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

Cynthia A. Challener

As understanding of the endocannabinoid system increases, interest in developing drug candidates that are either natural phytochemicals or synthetic derivatives of cannabinoids has risen. The endocannabinoid system consists primarily of two G-protein-coupled receptors known as cannabinoid receptor 1 (CB1R) and cannabinoid receptor 2 (CB2R) and their natural ligands anandamide and 2-arachidonoylglycerol. It also includes, however, numerous other components, many of which are present in most types of cells in the human body.

In fact, approximately 600 Cannabis metabolites have been isolated, with more than 100 of them classified as cannabinoids and seven specifically as cannabidiol (CBD)-type compounds (1). CBD has been shown to have antioxidant, anti-inflammatory, and analgesic properties. Other cannabinoids aside from CBD and Δ

-THC), the psychoactive ingredient in marijuana, include cannabichromene (CBC), cannabidiolic acid (CBDA), cannabigerol (CBG), cannabinol (CBN), cannabidivarin (CBDV), cannabidivarinic acid (CBDVA), cannabigerolic acid (CBGA), cannabicyclol (CBL), Δ

-THC, tetrahydrocannabinolic acid (THCA), and tetrahydrocannabivarin (THCV).

FDA has, to date, approved one cannabis-derived drug product, Epidiolex (GW/Jazz Pharmaceuticals, cannabidiol) for the treatment of certain types of seizures, and three synthetic cannabis-related drug products, Marinol (dronabinol), Syndros (dronabinol), and Cesamet (nabilone), all for the treatment of nausea associated with cancer chemotherapy and anorexia associated with weight loss in AIDS patients.

Sativex (nabiximols, GW/Jazz Pharmaceuticals), a formulated cannabis extract that contains the principal cannabinoids Δ

-THC and CBD, as well as other minor cannabinoids and terpenes, is now approved in more than 25 countries around the world for the treatment of spasticity due to multiple sclerosis.

Several small biopharmaceutical companies, along with Jazz Pharmaceuticals, are developing additional phytochemicals and novel synthetic derivatives as potential cannabinoid-based drug candidates.There are advantages and disadvantages to both approaches.

Regardless of whether cannabis-based medicines are extracted from plants or produced using chemical synthesis techniques, the goal is to develop treatments that are safe and efficacious. “At GW, which is now part of Jazz Pharmaceuticals, we have a passionate belief in the potential therapeutic benefits of the cannabis plant and the possibility of creating tried and tested, regulatory approved, and modern cannabis-based medicines,” says Chris Tovey, the company’s chief operating officer.

“Whether plant-derived or synthetics, we believe patients and their clinicians deserve access to effective and well-evidenced cannabis-based medicines with an acceptable safety profile.This can only be achieved if a cannabis product, synthetic or otherwise, is subject to rigorous high-quality clinical trials and approval by medicines regulators, in the same way that all other medicines are,” Tovey stresses.

The preference for chemical synthesis or extraction of phytochemicals depends largely on the strategy of the drug developer. For a company such as Corbus Pharmaceuticals, which is focused on novel compounds that target the endocannabinoid receptors, synthesis is the only option because these compounds do not exist in nature, explains the company’s CEO Yuval Cohen. “For us, chemical synthesis offers the same advantage that it offers all pharmaceutical products: high purity, high yield, and the potential to manufacture at a large scale,” he adds.

Some cannabinoids, notes Tovey, exist as two or more stereoisomers. Although these compounds have the same formula, the atoms are in a slightly different arrangement and thus the molecules can have different pharmacological effects. The stereochemistry of the phytochemicals is determined by the enzymes, or synthases, in the Cannabis plant, according to John A. MacKay, founder and CEO, Synergistic Technologies Associates. “It can be difficult to synthesize specific stereoisomers with high stereoselectivity,” he says. The earliest synthesis of stereospecific isomers was achieved in the early 1940s by Roger Adams at the University of Illinois in cooperation with the Treasury Department Narcotics Laboratory (2,3).

As a result, due to the differences in the stereochemistry of plant-derived and synthetic versions of the same target cannabinoids, the extracted and synthesized products can have different chemical profiles (4,5). The approach to manufacturing candidates may therefore be dictated by the desired properties of the product.

“While we have, to date, chosen to focus on plant-derived cannabinoids and cannabis-based medicines, we cannot say whether there are particular advantages or disadvantages to a plant-derived or synthetic medicine, except that the chemical profiles of these cannabinoids are almost certain to be different, coming from different manufacturing processes,” concludes Tovey.

In certain cases, the composition of the plant and, therefore, the plant-derived medicine may be advantageous in treating a specific patient population. However, Tovey points out that further clarity on this hypothesis is needed and can only be gained through the high-quality research GW and other responsible organizations are conducting in both plant-derived and synthetic forms of cannabis-based medicines.

The biggest challenge with isolation of phytochemicals is the number of diverse compounds in the natural plant, MacKay observes. Techniques for extraction of natural cannabinoids have improved during the past decade, with advances occurring at an accelerated rate outside of the United States where research on these compounds can be pursued with fewer restrictions, he comments. A review of recent developments can be found in reference (6). “One of the critical factors of this increased success has been adherence to scientific principles and access to modern tools,” he adds.

For those cannabinoids that are produced via chemical synthesis, Cohen emphasizes that there are no unusual challenges to synthesizing novel compounds targeting the endocannabinoid system that are not faced during chemical synthesis of other complex small-molecule drug substances. As a rule of thumb, he mentions that this class of compounds tends to have solubility issues, but they are addressed with formulation and are generally manageable.

Cohen goes on to remark that, “Corbus’ hypothesis is that for the purposes of cannabinoid-related medical therapeutics (in contrast to the ‘wellness’ sector), it is imperative to have intellectual property protection and potency that is high and targeted. We believe that only rationally designed, synthetic, novel small molecules can achieve that.”

For phytochemicals, controlling plant growth is crucial

Managing the complexity of natural compounds and the concentration levels of each that exist in Cannabis plants is an important factor in developing potential drug products based on hundreds of metabolites that are present.

GW, according to Tovey, has designed its growing protocols and manufacturing facilities to ensure the highest quality of medicines with the goal of achieving consistent, standardized processes and products that comply with required regulations.

