in the Department of Chemistry and Chemical Biology at Cornell University, has been focused for several years on developing new electrochemical processes that address important needs in organic synthesis. “Harnessing electrochemistry to solve the challenging transformations in organic synthesis is one of the most important research interests in our group,” he observes.

One of those areas relates to cross-electrophile coupling (XEC) of organic halides for C–C bond formation. Despite great advances in this area, Lin notes that the selective XEC of two alkyl halides for C(sp3)–C(sp3) bond formation remains elusive using traditional chemical approaches.

Inspired by the generation of C(sp3)–C(sp3) bonds via SN2 displacement, his research group, in collaboration with scientists at Caltech and Merck, envisioned a new approach to cross-coupling of two (one tertiary and one primary) alkyl halides by exploiting the disparate electronic and steric properties (1).

“This electrochemical approach enables XEC of a diverse range of unactivated and functionalized alkyl halides, including α-haloboronic esters, α-halosilanes, benzylic chlorides, and allylic/propargylic halides, under transition-metal-free conditions with excellent selectivity,” Lin states. It also operates by a mechanism different from that previously reported for cross-coupling reactions catalyzed by nickel complexes. “This new strategy circumvents the generation of Ni-alkyl intermediates that could often lead to unselective electrophile activation and various side reactions,” he explains.

As a result, the e-XEC reaction shows improved selectivity compared to known methods for C(sp3)–C(sp3) XEC. “This new reaction provides a robust and selective method for constructing C(sp3)-C(sp3) bonds, which remains a critical problem in modern organic chemistry. It will be particularly beneficial for drug discovery given that recent research in medicinal chemistry has demonstrated a correlation between higher fractions of sp3 carbons in drug candidates with improved success rates in clinical trials,” Lin says.

To date, the reaction has been scaled up to 20 mmol, providing multigram quantities of desired products in excellent yield. Lin hopes to further scale up the method to create a wide range of molecules for drug discovery. He does caution, however, that a wide gap remains between the current optimal laboratory conditions and GMP requirements for commercial pharmaceutical manufacturing. “We need to overcome several issues before further scale up the reaction, including using less of a safer electrolyte than the currently used tetrabutylammonium perchlorate and avoiding the use of a sacrificial magnesium anode.”

In addition to the e-XEC reaction, the Lin group has used an electrocatalysis strategy for the oxidative difunctionalization of alkenes (2). He and his colleagues are also working to further scale up the electrochemical diazidation of alkenes, another transformation developed by his group (3). This reaction provides an efficient and robust approach to the synthesis of precursors of vicinal diamines, a functional moiety that is widely prevalent in many natural products and a large number of bioactive compounds.

S. Lin et al., “Electrochemically Driven Cross-Electrophile Coupling of Alkyl Halides,” 

, Dec. 13, 2021. DOI: 10.26434/chemrxiv-2021-c2hd6-v2.

, 53, 547-560 (2020). DOI: 10.1021/acs.accounts.9b00529 .

, 357, 575-579 (2017). DOI: 10.1126/science.aan6206.

A more sustainable, safer, and less expensive alternative

“Electrons,” says Markus Furegati, a senior principal scientist at Novartis, “are ‘reagents’ that can be regarded as green, safe, and cheap. One mole of electrons costs less than one US dollar, and there is significantly less waste generated during manufacturing.” In some cases, he adds, an electrochemical approach offers better selectivity compared to classical procedures.

Electrochemistry offers a mild and efficient alternative to conventional chemical approaches specifically for redox transformations, according to Song Lin, associate professor in the Department of Chemistry and Chemical Biology at Cornell University. “Upon the application of an appropriate potential, organic molecules can lose or gain an electron at the electrode surface and generate highly reactive intermediates, such as radicals, carbocations, and carbanions. In the process, the potentially expensive, hazardous, and toxic oxidizing and reducing reagents employed in canonical redox reactions may be replaced by greener, less toxic, and milder electron donors and acceptors,” he explains. Lin also observes that many studies have demonstrated the potential of electrochemistry to achieve transformations that are challenging to realize using chemical redox reagents.

One of the keys to success for electrochemical reactions is for the functional group of interest to have an appropriate redox potential compared to the rest of the molecule, according to Claudio Bomio, principal scientists II with Novartis. Precise control of the applied potential can, Lin notes, provide the ability to selectively activate complex molecules and generate highly reactive intermediates in a controlled fashion. “The selectivity of electro-oxidations can be tuned using a set of parameters not usually used in organic chemistry, such as choice of voltage, electrode material, the use of redox mediators, etc., which expands the scope and utility of electrochemistry for organic synthesis,” adds Bomio.

In addition to the low cost due to the waste-free and sustainable nature of electrons, Frederic Buono, senior associate director of chemical development in the United States at Boehringer Ingelheim, points out that electrochemistry technology is also easy to manipulate and fast to assess. “Using process analytical technologies, it is possible to monitor the generation of specific impurities and the overall reaction conversion and selectivity in real time and adjust the power and/or voltage units as needed,” he explains.

With respect to chemical reactivity, Buono also comments that electrochemical methods often exhibit a high functional group tolerance as well as higher reactivity. The latter allows reduction of the quantities of some reagents needed and thus the production of less waste and toxic by-products. Electrochemical reactions are also typically performed under milder conditions (temperature and pressure) than those necessary with traditional approaches, according to Buono. Furthermore, to halt electrochemical reactions simply requires switching off the power, whereas with traditional organic chemistry an extra reagent must often be added.

Because of the many benefits that electrochemistry possesses over traditional approaches in organic synthesis, it could play a major role in chemical/intermediate production, Buono contends. Bomio agrees: “Electrochemistry is an area of tremendous growth in academic research, with new methods and reactions emerging every day.”

