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

Cynthia A. Challener

The COVID-19 pandemic remains a global threat due to continued transmission of the SARS-CoV-2 virus despite the availability of several intramuscular vaccines. Vaccines administered intramuscularly via injection can elicit strong systemic responses and thus prevent the occurrence of severe disease. These vaccines don’t generally, however, lead to the development of mucosal immunity, which neutralizes the pathogen at the point of entry into the body. Mucosal immunity reduces viral replication at the level of the mucosa, which results in lower carriage by the vaccinated individual, hence reducing transmission.

Effective vaccines delivered via inhalation can lead to both strong mucosal and systemic immunity. The first such product against COVID-19 (Convidecia Air)—a collaborative effort between pharma company CanSino Biologics (CanSinoBIO, Tianjin, China) and respiratory device developer Aerogen (Galway, Ireland)—received regulatory approval in China in early September 2022 (1).

However, developing these vaccines can be challenging. Active substance formulation and delivery-device development must be pursued simultaneously. Once delivered, the vaccination agent must penetrate protective mucosal barriers and survive long enough to stimulate the desired immune response.

Mucosal membranes line the respiratory, digestive, and urogenital tracts and form an interconnected network of mucosal compartments (2–4). Vaccination that leads to mucosal immunity at one of these mucosal sites thus leads to immunity at distant mucosal sites. Mucosal immune responses involve activation of antigen-presenting cells (APCs) and the production of many different types of immune cells, antibodies, and inflammatory cytokines/chemokines.

Targeting mucosal layers in the upper and lower respiratory tract and lungs is an attractive approach to vaccination because most airborne pathogens enter the body through this route and infect these tissues (5). “Generation of mucosal immunity results in activation of many parts of the immune system, not just antibodies. In addition, replication of viruses is prevented in the respiratory system, enabling interruption of the transmission cycle,” explains J. Robert Coleman, CEO of Codagenix.

Numerous advantages to inhalation vaccines

Vaccines delivered via inhalation enable targeting of the respiratory tract mucosa and generation of both humoral and cell-mediated immunity, according to Pierre A. Morgon, executive vice president, Portfolio Strategy and Supranational Affairs and managing director, CanSinoBIO Europe. It is also possible to achieve good drug absorption, fast onset of action, and high bioavailability with inhaled vaccines (6).

In addition, they eliminate the need for needle-based administration, reducing the risk of needle-stick injuries and allowing avoidance of invasive injections, fear of which may keep some people from getting vaccinated (7).

In fact, inhaled vaccines delivered as liquids using nebulizers can potentially be administered with lower requirement for extensive healthcare infrastructure or as many trained healthcare personnel within an immunization center (2). Delivery by inhalation should, therefore, make large-scale mass vaccination easier to conduct, according to Ronan MacLoughlin, associate director of R&D, science and emerging technologies at Aerogen.

Importantly, less vaccine is needed for delivery via inhalation—typically less than 0.2 mL vs. 0.5 mL for injection, says Morgon. “Lower doses means more doses can be produced per batch, and mass vaccination can thus be achieved more cost-efficiently,” he says.

Furthermore, administration via inhalation can be theoretically achieved through the mouth or nose. The preferred route is, says MacLoughlin, entirely dependent on the target receptors for the vaccine. “If nasally expressed, then nasal should be the preferred route. Inhalation into the lung via the nose would allow for both nasal and lung deposition. Oral inhalation allows for a larger dose to be delivered to the lung, given that avoiding the nasal passages will increase the available fraction of aerosol,” he comments.Aerogen’s proprietary aerosol-generating technology converts the liquid vaccine into fine aerosol droplets that are suitable for delivery of the vaccine deep within the lung.

Some are of the opinion that vaccination through the mouth may result in enhanced induction of mucosal immunity. Researchers at McMaster University, for instance, have shown inhalation of a tuberculosis vaccine to be more effective than delivery via nasal sprays, because the vaccine penetrates much deeper into the airway (8).

Because inhaled vaccines provide local immunity to the respiratory tract, they are seen to be ideal solutions for interrupting the spread of viruses with high transmission rates and the potential to lead to global pandemics (9).

The specific types of vaccines that can be formulated for inhalation delivery is not fully understood yet, according to Jean-Denis Shu, SVP of Medical Affairs with CanSinoBIO. “The critical factors are determined by the target receptor, the vaccine’s stability in the lung, and whether sufficient dose can be given to illicit the immune response,” he observes. CanSinoBIO’s inhaled COVID-19 vaccine, now approved in China, is based on an adenovirus-vectored vaccine (10).