As an example, GW has invested in state-of-the-art facilities that include high-tech glasshouses that allow the company to control the entire growing environment for its plants, including the temperature, light, air quality, and moisture levels. Using lights and blinds in the glasshouses, it is possible to artificially adjust light exposure to ensure that plants rapidly reach an optimal size and control their flowering time, Tovey explains.

Whether cannabinoids are isolated from plants or produced chemically, careful analysis is required to ensure that the right molecules are included in the final product. With such complex matrices coming from the Cannabis plant and the potential to produce complex mixtures of various structural and stereoisomers during chemical synthesis, it is essential to use an array of complementary, advanced bioanalytical tools to ensure full characterization as well as to gain insights into their bioactivities and medicinal properties and conduct risk assessments (1).

Nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS) methods have become increasingly useful in metabolomics analyses of cannabis-derived matrices (1,7). In addition, for phytochemicals, the modern analytical and concentration instruments used for natural product processing have application in natural cannabinoid product development, according to MacKay.

Supercritical fluid chromatography is, MacKay says, also well-suited to the challenge. “This technique allows high resolution of different compounds based on minor chemical properties differences. It is also used for the separation of enantiomers and diastereomers and is an essential medicinal tool for understanding enantiomeric excess.” He points to two papers as good examples of work done in this area (8,9).

Advanced analytics also facilitate structure-property relationship studies during development of cannabinoids with increased bioactivity and bioavailability. “Minor changes in the length of a carbon chain substituent or the addition of a hydroxyl group or hydrogenation of an unsaturated functionality can have different effects on the metabolism of cannabinoids. Analytical tools that can detect minor differences in chemical structures and different responses in the body are needed to perform these evaluations—a challenge that exists in drug development across all classes of compounds,” MacKay observes.

As previously alluded to, in the US, compounds that target the endocannabinoid system often fall under the auspices of the Controlled Substances Act (CSA). This issue is the biggest challenge to developing rationally designed cannabinoid derivatives, Cohen contends. “For compounds covered by the CSA, there are limited choices with respect to US-based contract research organizations (CROs) and contract development and manufacturing organizations (CDMOs) that have the permits necessary to handle and synthesize Schedule 1 compounds,” he explains. One way to avoid this problem is to use non-US-based service providers, because they typically do not have these issues.

It is important to remember that the cannabinoids being developed as therapeutic drug candidates are not the same as products developed for direct sale to consumers, whether those are CBD-based gummies or brownies containing Δ9-THC that are sold for medicinal use. The former must undergo the same rigorous development and approval process—whether plant-derived or synthetic—that is required for all regulated drug products.

“We believe that it is important to stress the difference between regulatory-approved cannabis-based medicines and other products that have not been through regulatory approvals, as non-regulatory approved cannabis-based products lack all or many of the important characteristics of a modern medicine since they are not rigorously tested and have not been scrutinised by regulators—we do not know their effect, side effects, correct dose, potential harm, and toxicology, amongst many other things,” Tovey notes.

Cannabinoids have been shown to have a wide range of pharmacological effects, including psychotropic, analgesic, anticancer, anti-inflammatory, antioxidant, antidiabetic, anticonvulsive, anti-glutamatergic, antibacterial and antifungal, and so on.

Consequently, the range of diseases for which cannabinoid-based drugs in development are targeting is expansive, and includes multiple sclerosis, scleroderma, Parkinson’s Disease, Huntington’s disease, cystic fibrosis, dermatomyositis, and systemic lupus erythematosus, and glaucoma, among others (10).

In addition to the few cannabinoids approved by FDA, there are several others progressing through the clinic. Corbus is developing synthetic candidates that target chronic inflammatory diseases that involve dysregulation of the immune system. Its lead candidate is lenabasum, which by binding to and activating the CB2 receptors on immune cells down-regulates the immune response and upregulates anti-inflammatory activity. This candidate is currently in a Phase III clinical trial in cystic fibrosis and Phase II study for systemic lupus erythematosus.

Emerald Health Pharmaceutical’s candidate EHP-101 for the treatment of systemic sclerosis completed a Phase I study in September 2019 and entered a Phase IIa study (delayed by COVID-19) in 2020. The company is also initiating a Phase II study in multiple sclerosis. The compound, an aminoquinone derivative of CBD, interacts with CB2 receptors and peroxisome proliferator-activated receptor γ (PPARγ), which is associated with neuroprotective effects (11). Emerald is also in preclinical development of a CBG derivative (EHP-102) for the treatment of brain disorders that affects pathways other than those that involve CB2 receptors.

Other cannabinoid derivatives in early development (preclinical or earlier) as potential therapeutics include SBI-100, previously THC-Val-HS (THCVHS, THC-valine hemisuccinate) and CBD-Val-HS (CBDVHS) from Skye Bioscience for glaucoma and possibly ocular, analgesic, neurologic, and anti-infective applications, respectively. SBI-100 is a Schedule 1 controlled substance, while CBDVHS is not. The addition of the valine hemisuccinate moiety helps improve the water solubility of THC and reduces the euphoria and intoxication associated with this psychoactive substance, according to a company spokesperson. The company is exploring the use of SBI-100 in eye drop formulations for the treatment of glaucoma and expects to complete its Phase I clinical trial evaluating the safety and tolerability of this candidate by mid-year. A Phase II clinical trial is anticipated to initiate there shortly after.

KLS-13019 from Neuropathix (previously Kannalife Sciences), a derivative of CBD designed to be more water soluble and thus more orally bioavailable, is intended for the treatment of certain types of pain and possibly chronic neurodegenerative diseases. In September 2021, Neuropathix received a $2.97 million Phase 2 STTR Study Grant from the National Institute of Neurological Disorders and Stroke (NINDS) of the National Institutes of Health (NIH) to support the further development of KLS-13019 for chronic and neuropathic pain, specifically chemotherapy-induced peripheral neuropathy (12).

K. A. Aliferis and D. Bernard-Perron, “Cannabinomics: Application of Metabolomics in Cannabis (Cannabis sativa L.) Research and Development,” 

C. Schaffer, “Medical Cannabis Pioneers Stake Their Claims,” Genetic Engineering News, March 1, 2020.

Neuropahtix, Inc., “Neuropathix, Inc. Wholly Owned Subsidiary Kannalife Sciences, Inc. Awarded $2.97 Million Phase 2 Study Grant from National Institute of Neurological Disorders and Stroke (NINDS),” Press Release, September 28, 2021.