Based on published literature, Jeff Song in Boehringer Ingelheim’s chemical development group in the US notes that there is a large variety of transformations for which electrochemistry is an attractive synthetic method, such as oxidations (e.g., oxidative intermolecular/intramolecular coupling, Pinnick/Anelli oxidation, Shono oxidation, etc.), reductions (e.g., Birch reduction, reductive cross-electrophile coupling), C=N formation (azines and oximes), methoxylation, and amination. “The benefits of electrochemistry for these transformations are relative to the functional tolerance of the complex molecular structures,” he adds.

The rising interest is driven in part by the fact that oxidation and reduction reactions are among the most important and frequently executed processes in organic synthesis, according to Lin. “In the pharmaceutical industry, the ability to replace oxidation and reduction reactions that involve hazardous or waste-generating reagents used for the synthesis of APIs or their intermediates with electrochemical processes is very attractive. Synthetic electrochemistry can realize similar transformations through electron transfer between organic molecules and electrodes, avoiding the use of hazardous or toxic stoichiometric redox reagents. Moreover, the controllable applied potential results in the potential ability of electrochemistry towards selective functionalization of complex molecules,” he says.

Electrochemistry is definitely not limited to small scale, and there are, according to Laurin Wimmer, senior expert, science and technology with Novartis, quite a few established processes in industry. He points to production of the fragrance compound lysmeral by BASF at more than 10 kilotonnes per year as one of the largest and most well-known industrial electro-organic processes. Lin adds that there are also many other chemicals, such as adiponitrile (a key intermediate in the manufacture of Nylon 6, 6 polymers) (4) and azobenzene (5), produced at industrial scale using electrochemical processes.

In the pharmaceutical industry, electrochemistry is widely used in medicinal chemistry labs and also in early development for the synthesis of intermediates on a gram scale, says Song. At Boehringer, electrochemistry is also applied for the synthesis of impurities.

Electrochemistry at large scale: continuous development

Inspiration from academic leaders in the field of electrochemistry and their achievements in organic synthesis is leading to the exploration of electro-organic technologies for process development and commercial manufacturing, Wimmer contends. While for small-scale laboratory applications stirred electrochemical cells are most common, industrial processes typically rely on a flow-chemistry approach (6).

In recent years, Jeff Song remarks that it has become more common to use electrochemistry in day-to-day operations as commercial laboratory reactors became more available in batch and flow mode. The throughput of these reactors is still very limited, however.

Indeed, Buono emphasizes that a key limitation to further scale-up and commercial manufacturing is the lack of both standard manufacturing equipment and the technical knowledge for scale-up, including understanding of the impact of electrode materials on reaction output, reaction parameters, and other aspects that can change with scale. In most cases, he notes that companies have been using in-house-built reactors to perform electrochemistry at the kilogram scale.

There are other challenges to implementing electrochemical processes for organic synthesis at large scale beyond the lack of knowledge and experience in scaling up electrochemical processes and the need for suitable equipment. Buono highlights the lack of specific regulations regarding the use of electrochemical technology in pharmaceutical manufacturing.

Lin, meanwhile, points to safety concerns related to the application of a high current in the presence of flammable organic solvents; the price, concentration, and safety of the electrolyte; the price, availability, and potential passivation of electrode; the generation of hazardous gases such as H

; and the current density and ratio between the electrode surface area and reactor volume.

Despite these issues, based on the evidence accorded by the numerous papers generated by academic/pharmaceutical collaborations, most major pharmaceutical companies are actively exploring potential applications of electrochemistry, according to Wimmer. For instance, Lin notes that companies including Merck, Bristol-Myers Squibb, and Genentech are interested in the development of electrochemical methods to synthesize APIs. Researchers at Merck, in fact, published an article describing the successful scale-up of an organic electrosynthetic method from milligram to kilogram scale (7).

“In general,” Lin states, “the widely accepted means to industrialize an electrochemical process is the combination of continuous flow technique with electrochemistry. The short distance between two electrodes results in a significant decrease in the ohmic drop of the system, enabling electrolysis with very low concentration of electrolyte. Moreover, the large ratio between electrode area and reaction volume compared to batch cells is beneficial for the electrolysis efficiency,” he explains.

There are efforts underway to establish the needed large-scale equipment that will enable commercial electrochemical processes. One example of note, according to Buono, is the Enabling Technology Consortium, which is working to design and build both laboratory- and manufacturing-scale electrochemical reactors that meet the safety, robustness, and productivity requirements for each scale.

Witnessing the renaissance of synthetic organic electrochemistry

The trajectory for electrochemistry in the pharmaceutical industry appears to be an exciting one. Furegati anticipates progress to occur along a pathway similar to that observed for modern photochemistry. “The availability of easy-to-use equipment and broader literature coverage will facilitate the use of electrochemistry for niche applications. Success in these areas will then stimulate the development of commercial solutions,” He adds that if contract research and contract manufacturing organizations actively pursed and implemented electrochemical processes, the barrier to adoption could be potentially lowered.

There is already, says Lin, a renaissance in synthetic organic electrochemistry underway. “Breakthroughs and advances in this area have led synthetic organic chemists to gradually realize the powerful potential of electrochemistry as a robust technique in organic synthesis, stimulating a tremendous surge of research interest in this field amongst the organic chemistry community. In this context, we believe more practical and efficient electrochemical reactions will be reported in the future,” he explains.

If certain steps are taken, the influence of electrochemistry in organic synthesis can be further strengthened, according to Lin. One would be the development of electrochemical methods for late-stage functionalization of complex bioactive molecules and biomacromolecules such as peptides, nucleotides, and sugars. Another would be the fabrication of novel electrodes to explore new reactivities and tune the selectivity of electrochemical methods. A more long-term goal would be the development of enantioselective electrochemical transformations.

A further area of interest for both Lin and Buono is the extension of electrochemical applications through the combination of electrochemistry with other promising synthetic techniques. Electrophotocatalysis (8) and bioelectrocatalysis (9), for instance, may provide avenues for the discovery of new reactivities and enable wider use of electrochemical solutions by process chemists. Buono also envisions the combination of artificial intelligence, automation, and electrochemistry enabling the rapid optimization of electrochemical reactions (10).