“Inhalation delivery offers the potential advantage of bypassing anti-vector immunity for viral vector vaccines for which people may have already developed some immunity through, for instance, exposure to cold viruses,” Morgon adds. The formulation of CanSinoBIO’s inhaled vaccine (Convidecia Air) is the exact same as that of its intramuscular vaccine (Convidecia) and there is no need for an adjuvant to boost immunogenicity.

Codagenix’s inhaled COVID-19 vaccine candidate is a live, attenuated virus that, according to Coleman, safely and stably brings not only the SARS-CoV-2 spike protein, but many other proteins of the virus to the immune system, allowing for broad protection.

Beyond these two approaches, there are inhalation vaccines under development based on attenuated influenza virus, parainfluenza virus (PIV) 5, lentiviruses, Newcastle disease virus (NDV), and vesicular stomatitis virus (VSV); bacterium vectors, nucleic acids (messenger RNA, DNA), and protein subunits (3). Unlike the CanSinoBIO vaccine, the latter require formulation with adjuvants to ensure sufficient immunogenicity (4,11). A range of adjuvants have been explored, such as polyethyleneimine, N-[1-(2,3-Dioleoyloxy) propyl]-N,N,N-trimethylammonium chloride, chitosan, toxoids, cytokines, saponins, lipids, and toll-like receptor agonists.

The need for effective delivery technologies remains the main challenge for realizing successful mucosal vaccines, including those administered via inhalation (12). Not only are strong adjuvants required for many types of vaccines, it can be difficult to maintain the integrity of the vaccines. The vaccination agent must resist degradation in the harsh mucosal environment in order to have time to reach the immune inductive tissue and elicit the desired immune response while also ensuring that oral tolerance does not develop (11). Overall, therefore, appropriate formulation of inhalation vaccines is crucial for their success (13).

Inhalation vaccines can be formulated as liquids or powders. Dry powders have the potential for long-term storage without cold-chain requirements, while liquid formulations are generally simpler to develop and implement, notes MacLoughlin. Dry powder formulations can be generated by various methods, such as spray drying and thin-film freezing (4,6). It is crucial to control the particle size and particle size distribution to achieve the desired release profiles and distribution in the lung.

The aqueous nature of liquid formulations, meanwhile, allows for rapid interaction with the airway epithelium. “Such interactions may be subdued for dry powders due to local conditions in the airway surface lining fluid,” Morgon observes. He also points out that the need for cold-chain management is not an automatic disadvantage for liquid vaccines, because storage and handling capabilities at 3 to 8 °C are generally available at most locations.

Certain attributes are essential for successful inhalation delivery

The physicochemical attributes of vaccine actives delivered intranasally must allow the vaccine to mimic the wild type natural virus that comes in through the airway, according to Coleman. “Such behavior is essential to induce very broad immunity and stop onward transmission,” he contends. He adds that Codagenix chose to use an attenuated live virus rather than a viral vector or protein subunit for this reason. “Using an attenuated virus allows for presentation of multiple viral proteins and avoids any underlying immunity issues, affording increased vaccine uptake,” states Coleman.

Development of optimized vaccine/device combination essential

A consideration equally important to the design of the vaccine active and formulation is the development of an optimized vaccine/device combination, according to Shu. “Picking any delivery device off-the-shelf and applying that increases the risk that the immunization is not effective. A solid understanding of how the vaccine and device interact are critical,” Shu avers.

Morgon agrees. “Understanding formulation, device performance, delivered dose, distribution of dose within the airways, etc. all impact the
likelihood of success.”

“Co-development, such as that conducted by Aerogen and CanSinoBio, has enabled an effective immunization approach that uses a fraction of the injected dose by volume or mg of drug substance,” MacLoughlin concludes.

Devices use for the delivery of inhalation vaccines should, ideally, be patient agnostic. “Taking the variability out of the patient input in the process is a key requirement in order to ensure consistent, reproducible, and reliable dose across a population,” MacLoughlin observes. To this end, Aerogen has focused on development of devices that require the patient to make only a single inhalation for their entire vaccine dose. “This way, there is no requirement for coordination of breath with aerosol generation, which is a key failing of pressurized metered-dose inhalers, and every patient has the same dose available to them for inhalation,” he notes.

Development of mucosal vaccines, including inhalation vaccines, faces an additional hurdle that vaccines administered by other routes generally do not. “The historic body of work in vaccinology has focused on the production of antibodies in the bloodstream. Successful vaccines are determined to be so because they result in high levels of antibodies,” Coleman says.