Volume 46, Number 2
February 2022
Page: 22–25

When referring to this article, please cite it as C. Challener, “Synthesis, Extraction, and Purification of Cannabinoids,” 

Articles

Interest in natural phytochemicals or synthetic derivatives of cannabinoids has risen.

Synthesis, Extraction, and Purification of Cannabinoids

Multispecific antibodies are of interest to drug developers because they provide an avenue for achieving more effective or safer treatments for a wider range of diseases. Those attributes result from their ability to simultaneously target or engage multiple protein targets. Unfortunately, a consequence of this enhanced capability is greater structural complexity, which is often accompanied by greater numbers of charge and structural variants and potentially a wider range of post-translational modifications (PTMs). Drug developers need to obtain deep understanding of these highly complex biomolecules as early as possible in the drug development process to avoid investing time and cost into nonviable candidates. Analytical techniques that provide better information, earlier, are needed to facilitate multispecific drug development.

Greater complexity than traditional monoclonal antibodies

Multispecifics are made up of similar domain structures as monoclonal antibodies (mAbs). They are more complex, however, and that complexity is increasing as antibody developers seek to tailor binding and half-life to specific biological requirements while also addressing steric considerations for multiple target engagement, according to Donmienne Leung, head of protein engineering at Absolute Antibody.

The challenge, says Susan Darling, senior director of capillary electrophoresis and biopharma product management and marketing at SCIEX, is that there are many more variants associated with these complex structures.

“Standard mAbs have identical heavy and light chains, but there are charge and sequence variants and many different post-translational modifications that afford many different isoforms. For multispecifics, the number of possible structural variants is greater and depends on how many different components are being combined together. Each one of those structure variants has its own range of isoforms. That makes the analysis of multispecifics exponentially more complicated,” she explains.

If multispecifics are produced with stochastic expression systems, inappropriate association of the different chains may occur. That makes even size assays for identifying intact mAbs inapplicable, because the different structural combinations may have the same size, but only the correctly assembled molecule will exhibit the desired activity. All of the rest are considered impurities. In addition, the greater structural complexity of multispecifics may result in a greater tendency to misfold and aggregate, comments Leung.

Furthermore, Darling notes that even if a strategy, such as the “knob-in-hole” approach, is used to afford multispecifics with the correct structures, the bioengineering process typically introduces more attributes to these proteins, such as different modifications, that make these biologics more complex not only structurally but from an affinity perspective.

The increased number of undesired side products may potentially make purification of the requisite therapeutic by traditional chromatography techniques more difficult, adds Leung. “Multiple chromatography steps and mass spectrometry (MS) become necessary for full characterization of the multispecific. The evaluation of multiple target binding will also be less straightforward when the geometry of the multispecifics and accessibility of each binding domain can be highly dependent on assay formats,” she states.

Similar characterization needs, plus much more

That greater complexity means that for multispecifics, all of the characterization and other assays required for monospecifics are required, plus many more. Specifically, it is crucial to confirm the correct folding and intramolecular pairing of various domains. As an example, Leung highlights the need to confirm the correct pairing of heavy chain to light chain for each specific antibody arm to retain affinity to the required target of interest.

“We can no longer assume that two heavy and two light chains will come together in a symmetrical molecule,” Darling agrees. “Many different combinations with minimal mass differences can be generated depending on whether the platform involves stochastic expression or not,” she says.

In addition, while the mispairing of both the heavy and light chains is often of critical importance for the potency and safety of mAb-based bispecific formats, it may not be a factor for other formats, observes James Graham, director of protein and process analytics within the Bio R&D group at Lonza. “Even though similar methods can be used, the range of multispecific formats means that ‘new’ attributes are specific to the individual format for the protein rather than broadly relevant for all multispecifics,” he notes.

Multispecifics built using Fc and antibody fragments, Leung continues, can be purified and analyzed similarly to traditional antibodies and proteins using affinity chromatography (Protein A, nickel, etc.). “The purity and homogeneity of multispecifics can be achieved and evaluated by a combination of chromatography (e.g., hydrophobic interaction, ion exchange, and size exclusion) and MS similar to traditional biologics, while PTMs on recombinant proteins and multispecifics are typically characterized by mass spectrometry,” she says.

Assumptions made for immunoglobulin (IgG) monospecifics don’t apply to multispecifics, Darling adds. “The assumption for monospecific IgGs that there will be Fc and target binding affinities is also no longer applicable. Two or three different binding affinities can be competitive against different targets,” she contends. In addition, she notes that there are often modifications in multispecifics that are not observed for mAbs because of the way the cell lines used to produce these biomolecules are engineered. There can be additional PTMs as well, and in particular more variability in disulfide bonds.

It is important to remember, though, Graham underscores, that mAbs themselves are already multispecifics, so there already is a good baseline in terms of knowing what needs to be monitored. “In addition to established attributes we monitor during mAb development (e.g., safety, purity, potency, and the range of product variants such as glycosylation), it is the specific modifications that have been made from the established mAb framework that need special attention. These may vary substantially depending on the protein architecture,” he concludes.

All of these variations, Darling asserts, can impact binding affinities and thereby make many other assumptions about monospecific IgGs also nonrelevant for multispecifics. “There can be different cross-reactivities, different catabolite forms, and different isoforms generated during the biotransformation process that can all have modified binding capacities. Simple ligand binding assays are no longer sufficient,” she says.

Leung also stresses that in addition to all of these issues, it is important to remember that multispecifics must be monomeric, soluble, and stable in nature to enable clinical development and downstream commercialization.

From a characterization standpoint, the big challenge for multispecifics is the increased possibility for greater higher-order-structure variability, according to Darling. “The molecular asymmetry and potential for multiple product-related impurities based on the combinations of chains being expressed is the source of this variability,” Graham notes.

The fact that protein structures, including multispecifics, when in solution are dynamic in nature with flexible structural isomers and potentially aggregated molecules further complicates bioanalysis, according to Leung. “Temperature can also influence the overall stability of the correctly folded molecule and the respective domains. Moreover, the surface interactions between molecules and between domains may have significant impact on self-association,” she comments.