Lin firmly believes that all of these developments and the adoption of electrochemical processes in general by the pharmaceutical industry will be expedited through the establishment of intimate collaborations between academia and industry. “In academia, we are more interested in the discovery of new reactivities and typically overlook the cost and safety issues related to reagents. We also in general lack the experience needed to efficiently scale up electrochemical reactions. By collaborating with industry partners, we gain greater knowledge of practical requirements for synthetic methods used in both drug discovery and
commercial manufacturing.”

The end result, Lin concludes, will be industrializable electrochemical processes that are more sustainable and scalable. Fortunately, many such collaborations are already in place and actively engaged in exploring electrochemical processes.

J. Liu, et al., “New Redox Strategies in Organic Synthesis by Means of Electrochemistry and Photochemistry,” 

Cynthia A. Challener, PhD, is contributing editor to 


Volume 46, Number 10
October 2022
Pages: 24–28

When referring to this article, please cite it as C. Challener, “Electrochemistry for API Synthesis,” 

Numerous benefits will eventually lead to large-scale applications in pharmaceutical manufacturing. 

Electrochemistry for API Synthesis

Numerous skin diseases and disorders that affect different layers of the skin can be treated with topical dermal drug delivery (TDDD) systems. Topical delivery is advantageous over systemic administration due to its ability to provide targeted treatment and avoid first-pass metabolism. Successful topical formulations deliver the API to the proper layer of the skin at an appropriate rate so as to maintain the necessary concentration over a period of time; in other words, they provide appropriate permeation kinetics. There are many factors that determine the delivery kinetics of TDDD systems, not the least of which is the need to overcome the protective barrier function of the skin. Thoughtful formulation development is essential to developing optimal topical drug products.

Topical drug delivery kinetics are a convolution of two factors: the rate at which the API is provided to the skin surface from the formulation—the API release—and the rate at which the drug diffuses into and through the skin.

 tests involve applying the product to the skin and then taking skin biopsies to measure the concentration of the active over time. Microdialysis and open-flow microperfusion are newer techniques that allow sampling of API concentrations in the skin without the need for biopsies.

The industry prefers in vitro, or ex vivo, testing. Ex vivo testing using synthetic polymers or cultured skin is generally not very predictive because these substrates do not have the same barrier properties as real human skin. Therefore, most ex vivo testing is performed using real skin. The closest animal model to human skin is pig skin, and many measurements are made using porcine skin samples obtained from animals bred and sacrificed for other purposes. When possible, tests are performed using human skin.

In vitro permeation testing (IVPT) is widely used to investigate the delivery kinetics of topical drug formulations. Excised human skin obtained from cosmetic surgeries or donated from cadavers is mounted within diffusion chambers designed to allow the test to be performed under conditions similar to those found within the human body.

The skin is prepared and its integrity evaluated to ensure that no defects such as pinholes exist that could allow excess API to pass through and thus skew the results. The skin is then placed into a diffusion cell on top of a buffer solution in which the API is known to have sufficient solubility. The topical formulation is applied to the surface of the skin and the penetration of the compound is measured by monitoring its rate of permeation into the receptor solution underneath the skin samples. This model also allows APIs and metabolites within the different layers of the skin (i.e., epidermis and/or dermis) to be measured.

IVPT is attractive because it offers a means for controlling the variables that may influence topical drug performance, including dosing volumes, humidity, temperature, API stability, skin thickness, and other factors. It also allows for performance of analyses that are designed with the specific indication in mind, John Newsam, CEO of Tioga Research says. If the product is intended for use on a wound, then compromised skin should be simulated by removing some of the test skin’s barrier function. Some dosage forms can complicate the measurement process, but it is essential to perform the test using the product as it will be formulated and used, he says. By taking this approach, IVPT makes it possible to appropriately examine the behavior of formulations depending on the dosage form and indication.

Over the years, the skin has become an accessible and practical site for the delivery of medicines, according to Gayathri Krishnan, associate director of 

 sciences with Tergus Pharma. Many different dosage forms enable delivery of actives to the skin, such as creams, ointments,
lotions, liquids, suspensions, gels, hydrogels, pastes, foams, suppositories, and nail lacquers.

Drugs in topical formulations can exhibit activity on the superficial layers of tissues, or via penetration and permeation into deeper layers, or through systemic distribution depending on the physiochemical qualities, targeted site of action, and formulation procedures, Krishnan observes. She adds that there are three main delivery mechanisms: transcellular, intercellular, and trans-appendageal.

In the trans-appendageal pathway, or the shunt route, the API is delivered through appendages such as hair follicles and sweat ducts, which provide entry through the protective stratum corneum. APIs delivered via the transcellular route leverage phospholipid membranes and the cytoplasm in the dead keratinocytes that make up the stratum corneum to directly cross the skin. This is the fastest route, but APIs must be able to pass through each cell’s lipophilic membrane, hydrophilic keratin-containing interior, and phospholipid bilayer membrane, repeating this process multiple times to cross the entire thickness of the stratum corneum. APIs that can do so effectively are rare, according to Krishnan.

The intercellular pathway comprises narrow crevices between skin cells that APIs can traverse. Despite therelative thinness (approximately 20 μm) of the stratum corneum, most APIs following this pathway must travel
another 400 μm or more as they diffuse this top skin layer.

Indication and delivery mode determine ideal delivery kinetics

In addition to these different delivery mechanisms, there are several possible modes of action of topical formulations, according to John M. Newsam, CEO of Tioga Research. Some are designed for the API to be active on the exterior of the skin, and thus operate via a superficial mode. Most are intended for the API to be active in the epidermis or dermis, which requires penetration into the skin. Some are designed for the API to be delivered through the skin but remain locally concentrated in an area adjacent to the site of application, such as a knee joint. Finally, transdermal formulations are engineered for the API to pass through the skin into the vasculature or lymphatic system to achieve systemic delivery.