Some mucosal vaccines, however, may generate lower levels of antibodies in the blood. That does not mean they are less effective, but regulators may not be aware of the differences and thus still compare the data for mucosal vaccines to those for injectable versions, Coleman adds. In the case of CanSinoBIO’s Convidecia Air, the humoral and cellular responses are higher with the inhaled booster than with the injectable one, Morgon comments.

One program designed to address this concern has been established by the World Health Organization (WHO). Its Solidarity Trial Vaccines program supports the development of second-generation COVID-19 vaccines with greater efficacy, greater protection against variants of concern, longer duration of protection, and improved storage and/or simplified delivery with needle-free administration (14). In the Phase III trial of Codagenix’s CoviLiv vaccine candidate, both antibody generation and actual efficacy (determined by comparing vaccinated and placebo groups) are being evaluated, so the actual efficacy rate will be measured. CanSinoBIO has also agreed to the request from WHO to include Convidecia Air in the Solidarity trial.

Several other inhaled COVID-19 vaccines in development

The same group of researchers at McMaster University that compared the delivery of a tuberculosis vaccine via nasal spray and inhalation also have an inhaled, adenoviral-vectored, aerosol COVID-19 candidate in a Phase I clinical trial (8,15).

Scientists at MIT have shown that peptide vaccines with albumin-binding lipid tails when formulated with the adjuvant CpG and administered via inhalation generate long-lasting immune responses (5). A group at University of North Carolina-Chapel Hill, meanwhile, used lung-derived exosomes, or nanoscale vesicles secreted from lung spheroid cells (LSC-Exo), to effectively deliver nucleotide and protein-based vaccines to the lungs via an inhaler (8,16).

In addition to the above inhalation vaccines under development for COVID-19, several groups are investigating the potential of intranasal vaccines intended to induce mucosal immunity. Some of these have been found wanting, most recently the intranasal version of the AstraZeneca viral-vector vaccine (17). These disappointing results, says Morgon, may drive a heightened focus toward the inhalation route.

At the same time, the approval of the CanSinoBIO inhaled COVID-19 vaccine provides evidence that inhalation delivery can be successful at generating mucosal immunity. “We now have significant data on the safety and efficacy of an inhaled vaccine. These data should give others confidence to proceed to trial with what is known to be a broad pipeline of vaccines that may have utility if inhaled,” Morgon says. “Indeed,” he adds, “significant activity has begun/restarted in the area, given that this new route of administration has been shown to be feasible, and on a global mass immunization scale.” Furthermore, if Convidecia Air is convincingly shown to materially reduce viral carriage in vaccinated individuals and therefore curb transmission, CanSinoBIO believes it will turn out to be a blockbuster product.

“The future of inhaled vaccines has just received a significant boost of more useful rapid methods, although further development is required to improve accuracy and repeatability,” he highlights. “Of the different ‘PAT [Process Analytical Technologies] methods’, ATP bioluminescence technology offers a higher sensitivity compared with colorimetric methods.”

In terms of management of cleaning validation processes, software developments are piquing Sandle’s interest, including some that are employing artificial intelligence. “Such packages can help to sort equipment into types where a matrix approach is preferred, as well as enabling scheduling and for setting re-validation targets,” he states.

At its very essence, cleaning validation will require greater consideration, simply as a result of the evolving therapeutic landscape, Kolbert asserts. “The increased use of biological therapeutic agents in pharmaceutical manufacturing will require increased complexity of analytical techniques as activity of the residue is as important as chemical presence,” he summarizes.

Pharmaceutical Cleaning Validation Market: Industry Analysis and Forecast (2021–2027) by Product Type, Validation Test, and Region. Report

CFR Title 21, 210 Part 133.4 (Government Printing Office, Washington, DC).

Validation of Cleaning Processes (7/93), Inspection Guide. 

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 


Vol. 47, No. 1
January 2023
Page: 26–29, 39

When referring to this article, please cite it as C. Challener. Inhalation Vaccine Development. 

Articles

Inhaled vaccines must resist degradation and penetrate the mucosal lining in the airways and lungs. 

Inhalation Vaccine Development

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.

Variety of oligonucleotide drug substances

The landscape of nucleotide-based therapeutics is based on more than half a century of research and development in these modalities, according to Julian Mochayedi, strategic marketing manager for mRNA solutions with MilliporeSigma, the Life Science business of Merck KGaA, Darmstadt, Germany. This knowledge translates to the clinic and beyond, with several approved therapeutic products on the market.