Where there are significant molecular weight differences between potential product variants, Graham adds that the assessment of aggregation can become challenging because a high-molecular-weight species could represent a protein aggregate or a mispairing variant.

This variability can also create hurdles during process optimization and scale up once a multispecific product is defined and an investigational new drug application (IND) has been filed. “With multispecifics, there simply are more chances because of the higher variability that any changes to the process will lead to the formation of the wrong structures and/or isoforms. That means the risk is greater. It also means that many more samples must be evaluated in a way that provides detailed information very quickly,” Darling says.

It is generally necessary, observes Darling, to use a combination of intact mass measurements and characterization with reductive techniques such as digestion prior to subunit analysis or peptide-level analysis to identify disulfide bonds.

A key trend in the biopharma industry affecting all drug development is the desire to compress development timelines down to just a few years. “Companies are looking to get development times down to 1000 days; they want to develop everything at pandemic speed, even when they have diverse portfolios that include highly complex next-generation products such as multispecifics, gene and cell therapies, and RNA-based treatments, all of which require new and different analytical capabilities,” Darling asserts.

The result is a unique situation in which a usually risk-averse industry, because it is rapidly moving to more complicated molecules, needs access to newer, perhaps initially slightly more experimental technologies. “The industry is really driving to be creative, not just in terms of analytical solutions, but in how they manage data and pull it together to knock the inefficiencies out of every step,” Darling concludes. “Our customers are challenging us to develop new analytical techniques that provide more information more quickly, and when we bring them new solutions, they are adopting them at a much faster rate than could ever be imagined even a decade ago,” she says.

The reason for this transformation within the industry is a new focus on the developability of biologics. “Developability is increasingly critical for downstream commercialization,” Leung states. Darling agrees. She says that the strategy of failing fast and early has been increasingly adopted at an accelerating rate over the past few years, with companies looking for technologies that enable more detailed characterization and stability assessments early on.

“Simply selecting the highest-yielding cell line without considering the product quality is no longer the typical approach. Developers want to find the right cell line and clone that produces a high yield of the desired product—mAb or multispecific—with the right structure and modifications from the outset to avoid costly late-stage failures,” Darling explains.

For instance, Leung points to the use of various 

 and pre-clinical screening techniques for the evaluation of non-specific binding, self-association, and chemical stability as means for identifying problematic molecules early on. She also notes that while sequence analysis and 

 assays have been recognized for the pre-clinical prediction of immunogenicity risks associated with traditional mAbs, these techniques have not been correlated for multispecifics with confidence.

Developers are also looking for high-throughput, highly informative, easy-to-use analytical solutions that support this strategy. For multispecifics, one of the challenges is the inability to apply platform analytical strategies because of the variety of formats. Product-specific methods become critical, and these methods are required early in development, according to Graham. “As well as ensuring that method performance is sufficient to monitor the attribute in question, throughput and turnaround times must be compatible with screening stages during cell line and process development,” he says.

Orthogonal approach to analysis essential

Comprehensive characterization of multispecifics, because these biomolecules are so complex, generally requires the use of several orthogonal methods. In addition to traditional antibody assays, many other methods are being explored.

“There are many different attributes of multispecifics that need to be determined, and because this type of biologic is relatively new, there is still work to be done to determine which methods are best,” Darling observes. There is a need, she adds, for iteration and customization of technologies to determine which will be most impactful and robust. As a result, multispecific developers are leveraging orthogonality, using multiple assays to confirm results and build more confidence in their data.

For instance, in addition to leveraging established techniques such as ultra-high performance liquid chromatography (UHPLC), capillary electrophoresis (CE), surface plasmon resonance (SPR), MS, and automated cell-based analysis platforms, techniques that can easily identify peaks within a profile are crucial, according to Graham. Native MS is one such technology that can be used to generate peak identity information in parallel to the primary analysis method, rather than having to perform subsequent follow-on testing. “Native MS is particularly interesting due to the information density of the technique and the fact that non-covalent variants or impurities may be present for some multispecific formats that can be challenging to characterize using other methods,” he remarks.

In general, adds Leung, a combination of high-resolution LC and MS techniques are necessary to fully characterize multispecifics. Thermal stability analyses such as differential scanning calorimetry (DSC) and differential scanning fluorimetry (DSF) allow developers to select for better-behaved molecules. CE and other electrophoretic techniques such as isoelectric focusing (IEF) and capillary IEF (cIEF) have improved resolution of traditional sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) methods in elucidating the correct pairing of polypeptide chains in multispecifics.

Intact mass analysis using Orbitrap-based LC-MS can resolve all theoretically expected species in a complex mixture, while using electrospray ionization time-of-flight (ESI-TOF) MS on quadrupole instruments enables quantification of various species with improved sensitivity and robustness.

While target binding can be confirmed using simple enzyme linked immunosorbent assay (ELISA) methods, biochemical characterization of multispecifics requires detailed kinetic analysis to compare the integrity of the final product. Association and dissociation phases of molecular interactions can be measured by established techniques including SPR, biolayer interferometry (BLI), and isothermal titration calorimetry (ITC).

Ultimately, though, Leung concludes that cell-based assays are necessary to confirm target engagement and the desired functional activity of the multispecific.

With so many options for analyses to perform, it can be quite challenging to determine which methods should be used for a given multispecific. Lonza has found that having a template library of different analytical methods based on the chemistry of the therapeutic protein provides a starting point for method development. “When combined with a design-of-experiment approach, development cycle for the deployment of a new method can be greatly streamlined and development times reduced from weeks to days or less!” Graham avers.

New tools for accelerating multispecific development

With an overall goal of accelerating biopharma development, SCIEX has focused on meeting the needs for easier-to-use MS solutions that provide rapid, detailed results. In 2021, the company introduced the accurate mass ZenoTOF 7600 system, which leverages both Zeno trap with electron activated dissociation (EAD) fragmentation technology.

In traditional TOF instruments, Darling explains, the fragment ions arrive at the pulser region with a spread of velocities, so only a small percentage of the ion population gets accelerated into the flight tube. This duty cycle problem is overcome because the Zeno trap enables all of the ions to arrive at the TOF pulser at the same time independent of their masses. More than 90% of the ions are injected, resulting in significantly improved MS/MS ion sampling for all m/z and greater sensitivity for MS/MS and high-resolution multi-reaction monitoring (MRM) acquisition scan modes and greater ion fragment coverage.