The kinetics of delivery depend on which of these four modes is being emphasized, Newsam says. For instance, transdermal delivery has an initial lag time as the API diffuses through the layers of the skin and then achieves relatively uniform provision of the active into systemic circulation over a period of time. “The ideal product will have a short lag time, moving to steady state delivery as quickly as possible and then enable sustained delivery for an extended period of time,” he notes. Ideally, a topical formulation will deliver the APIs to the right site at the correct therapeutic level over a few hours.

Delivery kinetics also depend on the type of skin and the area of the body to which the product is applied. Some topical drugs are, for instance, applied to areas of the skin that are compromised. These drugs are designed to not only treat the problem, but provide relief as quickly as possible, according to Newsam. In other cases, the API could be beneficial in treating a skin disorder but potentially give rise to systemic toxicity. Delivery to the epidermis or dermis with minimal API reaching the vasculature or use of an API with a short half-life so that it is rapidly cleared from the bloodstream would be important.

The nature of the topical formulation should also be designed to deliver the active to the appropriate part of the skin. Treatments of conditions involving the skin surface and very top layers should be designed to remain on the surface. Treatments targeting the epidermis should ideally deliver the active in a sustained-release fashion, while deeper delivery through the epidermis to the dermis is needed to treat inflammatory skin conditions.Options include film-formers for surface treatments and transdermal patches for deeper delivery.

Topical formulations targeting different indications and different patient populations must be designed to address the specific needs of those patients, according to Newsam. The condition of the skin and the location of application are two primary factors to consider. Others may include the age and skin color/pigmentation of the patients receiving the treatment. Blood supply to the skin along with skin moisture, pH, and temperature are also important, says Krishnan.

Patients with eczema or psoriasis might have cracked skin and, therefore, a reduced protective barrier. This reduced barrier could allow an increased rate of API delivery. Unlike the hands and feet where the barrier layer is thickest, barrier function is lowest in the eyelids and scrotum. Skin also tends to be more permeable for the very young and the very old.

The presence of structures such as hair follicles and sweat and apocrine glands can provide areas of ingress; a topical formulation applied to the scalp would likely exhibit different delivery attributes than the same formulation applied to the upper torso or thigh. Sebum, which is a thin, buttery film exuded from sebum glands located on the scalp, face, and underarms, has its own barrier characteristics that differ from those of the skin itself, leading to different delivery requirements.

The rate and degree of drug absorption can also be affected by a variety of illness conditions, with hepatic, cardiovascular, and gastrointestinal disorders having the greatest impact on API bioavailability, according to Krishnan. She adds that the area of absorptive surface, the level of vascularity, and the degree of skin hydration can impact delivery as well. For instance, stress reduces blood flow to the gastrointestinal tract, leading to less absorption. “As a result, patient-related factors have a significant impact on how well medications can be absorbed via the skin and thus on the ultimate efficacy of topical formulations,” she contends.

It is also important to consider the cosmetics and aesthetics of topical products, because they have a direct impact on patient compliance. People with acne want quick-drying products while those with psoriasis prefer soothing creams or ointments, for instance.

Transdermal absorption is influenced by an API’s physicochemical characteristics, such as charge, polarizability, hydrogen bonding, shape, and molecular weight. For delivery into the skin, the molecular shape of an API can, in fact, be a key determiner of bioavailability, according to Krishnan. “The skin acts as a physical barrier against particles on a macroscopic level, hence the physical state of the API has a significant impact on its permeation,” she says.

Most traditional drug substances chosen as transdermal delivery candidates fall within a relatively small molecular-weight range of 100–500 Dalton, Krishnan notes. “Due to its simplicity, molecular weight is typically used to approximate molecular volume with the implicit presumption that the majority of molecules are essentially spherical,” she explains.In that constrained range, the impact of molecular weight on drug flux seems to be rather small, Krishnan adds. For larger molecules, factors such as pH and particle size come into play as well.

A second important characteristic of APIs that determines their skin permeability is their lipophilicity/hydrophilicity. That is because the API most likely enters the stratum corneum through the lipid bilayers in between desiccated cells, and this initial partitioning occurs according to the API’s partition coefficient.

In practical terms, Newsam indicates that optimal skin permeability is realized for APIs that have logP (octanol-water coefficient) values between 2 and 3. “Lipophilic molecules such as cannabidiol get stuck in the lipid layers in the stratum corneum and don’t diffuse much further. Very water-soluble molecules with negative logP values also have intrinsically low skin permeability because they are typically charged or highly polar, which also prevents diffusion through the skin,” he explains.

It is also important to optimize the concentration or ‘strength’ of API in a topical formulation as the concentration gradient drives API diffusion from the formulation into the skin. If the API is poorly-soluble in the formulation, then the driving force for delivery into the skin will be low with only modest extents of delivery. Ideal formulations have the API concentration approaching, but no more than approximately 85% of the saturation limit, according to Newsam.

The potential for APIs to bind to proteins in different layers of the skin must also be considered because it can contribute to reduced activity. The increasing potency of APIs is also important, as the percentage of drug candidates in the pipeline that are highly potent is increasing, and higher potency may allow for topic formulations containing molecules with properties outside the typically acceptable ranges.

APIs must also be stable in topical formulations and in the skin. That means, comments Newsam, they must be resistant to isomerization, oxidation, or hydrolysis in the formulation itself and also to degradation by esterases, proteases, and other enzymes present in the skin. Finally, they should not be injurious to the skin, and therefore not be irritants or sensitizers, both as is and when exposed to photo-irradiation.

It is important to remember that in topical formulations, excipients comprise the bulk of the drug product. “Excipients affect the physicochemical properties of the API and the drug product, the sensory properties of the formulation, and the quality features of topical products,” agrees Krishnan.

The selection of the vehicle is influenced by the physiochemical properties of the drug, the disease, and the target patient population. The other excipients perform a variety of functional roles, including improving solubility to allow incorporation of the drug at the target concentration, controlling or improving API release and permeation, creating the desired aesthetics, enhancing API and formulation stability, and preventing microbial growth.