The different types of nucleotide-based therapeutics can be broadly classified by the type of nucleic acid, Mochayedi continues, such as those with DNA or RNA as a basis. Alternatively, they can be differentiated by their functional design, which determines the mode of action. “One can cite DNA-based adeno-associated virus (AAV) vectors for gene delivery, antisense oligonucleotides (ASOs), small-interfering RNAs (siRNAs) for gene expression disruption, and most recently, mRNA to express a protein of interest such as for immunization purposes, as the successful mRNA-based COVID-19 vaccines have demonstrated,” he explains.

mRNA instability particularly challenging

DNA-based oligonucleotide drug substances, such as AAV vectors and plasmid DNA, share the relatively stable characteristics of DNA, Mochayedi notes. The stability of shorter chemically synthesized RNAs such as ASOs and siRNA can be improved through chemical modifications during synthesis. Particularly long biologically synthesized mRNA molecules, however, are inherently unstable, and thus require additional management during manufacturing.

All nucleic acid-based platforms, however, must be formulated using delivery technologies that protect the therapeutic from degradation. Unique solutions such as LNPs help ensure stability in the patient setting, API stability and, ultimately, the efficacy of the therapeutic approach.

There are several reasons why mRNA is inherently unstable. The physiochemical properties of single-stranded mRNA molecules that lead to their instability include their very large size, high negative charge, sensitivity to rapid degradation by omnipresent enzymes such as endonucleases, shear sensitivity, and high viscosity through the formation of secondary structures in solution, according to Mochayedi.

Manufacturing strategies for managing mRNA instability

One of the main culprits causing mRNA degradation is the enzyme RNase. Avoiding it is difficult, however, because this enzyme is ubiquitous. “It is very difficult for mRNA to move freely in the environment,” Christian Cobaugh, CEO and founder of Vernal Biosciences, comments. “Extreme precautions must therefore be taken to avoid introducing this enzyme into manufacturing spaces or enclosures,” he stresses. This issue can often, he notes, be a barrier to entry into the mRNA market for laboratories not experienced in mRNA production.

Use of qualified processes, including sterile single-use technology, should be applied to mitigate contamination and carryover of endonucleases, Mochayedi says. Liberal use of RNase degrading chemicals for cleaning equipment surfaces, operator gloves, and so on is also essential, Cobaugh adds.

The instability of mRNA at room temperature creates further challenges, as the time available for holding generated mRNA sequences at room temperature is fairly restricted, according to Cobaugh. The drug substance must be moved seamlessly to downstream purification and formulation or quickly frozen.

“The choice of adopting a seamless manufacturing approach or having unit operations separated by controlled freezing and subsequent thawing steps must be considered early on, as the decision will impact the design and operation of the mRNA manufacturing facility, and once implemented, that design will essentially be locked in,” Cobaugh contends.

“Ideally, it is best to limit the number of freezing and thaw steps, as they can impact the quality of the mRNA drug substance and formulated mRNA-LNP products,” Cobaugh adds. Furthermore, gentle techniques must be employed. Continuous movement of the mRNA drug substance to purification and then LNP formulation avoids all but the final freezing step. “It is possible, though, with the right engineering solutions and validation procedures to surmount the challenges presented by either approach to mRNA manufacturing,” he concludes.

In addition, stringent optimization of reaction conditions and the process parameters for key unit operations, such as the synthesis step (in vitro transcription) and filtration (via tangential-flow filtration), can overcome viscosity challenges and ensure low shear during processing to maintain the stability of the mRNA at each stage of the manufacturing process, Mochayedi remarks. “Bespoke process development and optimization are necessary for each unique mRNA sequence to address and overcome the highlighted challenges,” he concludes.

While the concept of leveraging mRNA for the prevention or treatment of various diseases is straightforward, the safe and effective delivery of these fragile nucleic acids into cells where their message can be translated into proteins has proved to be a huge challenge, Mochayedi observes. “For the COVID-19 mRNA vaccines, the solution to this problem came through the development of LNPs. These particles, typically composed of four different lipids (ionizable lipids whose positive charges bind to the negatively charged backbone of mRNA, pegylated lipids that help stabilize the particle, and phospholipids and cholesterol molecules that contribute to the particle’s structure), provide a protective bubble for the delicate mRNA molecules, enabling their safe and efficient delivery into cells,” he explains.