EAD, meanwhile, provides for generation of electrons with tunable velocities and allows for different fragmentation mechanisms, including the hot ECD mechanisms ideal for peptide mapping of glycopeptides and disulfide-bonded peptides, according to Darling.

Importantly, EAD preserves valuable side chain information, such as for labile PTMs very common in multispecifics, that enables confirmation of various isomers and affords excellent MS/MS coverage for all charge states in one experiment. “We are finding real interest in this technology because it allows for much easier and more robust characterization of even highly complex molecules. In fact, for the first time it could be possible that electron dissociation could become the go-to-choice for fragmentation,” Darling asserts.

Another advance in MS technology is in the works at SCIEX and is based on the direct coupling of cIEF with MS. Initially developed by IntaBio, which SCIEX acquired in early 2021, the system enables high-resolution separation of charge variants combined with detailed MS data for each variant. “The combination of isoelectric point directly coupled with MS data allows for confident identity assignment during intact analysis in much less time than is required for conventional peptide mapping analyses,” Darling notes. The company expects to launch the system sometime in 2022.

On the CE front, SCIEX has introduced in 2021 a new system that allows for much higher throughput analysis. “The BioPhase 8800 system leverages a new cartridge that allows parallel processing of eight different CE samples (same or different molecules) simultaneously using CE-SDS and/or cIEF with UV or LIF detection, providing all the advantages of CE while delivering consistent, accurate results so that more samples can be analyzed in less time,” Darling explains.

Importantly, methods developed for SCIEX’s widely used PA 800 system can be transferred to the BioPhase 8800, avoiding any need to do bridging studies as projects move to late-stage development and commercialization. “That can save weeks to months of time,” Darling says. “In addition,” she emphasizes, “companies can rapidly perform design-of-experiment studies to quickly optimize both assays and manufacturing processes.”

More complex molecules and the need to perform more orthogonal analyses means that more samples must be prepared and much larger data sets are generated that must be analyzed. Automation of both of these aspects of multispecific analysis is becoming another crucial strategy for speeding up the development process. “Biopharma companies are looking for any opportunities to remove unnecessary manual processing, whether with respect to sample preparation or data processing,” comments Darling. That includes investing in more specific software products, many of which leverage artificial intelligence and machine learning, to help with data integration.

While standardizing assays for multispecifics is not possible given the unique nature of each of these biologics, automation of sample preparation and data acquisition and analysis can help simplify these solutions, Darling insists. High-throughput data analysis platforms, agrees Graham, are critical to the deployment of newer analytical methods for multispecifics into process development, because otherwise the data analysis time quickly dominates the overall time to result.

The challenges associated with multispecific analysis, while real, are not so great that the bioanalytical experts with experience in biologics development cannot manage them. “Multispecific analysis is definitely an interesting field, but it should not be considered a major risk,” Graham asserts. “Rather, analytical development for these complex biomolecules should be approached with a structured but flexible workflow combined with a good understanding of each molecule’s chemistry,” he concludes. Having multiple answers on hand for multiple multispecifics works!

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

The multispecific analysis of biologics is a complex task that requires appropriate strategies.

Multispecific Analyses of Biologics Pose Multiple Challenges

Medication adherence is a crucial issue today, and drug manufacturers are seeking to develop drug products that meet the needs of patients with respect to ease of use and palatability. For oral formulations, taste, appearance, and smell have a direct impact on medication adherence. Choosing the right excipients to provide optimum performance is therefore an important part of the drug development process. Various factors must be considered, from the API properties to the dosage form and the characteristics of the patient population.

Multiple approaches for different dosage forms

Oral dosage forms available today include traditional and fast-disintegrating tablets; capsules; chewables and gummies; lozenges and other suckable products; effervescent, buccal, sublingual, and thin-film formulations; suspensions, syrups, sprays, gels, sachets, stickpacks, and chewing gum; among others. The large number of different formulation types creates the need for many different approaches to improving taste and appearance. For solid formulations designed to be swallowed, particularly tablets, taste-masking and appearance are generally achieved using film coatings, according to Alen Guy, technical director IMCD Pharmaceuticals.

Such coatings prevent the API from being dissolved in saliva and interacting with the taste buds in the mouth before the drug product is swallowed, adds Nasrin Mahmoudi, AD&I scientist, Pharma Solutions with IFF. She also notes that coatings can help with appearance by providing a shiny, matte, or colorful look and hiding undesirable surface properties.

Capsule formulations are a little different from tablets. “Capsules generally don’t require taste masking unless they are designed to be opened and sprinkled onto a food or poured into a drink. Appearance is important for this oral dosage form, however, and color and design are often used to distinguish different products,” he observes.

For oral dosage products in which a bitter API does come in contact with saliva and taste buds in the mouth, different sweeteners are often used depending on the dosage form, Guy says. For instance, Elizabeth Tocce, AD&I scientist, Pharma Solutions at IFF, points to the use of sweeteners, often in combination with texture modifiers, in orally dissolving tablets (ODT), chewable tablets, oral thin films, and sachet formulations that stay longer in the mouth.

“Taste modification using flavors and other additives is more complicated, though,” Guy comments. Even so, it is often needed. “In many cases, and for very bitter high-dose drugs in particular, just changing the organoleptic properties is not sufficient, and other methods to create a physical or chemical barrier around the drug particles, such as microencapsulation and complexation, are used,” Tocce explains.

The method employed largely depends on the API characteristics and the intended dosage form, agrees Krizia M. Karry, head of global technical marketing for BASF Pharma Solutions. “Poorly water-soluble APIs with adequate dissolution rates (i.e., BCS [Biopharmaceutics Classification System] II APIs) tend to be micronized and compressed into tablets. At low dosages, adding flavors or sweeteners can suffice to ensure palatability as most tablets are not meant to disintegrate in the oral cavity, but flavors will not suppress bitter tastes,” she says. At high drug loadings and for orodispersible products, Karry adds that film coating with methacrylate copolymers that are insoluble at saliva pH is the most effective technology.