By looking at an API’s physicochemical characteristics—molecular weight, logP, polar surface area, number of hydrogen-bonding donors and acceptors, melting and boiling points, etc.—Newsam indicates it is possible to estimate how readily it will diffuse into the skin. But once an API is put into a real formulation, “all bets are usually off”. As a result, excipients play a very important role in topical formulations.

Excipients that affect the permeability of the skin include molecular penetration enhancers such as alcohols, fatty acids, and fatty esters. However, Newsam cautions that because of cross interactions between different diffusion pathways, the effect of these excipients is rarely simply additive or subtractive, making it difficult to predict formulation performance.

Other excipients are used to create the desired appearance and texture of topical formulations depending on the dosage form. Emulsifiers or surfactants are needed to stabilize lotions and creams, which typically comprise two immiscible phases, while rheology modifiers are needed to achieve the desired viscosity for products that must flow. Bioadhesives make it possible for patches to adhere to the skin even when wet, but still allow them to be removed without causing too much pain. Antioxidants and antimicrobials protect formulations in storage and in use.

Permeability and solubility enhancers must be compatible with not only the API, but all of these other excipients and the carrier, Krishnan comments. They must adhere to pharmacopeial quality requirements, and not irritate, sensitize, or irreversibly perturb the stratum corneum barrier. “Often extensive screening is required to identify the right combination of excipients for a given API that affords the optimum solution with respect to permeability, applicability, and appearance,” states Newsam.

Formulation success requires more than achieving the right level of permeation

Topical formulations are highly complex medicines intended to interact with the skin, which is designed to serve as a protective barrier and therefore by its nature limits the delivery of APIs into and through it.

“Achieving the appropriate level of delivery or permeation is usually the greatest challenge in topical drug formulation,” says Newsam. “The number of approved drugs that actually deliver API through the skin is few, because even fairly small molecules can have low skin permeability, and that permeability tails off exponentially as molecular weight increases above about 500 Da,” he says. APIs in transdermal products on the market today were, Newsam adds, initially approved for another mode of administration and developed as topical formulation in a next-generation approach.

Even with the challenge of achieving appropriate levels of delivery and permeation, the failure of many topical drug candidates that make it into clinical development can be attributed not to efficacy or safety, but actually to chemistry, manufacturing, and controls (CMC) issues, according to Newsam. “To achieve the desired level of permeability, topical formulations are often complex, which can complicate their preparation in a robust, reproducible, and uniform fashion,” he explains.

In addition, the API must be intrinsically stable in the formulation and not react with water or other excipients at the formulation pH or undergo a structural conversion such as isomerization. It could also degrade when coming in contact with the skin, and the degradants might be irritating to the skin. “As a consequence, while delivery and permeation are often the governing concern, there are many other factors that must be addressed when developing topical formulations,” Newsam concludes.

Topicals have received a lot of attention recently and are becoming a greater focus in the pharma industry, but often, says Krishnan, issues arise because drug developers do not apply formulation principles correctly and safely. “These problems most likely arise due to a lack of comprehension of the concepts relating to skin permeation mechanisms and kinetics and the complex requirements for successful topical formulations. Effective development of safe, efficacious, patient-acceptable, and manufacturable topical products cannot be realized unless these concepts are applied and formulators understand the full range of characteristics and qualities that impact topical drug delivery and patient acceptance,” she concludes.

Several potential strategies for optimizing topical drug delivery

Topical drug formulation development is a multistep process, according to Newsam. The first step should be to understand the physicochemical characteristics of the API using 

 computational methods to identify the various challenges that must be overcome. Analytical method development should come next so that API quantitation is possible. Determination of solubility and compatibility properties follows. With this information it is possible to narrow down the potential tactics for achieving required levels of delivery.

Quality by design for topical product development with an emphasis on determination of the quality target product profile, critical process parameters, critical quality attributes, and critical material attributes is essential, Krishman underscores. “Product development at Tergus focuses on quality and compliance in topical formulation development, analysis, in vitro release and permeation testing, and all the way through clinical supply manufacturing,” she says

Tioga Research uses a three-pronged approach. First, the company has a set of established formulation systems designed and built for particular classes of molecules, such as with logP values in a particular range. A new API is introduced into the appropriate starting formulation, potentially with other compatible excipients, and the extent of delivery and permeation measured. Second, Tioga’s formulation scientists mine the company’s databases for results with similar molecules over their 20 years of experience in topical drug development to be guided towards optimum formulation systems.

If these two methods do not yield an optimal formulation, Tioga leverages its high-throughput experimentation platforms and proprietary Cascading Screening methodology to evaluate large numbers of different formulations. An added benefit of Cascading Screening, particularly for 505(b)(2) products, Newsam points out, is the potential to establish a solid basis for composition of matter patentability for the best formulations. “Screening the entire useful formulation space means that it is unlikely that there are other formulations that will perform as well, which makes it difficult for competitors to circumvent such a patent,” he explains.

Overall, Newsam stresses that topical formulation development strategies should be geared toward the desired dosage form, and formulation innovation should focus on the key requirements of the specific product as quickly in the development process as possible, whether that is a transdermal patch, a film-forming matrix, a foam, a spray, a gel or a cream. “At Tioga, we might run some initial screens using simple solutions, but as quickly as possible we progress to preparing libraries of prototype formulations and measuring the performance of each, because, for instance, results with simple solutions may be poorly predictive of behavior in a practicable patch formulation,” he observes.


Volume 46, Number 9
September 2022
Pages: 22–27

When referring to this article, please cite it as C. Challener, “Delivery Kinetics for Topical Drugs,” 

API permeation into the skin modulates the efficacy of topical treatments.

Delivery Kinetics for Topical Drugs

Autologous chimeric antigen receptor (CAR) T-cell therapies have been shown to be effective for the treatment of many types of cancers. Challenges with scalability and manufacturing associated with autologous cell therapies have led to rapid advancement of allogeneic off-the-shelf cell therapies. However, allogeneic cell therapy products have their own challenges including problems with excessive immune responses and concerns about graft-versus-host disease (GVHD).