The structure of the nucleotide API has a strong impact on the selection of the excipients for the formulation. The manufacturing process must, in addition, be highly controlled. “The buffer solution containing the mRNA drug substance used to produce the LNPs is of low pH (typically aqueous sodium citrate) that places additional stress on the nucleotide’s stability, leading to even more rapid breakdown than occurs at neutral or slightly basic pH,” Cobaugh says.

A low pH of approximately 4.5 is needed to ionize the mRNA so that it forms nanoprecipitates with the lipids. The use of stabilizing process aids is, Cobaugh adds, generally avoided because they would interfere with the ionization of the mRNA and could also potentially end up in the product. Most molecules that would stabilize mRNA against pH-mediated degradation would, furthermore, likely be hazardous chemicals.

The key to successful LNP production, therefore, is minimization of the time required for the LNP manufacturing process. That, Cobaugh emphasizes, requires extensive understanding of the process from a quality-by-design basis combined with thorough process validation. “It is absolutely essential to have a deep awareness of what levers can be pulled from both process and engineering perspectives to minimize the time for all aspects of the formulation process,” he states.

With respect to fill/finish of mRNA therapeutics and vaccines, the strategy is generally no different than that of any other biological API, according to Mochayedi. The goal, agrees Cobaugh, is to fill the product—whether drug substance in sterile single-use plastic bags and containers or formulated drug product into sterile glass vials—and then freeze them as rapidly as possible but in a controlled manner. In this case, the challenges are largely engineering and equipment-related.

For storage and shipment of mRNA, the requirement to maintain the cold chain to ensure its stability and integrity all the way to the patient is of utmost importance, Mochayedi says. “Central storage is usually performed at very low temperatures (-80 °C). Similarly, formulations containing nucleotides require shipment at low temperatures, which is typically achieved using dry-ice packaging,” he notes. In some cases, mRNA drug products may require the addition of a cryoprotectant to protect the particles. The adjustment of excipients to protect the nanoparticles may also enable some formulations to be shipped at approximately 4 °C.

The production of mRNA-LNP formulations that are stable at warmer temperatures is a key focus of developers of candidate therapeutics and vaccines. One approach noted by Cobaugh involves the development of higher-quality DNA templates that are free of breaks (commonly referred to as nicks) that result in the shorter, more unstable mRNA sequences. Mochayedi points to new types of modified nucleotides and new capping technologies to physically altered forms of mRNA, such as circular RNA. “These new approaches may overcome some of the stability issues and enable a broader application of mRNA as a technology platform,” he believes.

Lyophilization of mRNA products as an alternative to freezing is the other main area of investigation. “Theoretically, there is no reason why freeze-dried mRNA products cannot be stored at room temperature as long as water is kept out of the storage containers,” Cobaugh says. He notes that there has been preliminary success in this area, and he is hopeful that lyophilization will be applicable for mRNA products in the not-too-distant future.

Mochayedi agrees, noting that significant investigations into formulation optimizations to overcome the ultra-low temperature requirements of mRNA drug products during shipment and storage are underway across the industry, with lyophilization of formulated mRNA-LNPs as an extremely promising alternative for liquid-based products. “The complete drying of formulations afforded by freeze-drying would unlock the possibility of much more easily providing mRNA therapeutics in countries with higher temperatures,” he remarks. 


Vol. 46, No. 12
December 2022
Pages: 22–23, 27

When referring to this article, please cite it as C. A. Challener, “Managing mRNA Instability During Formulation, Manufacturing, and Shipment,” 

Unique solutions are required to protect inherently unstable messenger RNA.

Managing mRNA Instability During Formulation, Manufacturing, and Shipment

Poor solubility issues are often associated with small-molecule APIs formulated for oral administration. Solubility is also important for parenteral products as well. Injectable products need to be free of particles (foreign substances and active ingredients) and often are high-concentration formulations, and both can be difficult to achieve when drug substances have poor water solubility. Special excipients can help overcome these solubility problems. Top candidates include polymeric micelles and lipid-based formulations.

The online version of this article has been updated from the print version to include additional content.

Poor solubility is a general issue in drug development

Solubility is a challenge that most newly discovered drug candidates face, irrespective of the intended route of administration, according to Philip Schäfer, head of process and formulation materials at MilliporeSigma, the Life Science business of Merck KGaA. In fact, approximately 40% of APIs on the market and around 90% of those in development are poorly water soluble (1).