If the unpleasant taste is related to grittiness or chalkiness in chewable tables, ODTs, or suspensions, a common method is to use finer grades of the APIs and excipients, according to Tocce.

Liquid prescription drugs are particularly popular in the EMEA (Europe, the Middle East and Africa) market, where there is a preference for the dosing flexibility and a perception that they offer rapid relief, according to Karry. In North America, meanwhile, liquids are mostly offered for over-the-counter cold and flu medications. In most of these liquid formulations, she observes that highly concentrated sweeteners and flavors are typically employed to mask the bitter API taste. In formulations where multiple APIs are combined including some that are bitter or where product shelf-life is of concern, however, Karry observes that insoluble polymers are used to envelop API particles and allow their suspension in the continuous liquid phase.

For liquids, other options if sweeteners aren’t sufficient, in addition to microencapsulation and complexation, include using an insoluble form of the API and adjusting the pH of the solution to lower the API solubility, according to Mahmoudi. She adds that viscosity enhancers and texture modifiers are also used. Alternatively, taste and appearance issues with liquid formulations can be overcome by containing them in soft or hard capsules.

Many different types of excipients are used to improve taste and appearance. Because the most common taste challenge is the bitterness of APIs, sweeteners, both natural and artificial, are the most commonly used taste-modifying excipients. Beyond compounds such as sugar, mannitol and sucralose, actual flavoring agents (orange, mint, etc.), flavor-maskers, particularly bitter-maskers, texture modifiers (co-processed microcrystalline cellulose and guar gum), and polyols are used. Complexing agents such as cyclodextrins and ion-exchange resins are often used for improving the taste of liquid formulations and orally dissolving dosage forms, Mahmoudi states.

“Depending on the formulation, different approaches to taste and flavor modification are needed,” Guy says. In fast-dissolve forms, short intense flavor bursts are needed, while in longer lasting products such as chewables, longer-lasting flavor effects are needed.

Hydrophobic (ethyl cellulose) or hydrophilic (hydroxypropyl methylcellulose, polyvinylpyrrolidone) polymers, lipids, and sweeteners can be used as coating materials, alone or in combinations, to modify both taste and appearance, Mahmoudi adds.

In addition to preventing an unpleasant taste, Guy notes that some coatings may also include pore-formers to help promote dissolution of the drug substance to meet standard immediate-release requirements for the API. “In such formulations, a careful balance must be achieved between taste-masking and efficacy. It is therefore a rich area of development given the complexity of drug particles and granules, varying solubilities, the need to often achieve a specific location of delivery (enteric needs for instance), and excipient compatibility concerns,” he comments.

For appearance, color is most widely used to differentiate products and mask undesirable product properties. For solid dosage forms, color is applied within coatings. For liquids, water-soluble dyes are generally employed.

Ultimately, Guy contends that the choice of excipients for taste and appearance improvement comes down to the preferred dosage form and the external factors that can impact the effectiveness of the choice, which include excipient compatibility, resistance to mechanical influence (granulation and tableting), solubility, stability, etc.

Impact of need for patient-centric formulations

Because taste and appearance are important factors influencing the patient experience, it is essential for formulators to understand the preferences of different patient populations for drug formats and, within those preferred formats, taste and appearance qualities that will encourage good medication compliance. “Different patient groups based on their ages, disease states, cultures, and demographic locations may require certain taste, flavor, texture/mouthfeel, and drug product appearance properties, such as tablet size, color, and shape for solid products and the viscosity and grittiness of liquid formulations,” Tocce asserts.

Flavor companies, adds Guy, are today much more attuned to patient and consumer preferences by gender, age, and other factors. “This information can and does help formulators and brand/product managers to make smarter decisions for taste and appearance as it relates to the intended patient or therapeutic group,” he says.

In particular, Guy notes that the need to create fit-for-purpose formulations for aging populations and pediatric patients has put pressure on appearance as it relates to the size of oral solid dosage forms. For instance, important research conducted by academics and members of the European Pediatric Formulation Initiative has revealed that young children are more accepting of small or mini-tablets.

“Such a patient-centric approach definitely benefits patients, but it does present manufacturing challenges,” Guy states. “For example, it can be difficult when producing tablets that are 2 mm in diameter or smaller to achieve good flow through production equipment for APIs and excipients, which in turn impacts the ability to achieve good content uniformity and acceptable taste,” he says.

From a personalized medicine standpoint, it is also important to understand specific age-related elements of physical condition that might impact drug performance and the appropriate excipient choices. “For pediatric medicines, factors to be considered include the fat and water content along with relevant genetic information. It is a tricky area from a regulatory standpoint, but it does help guide the selection of excipients by taking into consideration their potential impacts on young developing bodies,” observes Guy.

Age is indeed one of the most important factors guiding excipient selection. If a medication is intended to treat a wide patient age range, Guy recommends developing modified versions formulated for subsets of the population based on reasonable age groups, such as pediatric through adult and then elderly or long-term care patients.

The first consideration, though, Guy emphasizes, is therapeutic benefit and efficacy, followed by safety, ease of use for the patient, and manufacturability. “We need to understand the target drug product profile when selecting a taste-modifying agent/approach to ensure drug performance is not negatively affected,” Mahmoudi agrees. The characteristics of the API and the desired properties of the final product directly impact excipient selection, she states.

The extent and type of unpleasant taste associated with the API (bitter, sour, irritating) and its aqueous solubility determine which excipients will be appropriate. “It can be more challenging to cover the poor taste of a highly water-soluble API than that of a less-water-soluble active,” Mahmoudi says.

In addition, the particle size and shape of the API can influence the approach for taste-masking and appearance improvement. Specifically, Mahmoudi notes that spherical particles are easier to coat than irregularly shaped particles. The dose must also be considered; it is usually easier to taste-mask a low-dose formulation.

In film-coating approaches, adds Karry, the formulator should ensure complete drug solubilization and absorption to achieve optimum bioavailability. “Doing so is especially important for reverse-enteric polymers, as they should quickly dissolve in the acidic stomach media to allow drug diffusion out of the coated core and absorption in the small intestine. Fast polymer solubilization is critical because the tablets and pellets have a short residence time in the stomach, and these polymers are not soluble in non-acidic media. In other words, if the film does not dissolve in the stomach, it will not dissolve anywhere else in the body and as such, the drug will not diffuse out of the protected core,” she explains.