T cells are not the only immune cells, however, and there is significant interest in leveraging natural killer (NK) cells in a similar manner. NK cells are lymphocytes that have the ability to target tumor cells, releasing chemokines and cytokines that activate the innate and adaptive immune systems. They can be isolated from a variety of sources and do not have to be patient-specific. Progress is being made in the development of scalable production processes. Clinical trial data from engineered allogeneic NK cells is already in the public domain, additional clinical data continues to emerge from early-generation products, and data read-outs will soon begin for next-generation engineered products.

NK cells are important lines of defense the body has against infectious agents and tumors. They target any cells lacking a major histocompatibility complex (MHC), a group of genes that code for proteins found on the surfaces of cells that are unique to each individual and part of the human leukocyte antigen (HLA) system, according to Heather Stefanski, vice president of medical services at the National Marrow Donor Program (NMDP)/Be The Match. “Each T cell has a unique T cell receptor that recognizes a cognate antigen presented in the MHC complex; the repertoire of human T cells includes millions of unique T cells. Tumor cells can evade the immune system as tumors often do not have antigens that T cells can recognize, and therefore, the T cells do not attack them,” Stefanski says.

NK cells have the intrinsic ability to mediate tumor killing through innate recognition pathways without prior sensitization, comments Ryan Larson, vice president and head of translational science at Umoja Biopharma. “These intrinsic anti-tumor properties can be augmented by engineering NK cells to express synthetic tumor targeting proteins, such as CARs or adapters compatible with tumor-specific monoclonal antibodies that can be administered in combination,” he notes.

Next-generation CAR cell therapies, contends William Rosellini, co-founder, president, and director of CytoImmune, are focused on NK cells because they are one of nature’s best killing machines. “The NK cell has unbelievable tumor-killing activity with significantly reduced safety issues like on-target/off-tumor effects, GVHD, and cytokine release syndrome (CRS),” he explains.

NK cells do not have endogenous T cell receptors, nor do they cause the secretion of interleukin 6 and other cytokines that can cause undesired immune responses, Stefanski notes. As a result, NK cells typically have shown reduced cytokine production and expansion, and thus reduced rates of some of the serious side effects of CAR T-cell therapies, such as CRS and immune effector cell-associated neurotoxicity syndrome (ICANS), according to Larson. In addition, NK cells do not express antigen-targeting receptors that are tuned specifically to their host, thus eliminating the graft-versus-host risk that exists with allogeneic T-cell therapies.

That latter attribute also makes the development of NK-cell therapies less complex than the development of allogeneic CAR T-cell therapies, Larson adds. “For any allogeneic T-cell product, a genetic manipulation must be made to eliminate T-cell receptor expression. This step is not required for NK cells, and thus simplifies their engineering and manufacturing processes, as well as removing a potential safety issue,” he observes.

The ability to produce allogenic products from healthy starting cells is another advantage. “Autologous, or patient-specific, CAR T-cell therapies are produced using cells collected from patients who are very sick. Those T cells are potentially not ideal for a cell therapy product as some may not be good at expressing CAR proteins. In addition, the types of T cells present in each person will vary, and those differences can have a direct influence on the ability to modify those cells and generate an effective therapy,” Stefanski explains.

Overall, concludes Chris Nowers, CEO of ONK Therapeutics, allogeneic NK-cell therapies have the potential to deliver treatments with compelling clinical profiles that are better tolerated and more easily administered, logistically simpler to manufacture, and produced at lower cost. “We believe that through the reduction of costs and a growing accumulation of clinical data demonstrating the positive longer-term benefits, we will be better able to demonstrate measurable value,” he states.

NK cells are present throughout the body and can be harvested from a number of sources. They can be separated from peripheral blood or isolated from umbilical cord blood or the placenta for the development and production of allogenic therapies.Another option involves differentiation of human pluripotent stem cells (iPSCs).NK cells may also be generated from CD34+ stem cells present in bone marrow.

According to Rosellini, NK cells isolated from umbilical cord blood samples have been shown to have a higher cytotoxic capacity and span as much as 30% of white blood cells recovered, while NK cells isolated from peripheral blood tend to be a more mature population exhibiting more refined cytotoxic capabilities with a lower 15% of white blood cells recovered.

The lymphoma-derived NK-92 cell line is one established cell line designed to be easy to maintain and expand, as well as easily genetically modified using both viral and non-viral methods. It does require irradiation prior to clinical use, however, which can impact activity.

All of these different sources have been used to produce CAR NK-cell therapies undergoing clinical development. ONK Therapeutics, for instance, uses umbilical cord blood provided by Anthony Nolan Cell & Gene Therapy Services for development of its engineered NK-cell therapies. “We believe that cord blood offers a widely available source of NK cells that has been clinically validated and provides cells with a potential attractive phenotypic makeup,” Nowers observes.

Larson’s perspective is that we don’t know yet whether the source will affect the application. “It is likely that some combination of source, CMC [chemistry, manufacturing, and controls] processes, engineering/synthetic biology, and tumor-targeting approach (e.g., CAR) will determine the best therapeutic applications,” he states.

As with CAR T-cell therapies, once the NK cells for development of the therapy have been isolated, they must be expanded and genetically modified to express the CAR protein. The methods for cell expansion and cell engineering are similar for NK cells and T-cells, with some specialization accruing to each cell type, according to Larson. In most cases, large-scale expansion using cytokines and feeder cells is required to produce sufficient NK cells for clinical applications, notes Nowers. This approach facilitates efficient engineering.

Rosellini adds that retroviral, lentiviral, or non-viral delivery of genetic material is used, with viral-vector gene delivery systems currently considered the most effective. ONK Therapeutics is an exception. The company uses a combination of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) editing and transposition of transgenes employing a non-viral TcBuster transposon approach (Bio-Techne) to produce large numbers of edited CAR-NK cells, according to Nowers.