“Today’s approach of target-oriented high throughput screening aimed at identifying more lipophilic compounds combined with the trend towards larger and more complex molecules both contribute to the development of APIs with reduced solubility,” says Schäfer. More specifically, during drug development, explains Shreya Shah, associate director of formulation services at LLS Health, many molecular targets, such as drug/enzyme binding sites, are hydrophobic. “As like attracts like, the main structure of candidate APIs is also often hydrophobic, making them poorly soluble in aqueous media,” she observes.

Molecular weight, chemical structure, and chemical-group lipophilicity are key factors affecting the solubility of small-molecule APIs, adds Philipp Heller, project manager innovation at Evonik Health Care. He notes that some peptide-based drugs can also suffer from poor solubility depending on their amino acid composition, while proteins have solubility challenges as well.

In the case of peptides and proteins, poor solubility generally occurs when the tertiary structure of these molecules includes large hydrophobic portions on the surface and thus exposed to aqueous media, according to Vincenza Pironti, senior staff scientist, research and development, pharma services at Thermo Fisher Scientific. “Often this aspect can be circumvented by conducting pH or ionic strength studies to find the right window where the protein structure contains exposed charged portions,” she comments.

Solubility is important not just for orally administered drugs; it is also essential for parenteral formulations because these product solutions must be particle-free, Schäfer notes. That must be achieved even for high-concentration products—a growing formulation trend, which further increases the need for high solubility.

“There are several strategies used to improve the solubility of hydrophobic compounds and each has its limitations. Safety is always a key concern in the formulation of novel therapeutics, but it is even more crucial for parenteral formulations that bypass the gastrointestinal tract and are delivered directly to receptor sites via the bloodstream. Therefore, not all strategies are practical, especially when formulators are trying to achieve high drug loading, for example, to reduce the frequency of administration,” comments Shah.

Aqueous parenteral formulations must also have the right right tonicity level to avoid the insurgence of pain after administration, Pironti stresses. This requirement has limited the number of excipients currently approved and reported as generally regarded as safe (GRAS) by FDA. Nonstandard formulations leveraging oil-based solutions in lipophilic media present an entirely different set of issues, she adds.

Excipients must provide a shielding effect

Circumvention of the blood-brain barrier with parenteral formulations also means that excipients used in these formulations must meet high quality requirements with respect to bioburden, endotoxin levels, etc. “Due to this fact,” Schäfer says, “not all types of excipients that may increase solubility are also suitable for parenteral administration.”

In addition, because many parenteral formulations are high-concentration products, there are challenges to increasing solubility with the limited amounts of excipient that can be used, according to Heller. “For each specific API, the hydrophilicity and hydrophobicity of an excipient or excipient mixtures must be balanced to achieve high loading efficiency and formulation stability, while at the same time ensuring efficient release of the API from the formulation,” he remarks. The limited number of excipients available for parenteral use only exacerbates this challenge.

For instance, Shah explains that when surfactants or organic co-solvents are used to increase solubility to enable high drug loadings, toxicity issues and/or immunogenic responses can occur. Cyclodextrins and other excipients that boost API solubility by forming inclusion complexes, a process that is governed by thermodynamics, also must be used at high concentrations that can cause excipient toxicity issues.

Physical modification such as particle-size reduction via nanomilling may in some cases be an effective approach to reducing the concentrations of excipients required to enhance solubility, Shah adds. She also notes that reduced excipient concentrations may enable higher drug loadings because the API remains in a solid-liquid dispersion rather than being solubilized in an aqueous solution.

In general, because poor water solubility is usually driven by the presence of highly hydrophobic regions within APIs, avoiding contact between these hydrophobic areas and the aqueous environment within parenteral formulations has proven to be a potent measure for increasing solubility, Schäfer observes. “Many solubility-increasing excipients consequently share the same mode of action—a shielding effect of the hydrophobic areas,” he says. Examples include silica carriers and polymeric micelles.

Amphiphilic excipients with both hydrophilic and hydrophobic moieties that can be fine-tuned work best to enhance the solubility of APIs in parenteral formulations, notes Heller. In addition to polymeric micelles, he points to nanoparticle preparations and liposomal formulations as having been proven useful for the solubilization of hydrophobic, small-molecule APIs. He adds, though, that higher API loadings are typically achievable with polymeric micelles and nanoparticles.

Generally, extensive experimental screening is performed using a variety of excipients to find the right match, according to Shah. Starting from the structural properties of the drug substance, Pironti adds that it is possible to define a strategy for solubility enhancement using 

 models. “Such models enable scientists to start with a limited panel of excipients to increase solubility with a design-of-experiment approach,” she explains.