Appearance factors such as shape and color can play a role in determining the correct identification and/or correct dosing material (syringe or spoon, etc.). In some cases, a certain format can address appearance concerns. For instance, Guy points to single-unit doses such as stickpacks as a potential means for enhancing identification through improved naming and ‘branding’ of the unit itself. He does stress, however, that care must be taken to ensure that these adult/senior-friendly products are child-resistant.

Experience and knowledge of patients best guides

For some excipients, such as those designed to improve API solubility, formulators have developed predictive algorithms that facilitate more rapid formulation development. Taste and appearance are more challenging, however, because often these qualities vary from one individual to another.

“Structuring data from humans and how they respond takes a great many data points and statistical analysis and remains subject to less-well-understood error knowledge. While augmented intelligence approaches can certainly help formulators make better choices sooner rather than later, there are too many unknowns, and the need for structured data is too overwhelming for good models to work at this point,” says Guy.

The fact that there is no one method that can address taste and appearance properties in every oral medication makes predictive approaches difficult, Mahmoudi agrees. She does note, however, that evaluating literature reports and patient and caregiver surveys has revealed some general “dos and don’ts” that should be followed to achieve desirable taste and drug appearance.

For example, Mahmoudi says that the use of a coating on tablets is preferred among older patients because it can help to optimize both medication identification and taste. “Elderly patients generally take multiple medications, and having tablets that are visually appealing and easy to identify and handle, such as with a score line for easy breakage and optimized dimensions and shapes that address dexterity issues, is important,” she explains.“There are more preferences towards brightly colored tablets and identification based on shape and color for different indications to avoid medication errors,” she adds.

Karry disagrees with Guy and Tocce with regard to the ability to predict bitterness and believes the process for identifying if an API is bitter has rapidly progressed in the past few years. She points to BitterDB, a free database available to formulators with more than 1000 naturally bitter and synthetic compounds and the use of machine learning algorithms trained with known bitter API molecules that can predict the bitterness of new compounds. “Based on these predictions and additional physiochemical API properties, pharmaceutical formulators can proactively plan for their choice of taste-masking technology,” Karry contends.

The use of electronic tongues, however, has not been successful to date, says Karry, and the process to test and optimize taste-masked formulations continues to be reliant on trained human panels. E-tongues are expensive instruments based on capacitance sensors that are used to rank the bitterness of formulations, but have been shown to exhibit no correlation to the results obtained by human taste panels (1).

The use of human test panels presents its own set of challenges, including a new one that has recently arisen. A study published in 2017 by Dagan-Wiener et al. showed that 66% of drugs in clinical and experimental stages at the time were classified as bitter compounds (2). The situation remains the same today, according to Karry. Many of the drug candidates under development today, however, are different because they have much smaller differences between their therapeutic and toxic doses (NTI-drugs), she says. “For these drugs, recruiting healthy volunteers for taste panels and ensuring no side effects after evaluation of test formulations has become more complicated,” Karry remarks.

Currently, conventional film coating and the addition of sugars, flavors, and sweeteners remain the common approaches to taste masking, but numerous other taste-masking techniques have been developed to address this challenging task, according to Mahmoudi. “Microencapsulation, high shear mixing, freeze-drying, fluid-bed coating, and spray drying have been successfully used to modify taste and making various dosage forms,” she says.

In fact, Karry notes that there is a move away from the use of flavors and sweeteners to mask bitterness because studies have shown that sucrose-sweetened medicines are associated with increased dental issues in children. Addition of acids to generate sour liquid formulations rather than bitter medicines, meanwhile, affects tooth enamel and causes dental erosion.

“For these reasons,” Karry says, “film-coating approaches are increasingly used for taste masking of bitter compounds. Commonly used polymers include methacrylate copolymers such as Kollicoat Smartseal or Eudragit E-PO since they are insoluble in saliva (pH 6.2 –7.4), but readily soluble in the stomach.”

Hot-melt extrusion and 3D printing for personalized medicines are emerging pharmaceutical technologies that Mahmoudi believes can be efficiently used to mask the taste of very bitter APIs. In particular, Mahmoudi notes that 3D printing is a powerful technique with the potential to enable the design of visually appealing tablets with modified taste.

With respect to advances in excipient technology, Mahmoudi points to the introduction of several taste-masking polymers with pH-dependent and independent solubility, new excipients such as co-processed microcrystalline cellulose (MCC) and guar gum, colloidal MCC, and many different flavors and taste-masking agents developed by excipient manufacturers as being tremendously beneficial in modifying taste and drug appearance.

For Guy, the use of taste-masking agents as ingredients, rather than simply applying a coating as a physical barrier, is transforming flavor and flavor perception. “Such an approach has allowed us to tackle ‘off-notes’ in products, particularly in nutraceutical formulations. Specifically, combining bitter-maskers with an appropriate flavor combination helps to reduce or even eliminate off-notes to a background level that is tolerable,” he says.

Orally disintegrating and dispersible solid-dose formulation have increased in popularity in recent years, creating challenges for taste masking and appearance. “It can be difficult to navigate the possible excipient choices and strategies that will ensure the desired product performance, for which there are numerous requirements and constraints,” Guy observes. “Facing challenges is not unfamiliar territory, though, and formulators do have an extensive and growing toolbox of excipients that can be applied for these and other novel oral dose forms,” he concludes.

When formulating any type of drug and using excipients of any kind, including
for the modification of taste and appearance, the safety, regulatory acceptance, and daily allowable intake of each excipient must clearly be considered in addition to process and cost, according to Mahmoudi. “Beyond that, patient-centric drug design is paramount. There are great taste-masking excipients/texture modifiers and techniques that should be considered from the early formulation development steps when pursuing patient-centric solutions,” she says.

In addition to ensuring safety and efficacy, Guy reiterates that the most important thing formulators must do for any drug product is to ensure that the patients and care-givers that will be using/administering the medicine will not be afraid or put-off by the nature of the drug. “It is essential to have and apply good people knowledge and consider different formulation options for different patient populations as dictated by their differing needs. Patients are people, and that must always be top of mind,” he insists.

1. Senopsys, “Case Study: Correlation of Electronic Tongue (E-Tongue) and Human Taste Panel Data,” Senopsys.com, Accessed Nov. 24, 2021.