Larson believes that the method that gives the largest scale possible will be the best to commercialize, as long as it can accommodate some engineering to make sure that the cells will perform well, including in the tumor microenvironment (TME) for solid tumors. He does note, however, that there are schools of thought that think it best to use minimally expanded cells.

While the theoretical unlimited expansion capacity of iPSCs makes them an attractive option for the production of CAR NK-cell therapies, Larson also points out there have been challenges to translating this feature to downstream yield of relevant immune cell progenitor populations in an efficient and scalable format. “Solutions for scaling a directed differentiation manufacturing process will realize the full value of iPSC as a starting material from a CMC perspective-in terms of simplicity, reducing cost, and patient access,” he remarks.

In this vein, Umoja recently entered a collaboration with TreeFrog Therapeutics to develop CAR NK-cell therapies (1). Umoja’s iPSCs are engineered with a synthetic rapamycin-activated cytokine receptor (RACR) to drive differentiation to, and expansion of innate cytotoxic lymphoid cells, including but not limited to natural killer (NK) cells in the absence of exogenous cytokines and feeder cells. TreeFrog’s proprietary C-Stem technology relies on the high-throughput encapsulation (>1000 capsules/second) of iPSCs within biomimetic alginate shells that promote in vivo-like exponential growth and protect cells from external stress.

One of the less-desirable attributes of CAR NK-cell therapies when compared to CAR T-cell therapies is their lack of persistence in the body. NK cells have a much shorter half-life on the order of weeks. Administration of cytokines such as interleukin 2 to patients can prolong NK cell lifetimes, but Stefanski notes this approach often comes with harmful side effects.

The need for lymphodepletion is another important issue associated with allogeneic CAR NK-cell therapies. Prior to administration, patients receive a short course of chemotherapy to kill their existing T cells as a means for increasing persistence. “Lymphodepletion adds a veneer of immune system damage that provides short-term benefits in enabling engraftment of therapeutic cells but is detrimental to immune system function and may contribute to recurrences or other problems in the long term,” Larson says.

It is important to employ strategies to improve drug exposure through engineering strategies and be able to dose more than once at high dose levels. “Scalable manufacturing solves a lot of problems,” Larson states. Developing NK cells that secrete their own cytokines, such as interleukin 15, is also an approach to improving NK persistence, according to Nowers. He notes that this strategy can be synergistic with the knockout of the cytokine inducible SH2-containing (CISH) protein.

The development of stealthy NK cells that are edited to evade the immune system is another strategy being pursued by several companies, according to Nowers. ONK Therapeutics is also looking to increase the ability of its NK cells to target specific tumors by using cells that express high levels of favorable chemokine receptors such as CXCR4 and CXCR3 and E-selectin ligands. “These features ensure a higher likelihood that the NK cells will traffic to the site of the tumor within their natural lifetimes,” says Nowers.

Other challenges for CAR NK-cell therapies, according to Stefanski, are similar to those faced by CAR T-cell therapies: difficulty infiltrating the TME of solid tumors, the use of lentiviral vectors for delivery of genetic material, and the need to know the target antigen. In addition, although CRS is avoided by using NK cells, other off-targets are possible that must be avoided. There are also complexities associated with garnering regulatory approval for cell therapies, Rosellini adds.

As next-generation treatments, the development of CAR NK-cell therapies is not as far along as that of CAR T-cell therapies. Most programs are preclinical to early-phase clinical, including both Phase I and II trials. Cancer is the focus, with the greatest number of candidates targeting blood cancers including various leukemias, lymphomas, and multiple myeloma, but there are trials underway in solid tumors such as glioblastoma, non-small cell lung cancer, head and neck squamous cell carcinoma, and prostate cancer.

Rosellini points out that academic research groups are hard at work understanding how CAR-NK cells could be used on other diseases too. “Similar to cancer cells, we could train NK cells to target other malfunctioning cells in inflammatory diseases,” he notes. Stefanski suggests there is a potential for genetically modified NK cells to fight viruses that infect patients following stem-cell transplants and to treat autoimmune diseases driven by malfunctioning B cells. “Theoretically, CAR NK-cell therapies could be used to treat any disease that involves proliferation of abnormal cells,” she concludes.

The CAR NK-cell therapy field is still quite new. Additional clinical data from Affimed and Fate and readouts for some of the hypo-immune cells from Century are expected during the next 6–12 months, according to Nowers.

Stefanski anticipates seeing the number of Phase II trials continuing to grow. Within the next year, she believes at least one hematological malignancy candidate may advance to Phase III. Five years from now, she hopes there will be at least one CAR NK-cell therapy on the market.

CAR NK-cell therapies, Stefanski also suggests given their limited persistence, may have the potential to get patients into remission and in a position to undergo stem-cell transplants they wouldn’t be eligible for otherwise. The limited persistence of NK cells may also be an advantage from a safety point of view, according to Nowers, because it could lead to reduced risk of prolonged on-target, off-tumor toxicity.

Improvements in manufacturing technologies are also anticipated, whether using NK cells isolated from umbilical cord blood or differentiated from iPSCs. “Enhanced manufacturing procedures and the increasing use of automated closed platforms both have the potential to significantly reduce cost of goods,” Nowers notes.

More advanced cell modifications are also being explored. Approaches that include multiple receptors and different ligands on the NK cells have shown potential. “Researchers are beginning to figure out what is best for a given application, because solutions that work well for one type of cancer aren’t necessarily optimal for others,” Stefanski says. As information is gained about the current targets and how existing CAR NK-cell therapy candidates behave clinically, Rosellini believes that much will also be learned about how to improve their safety and potency.

For instance, Larson observes that organizations including Umoja will introduce second-generation approaches that avoid lymphodepletion, include engineering strategies to enhance engraftment and pharmacokinetic profiles, and enable the production of larger numbers of doses with higher cell numbers per dose. “As these advances are layered on, I expect we will see better and better clinical results,” he states.