For instance, if the API is hydrophobic and the excipient is an emulsifier, the API will sequester into the hydrophobic core of the emulsifier. Indeed, she points out that excipients with a hydrophobic group and sufficient hydrophilicity to coexist in an aqueous environment are effective emulsifiers and useful solubilizing agents. Polysorbate 20 and polysorbate 80 are popular excipients in the formulation of parenteral therapeutics. Polyethoxylated surfactants and polyethylene glycols have also been evaluated regarding their ability to improve solubility in these applications.

One overarching consideration for the formulation of poorly soluble APIs for parenteral administration, stresses Shah, is that there is no one-size-fits-all excipient. “Studies are still ongoing that aim to understand which API properties contribute to optimum interactions with different excipients,” she notes. Heller agrees. “Excipients must be carefully chosen and fine-tuned for each API.”

Polymeric micelles work by encapsulating poorly water-soluble APIs within their inner cores, thus shielding the drug substances from the aqueous environment. They are attractive for several reasons, notes Shah, including the fact that polymeric excipients tend to have fewer toxicity issues and are relatively safe at higher concentrations compared to small-chain surfactants. Many are also biocompatible and formed from amino acids or other building blocks that exist within the human body.

According to Schäfer, they can be assembled from a variety of different polymer types, with the most popular choices including polyethylene glycol, polyethylene oxide, stearic acid, and G-chitosan. “All of these polymers have hydrophobic regions that orient towards the cores of the micelles and hydrophilic regions that orient towards the external aqueous medium,” he explains. The polymers exhibit self-assembly behavior triggered by forces such as hydrophobic interactions, electrostatic interactions, and complexation. The resulting micelles are typically smaller than 100 nm in diameter.

Amphiphilic bioabsorbable diblock copolymers such as poly(ethylene glycol)-methyl ether-block poly(DL-lactide) (mPEG-b-poly(DL-lactide) and poly(ethylene glycol)-methyl ether-block poly(DL-lactide-co-glycolide) are commonly used, according to Heller. When formulated with these polymers, hydrophobic APIs associate with the hydrophobic lactide/glycolide polymer segments, while the hydrophilic PEG polymer segments arrange themselves into the surrounding aqueous environment.

“Micelles or nanoparticles are formed based on the molecular weight of the block copolymers,” Heller continues. In the end, extremely small, high-surface area nanomicelles or nanoparticles are generated that are colloidally stabilized by the outer hydrophilic polymer PEG segments. The size and physical properties of polymeric micelles and nanoparticles improve API solubility and enhance API exposure to patients, he comments.

Genexol PM (Samyang Holdings) is an example of a recently introduced commercial paclitaxel product formulated using 20- to 50-nm mPEG-b-poly(DL-lactide polymeric micelles. It offers, according to Heller, sustained release over 48 hours with improved solubility and efficacy and reduced toxicity and hypersensitivity compared to more conventional formulations containing surfactants.

“Polymeric micelles and polymeric nanoparticles are attractive preparations because they combine high loading efficiency, favorable pharmacokinetics properties, and improved clinical efficacy,” Heller concludes. “We see future potential for polymeric micelles and polymer nanoparticles made with biocompatible, bioabsorbable mPEG-b-polylactide diblock copolymers,” he predicts. "These diblock copolymers are part of that class of lactide/glycolide polyester functional excipients that have a proven safety record as evidenced by the number of long-acting injectable products (60) and bioabsorbable medical devices that have been on the market for decades,” Heller explains.

Shah also sees polymeric micelles as an exciting technological advancement for parenteral formulations. In addition to block co-polymers such as PEGylated polylactic acids, PEGylated polyglycolic acids, or even a combination of lactic and glycolic acids, she finds the polaxamers, or pluronics, have also been used for micelle formation and solubility enhancement. “These triblock co-polymers [have] a more hydrophobic central portion of the chain that forms the core, with more polar, hydrophilic ends extending out into the aqueous milieu.”

An example of a new excipient technology for increasing the solubility of parenteral formulations based on polymeric micelles comes from LLS Health. Its Apisolex excipient is a multiblock co-polymer comprising a polysarcosine block and a D-/L- mixed poly amino acid block, according to Shah. “Sarcosine is an amino acid derivative that is naturally found in human muscles and tissues, making it a biocompatible alternative to existing excipients used in parenteral formulations, especially where high drug loading is desired,” she says. In the polymeric micelles formed by Apisolex polymer, the mixed poly amino acid block forms the hydrophobic cores while the polysarcosine block forms the hydrophilic coronas.

LNPs also encapsulate APIs, but they are structurally different from polymeric micelles. “Solid LNPs house hydrophobic APIs within the lipids in a solid core that remains solid even when the particles are dispersed in the aqueous environment. Nanostructure vesicles, meanwhile, consist of both liquid and solid lipid components/compartments within the solid particles,” says Shah.

LNPs have recently become well known for their use in protecting messenger RNA (mRNA) from degradation and aiding in its cellular uptake, but they can be advantageous for many other drug substances. While nucleic acids such as mRNA and small interfering RNA (siRNA) do not have poor solubility properties, they can enhance the solubility of more conventional small-molecule APIs. “LNPs can provide improved bioavailability and controlled release for poorly soluble small molecules, and there have been many examples in the oncology area reported in the literature,” Pironti observes. “The future for LNPs lies in the development of new functional lipids to navigate the patent landscape and enable targeting of specific organs, tissues, and cells,” contends Heller.

Given that most new drug candidates suffer from poor solubility to some degree, much work has been focused on identifying a wider array of solutions for solubility enhancement. For parenteral drugs, cyclodextrins are an example of a type of excipient that has been re-discovered and used with increasing frequency in recent years.

These cyclic oligosaccharides are derived from natural starch and arranged in the form of a hollow cone with a hydrophilic exterior and hydrophobic interior and thus can shield a broad range of hydrophobic, poorly water-soluble APIs from aqueous media, according to Schäfer. He adds that they exhibit a very good safety profile, which makes them suitable for parenteral administration. In addition, cyclodextrins may also protect APIs from chemical degradation, reduce protein aggregation, and mask odors and tastes. One example highlighted by Schäfer is the use of sulfobutylether-β-CD for the formulation of remdesivir, a parenteral drug used for the treatment of COVID-19.

Heller points to albumin nanoparticles and synthetic polypeptides and other excipients that have attracted attention as solubility enhancers for parenteral formulations.

Using excipients to optimize solubility presents some challenges, because this type of formulation work typically takes place during the latter part of the development cycle. “Often developers will find that formulation optimization alone is not sufficient to enhance API solubility. It is therefore recommended that API solubility issues be tackled sooner in the development process,” states Schäfer.

That entails looking at API processing to improve drug solubility. In addition to particle-size reduction by, for example, nanomilling, salt formation, cocrystal formation, and polymorph screening are also important avenues to pursue, according to Schäfer. “These methods not only offer the possibility to significantly enhance solubility, but can also tackle characteristics like physical and chemical stability, dissolution rate, and processability,” he says. Further optimization by formulation can then be layered on if needed. In fact, Schäfer believes that API processing and tailored formulation should go hand in hand to achieve the optimum probability of success for drug candidates.

Traditionally, there has been limited development in novel excipients for solubility enhancement. Developers tend to focus, Shah observes, on creating new formulation technologies using excipients with a known safety profile rather than developing new excipients for solubility enhancement. “For example, there is significantly more [effort] to develop new technologies using existing polymers with known toxicity profiles, such as amorphous solid dispersion technologies, than [to develop] novel excipients for improving solubility of poorly soluble drugs,” she says.

More recently, however, Shah observes that novel excipients are becoming an attractive route as well. “Novel excipients present a way to leverage existing techniques and achieve the same, if not better, results,” she insists. They also provide increased IP protection and, because the IP is new, longer patent life for drug developers and marketers to leverage. Novel excipient IP may, in particular, be useful for older clinical candidates that have previously been shelved due to solubility issues, she notes.

Polymeric excipients, especially those which enable micelle formulation, are a good example. “These new excipients provide a significant means for achieving better solubility and high drug loading for APIs that previously failed during the drug-development process. Using the FDA’s [US Food and Drug Administration’s] 505(b)(2) regulatory pathway also allows reformulation of previously approved drug substances with novel excipients for enhanced biological performance but lower application risk and expedited time to market,” she concludes.

., 62 (11), pp. 1607–1621 (November 2010).

Cynthia A. Challener, PhD is contributing editor to Pharmaceutical Technology.


Volume 46, Number 11
November 2022
Pages: 20–23

When referring to this article, please cite it as C.A. Challener, “Excipients for Solubility Enhancement of Parenteral Formulations,” 

High-concentration injectable formulations present unique challenges.

Excipients for Solubility Enhancement of Parenteral Formulations

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 

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.

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