Similarly, companies that are exploring gene editing to benefit the cytotoxic potential, metabolic health, and durability of engineered cells in-vivo will demonstrate enhanced persistence and metabolism, improved tumor homing, reduced exhaustion and immune invasion, and maximization of antigen-independent killing, according to Nowers.

Longer follow up and more mature data from ongoing studies should give greater confidence in the durability of CAR-NK approaches as well as in their safety, says Nowers.“The breadth of ongoing research and an increased move to better tolerated, logistically simpler allogeneic NK cell-therapy platforms will allow evolution toward a more traditional treatment model for advanced cell therapies. Simultaneously, the use of big data and artificial intelligence to direct selection of the best patient candidates will lead to better efficacy, greater patient benefits, and increased understanding,” he contends.

The end result will potentially be significantly reduced list price costs combined with a growing clinical database demonstrating the positive longer-term benefits of CAR NK-cell therapies, according to Nowers. “Stronger demonstration of value will help support the willingness to pay of payors and subsequently drive broader patient access and adoption,” he reiterates.

1. Umoja Biopharma, “Umoja Biopharma and TreeFrog Therapeutics Announce Collaboration to Address Current Challenges Facing Ex Vivo Allogeneic Therapies in Immuno-Oncology,” Press Release, June 10, 2022.

Cynthia A. Challener, PhD is a contributing editor to 


Vol. 46, No. 8
August 2022
Pages: 24–26

When referring to this article, please cite it as C. Challener, “Progress in the Development of CAR NK-Cell Therapies,” 

Further advances in construct design and manufacturing scalability are still needed.

Progress in the Development of CAR NK-Cell Therapies

With many blockbuster biologic drugs soon to lose patent protection—Humira and Keytruda, among others—the number of biosimilars under development is increasing. In jurisdictions with robust regulatory standards, such as the United States and European Union, the approval pathways for biosimilars, while distinct from the pathway for originator biologics, are rigorous and scientifically appropriate, according to Jeff Yant, executive director of biosimilars operations at Amgen.

These approval pathways require demonstration of biosimilarity between the biosimilar candidate and the approved innovator drug in terms of efficacy, safety, and purity. To meet this requirement, biosimilar developers must evaluate the physiochemical and structural properties of the biosimilar candidates and reference products and conduct clinical studies to compare the safety and efficacy profiles of the products.

Biosimilars must have the same route of administration, the same dosage, and the same side effects profile as the innovator drug, notes Todd Stawicki, global marketing manager for biopharma at SCIEX. Therefore, to develop a biosimilar, it is necessary to fully characterize the reference product so the biosimilar can be built to be highly similar in all of these respects, including the different variants and impurities that are present, adds Susan Darling, senior director of product and market management for CE and Biopharma at SCIEX.

Biosimilarity is first demonstrated with analytical data supporting structural and functional similarity, then through clinical studies assessing immunogenicity and pharmacokinetics (PK), and in some cases pharmacodynamics (PD). Additional comparative clinical studies, says Ricardo Infante, global medical lead for Organon Biosimilars, are conducted between the reference product and the proposed biosimilar to ensure there are no clinically meaningful differences in efficacy and safety.

“The goal of a biosimilar development program is to demonstrate that the candidate biosimilar is highly similar to and has no clinically meaningful differences from its originator/reference product, not to independently establish de novo the safety and efficacy of the biosimilar,” adds Yant.

The array of data the manufacturer of a proposed biosimilar product must generate for the proposed product and the FDA-approved reference product in order to demonstrate biosimilarity include, according to Yant, analytical studies demonstrating that the biological product is highly similar to the reference product, notwithstanding minor differences in clinically inactive components; animal studies, including an assessment of toxicity; and a clinical study or studies sufficient to demonstrate safety, purity, and potency of the proposed biosimilar product in one or more of the indications for which the reference product is licensed.

Yant also emphasizes that no single study or endpoint should be considered “pivotal” in a biosimilar marketing application. Rather the totality of data and information submitted should support the demonstration of biosimilarity (1).

Developing a biosimilar is more nuanced than developing a small-molecule generic product. With small molecules, the API is a single molecule with a highly defined structure and chemical composition. There may be different synthetic routes to producing the compound that could potentially lead to different impurities, but the actual molecule itself will be identical in the branded and generic versions. There are no possible variants to consider.

Like all biologics, Yant notes that biosimilars are produced through an intricate, multistep process using living cells. The molecule is in fact determined by the process, Stawicki stresses. Every step in the process and every change in process conditions affects the nature of the biologic product. “There is a significant change in mentality needed when developing biosimilars vs. generics,” he says, “with the focus shifting to defining and controlling and characterizing the process to make the highly similar product.” There is also a need, Stawicki notes, for biosimilar developers to have a good understanding of how potential changes to the molecule can change side effects.

Complicating this issue is the fact that for most biologics, the majority of details about the production processes for these reference products are not publicly available. “The cell line and manufacturing process of the reference product are proprietary and belong only to the reference product
manufacturer,” says Yant.

As a result, biosimilar developers basically need to start from scratch and use their own processes. “Characterizing the reference molecule, figuring out which cell line to use, and essentially reengineering the manufacturing process is tremendously expensive. For this reason, some of the companies with the most demonstrated success in biosimilars are also developers of novel drugs—they are able to leverage the same expertise, processes, and toolset for both,” Darling observes.

While the same analytics are used in biosimilar development that are used for general protein-based new-product development, it is even more important to link structure to function, Darling remarks. “It is unlikely that a biosimilar production process will yield the same variants in the same percentages as the process developed for the innovator drug, and it is therefore necessary to be able to demonstrate that the differences in variants and variant percentages don’t impact the function of the molecule,” she explains.

Analytics, adds Infante, are the foundation of biosimilar development. “Analytical testing occurs during initial development to characterize the structure and function because analytical data [are] more sensitive than clinical data in detecting differences between a biosimilar and its reference product should they exist,” he says.

Biosimilar data analysis must demonstrate a correlation between structure and function.