2. A. Dagan-Wiener, et al. Sci Rep 7, 12074 (2017).

Cynthia A. Challener, PhD, in contributing editor to 

 
Vol. 46, No. 1 
January 2022 
Pages: 26–27, 45

When referring to this article, please cite it as C. Challener, “Taste and Appearance: Selecting the Right Excipients,” 

Dosage form and patient needs drive excipient choice.

Taste and Appearance: Selecting the Right Excipients 

A large percentage of small-molecule APIs in development today suffer from poor solubility and bioavailability. Stabilizing these APIs in their amorphous forms can help overcome these issues because a supersaturated state is achieved upon dissolution and delivery. Such a state can result in enhanced absorption across gastrointestinal membranes and improved bioavailability, according to Lindsay Johnson, global technical marketing manager for BASF’s Pharma Solutions business.

“The high thermodynamic energy of an amorphous, supersaturated state is inherently unstable, however, and such systems are driven towards crystallization, liquid-liquid phase separation, or non-descript precipitation,” Johnson says. Tendencies to recrystallize, absorb moisture, and participate in side
reactions that generate impurities must also be overcome.

Solid dispersions lead the stabilization way

Depending on the chemical nature of API candidates, there are a variety of mechanisms that can be used to overcome solubility challenges. Amorphization of the API is one mechanism that can be pursued, observes Johnson, and commonly amorphous APIs are stabilized in amorphous solid dispersions (ASDs) formed via solvent evaporation processes such as spray drying (SD) or fluid-bed coating or melt processes such as hot-melt extrusion (HME).

These methods for stabilizing amorphous formulations are polymer-based, notes Daniel Joseph Price, strategic marketing manager, excipients for solid dosage at MilliporeSigma. “Although different processing methods are used and formulations vary, the end result is the same: a polymeric matrix, with hopefully a homogenous distribution of API throughout the polymer,” he adds. The distribution of the API in the polymer essentially immobilizes the API in the amorphous form, preventing re-crystallization.

Currently, there are 17 commercially available spray-dried dispersion amorphous drug products and 13 approved HME products (1,2). “Both of these technologies are established in the industry, easily scaled-up, and continuous,” comments Molly Adam, an R&D engineer at Lonza.

Both methods have advantages and disadvantages, too. Spray drying does not expose the API to high temperatures, but requires the use of large quantities of organic solvents and nitrogen gas and can have low throughputs, all of which make the process costly, Adam says. While extrusion has a lower footprint and a higher throughput, the process involves high temperatures and shear stresses that can degrade some APIs.

Generally speaking, the selection of amorphous formulation technologies should be guided by the glass-forming ability of the API, according to Price. “The glass-forming ability essentially describes how likely it is for a molecule to re-crystallize,” he explains. “Poor glass formers are molecules that have very poor stability in the amorphous form, and so have an inherent re-crystallization risk. It is therefore recommended to only use polymeric amorphous solid dispersions when working with good or moderate glass formers,” he continues.

The choice of spray drying or HME depends on several factors, including processing space, scale-up needs, performance drivers, and the properties of the API, says Gereint Sis, an R&D scientist with Lonza. Important physicochemical properties that drive API stabilization are the melt and glass-transition temperatures (Tm and Tg, respectively). APIs with significantly high melting points are not typically amenable to HME due to degradation, while APIs with poor solvent solubility are challenging for spray drying.

Ultimately, the amorphous system (API-polymer interactions, weight percent of API, temperature, humidity, and the region of the phase diagram the formulation occupies) will drive the extent of stabilization (3).

“The optimum selection strategy involves cooperatively conducting high-throughput, small-scale manufacturing, iterative in silico modeling, bench-top testing, polymer screening, and discriminating in vitro testing,” Sis states.The resulting optimum stabilization method should ensure performance, manufacturability, physical and chemical stability, and enable patient compliance, he adds.

One challenge, Sis observes, is the difficulty of striking a balance between these metrics while alleviating complexity and lowering costs. “Strategic and agile technology selection methodologies can help manage these issues,” he says. In addition, adding robust data sets coupled with machine learning to current selection strategies will likely ensure shorter timelines and alleviate cost burdens to drug-product-development lifecycles (4).

Common excipients include polyvinylpyrrolidones (PVPs), their copolymers, and their cross-linked derivatives, polyvinyl acetates (PVAs), and some functionalized cellulosic materials such as hydroxypropyl methylcellulose (HPMC) and hydroxypropyl methylcellulose acetate succinate (HPMCAS).

Typically, a binary API-polymer system that affords favorable API-polymer interactions to provide the stabilization is used in an ASD (3). These excipients are usually required to have specific material properties, including a relatively high Tg (usually 70 °C or higher), according to Johnson. Polymers with high Tg values are great at stabilizing amorphous APIs because they increase the Tg of the dispersion and decrease mobility, Adam remarks.

In addition to the Tg, other excipient properties to consider include the degradation temperature, solubility in organic solvents, water uptake, and typical sustainment performance during dissolution, according to Adam. She says that polymers with a wide processing temperature range are great for HME because they have a lower chance of degradation.

Poloxamers or polyethylene glycol (PEG) are also common in HME to improve processability by plasticizing the system, which decreases its melt temperature and melt viscosity. For SD, polymers with good organic solubility and low solution viscosity when dissolved are crucial to enable sufficient solubility for both the API and polymer.

Depending on the stabilizing mechanism, Johnson also notes that excipients for ASDs may contain functional groups that can participate in specific polymer-drug intermolecular interactions or have a rough solubility parameter that matches the hydrophilic/hydrophobic balance of the API in order to offer non-specific interactions that can be stabilizing.

Furthermore, polymers can aid in stabilizing the drug in gastrointestinal fluids in vivo and in the solid state during storage, according to Adam. Neutral polymers like PVP grades and HPMC can maximize dissolution rates throughout the whole range of pH of the GI tract. Enteric polymers such as HPMCAS and Evonik’s Eudragit L functional delayed release polymer can enable acid-sensitive APIs to be released in the intestine rather than the stomach if they degrade at low pH or have low solubility in the stomach.

For poor glass-forming APIs with inherent mobility, Price points out that mesoporous silica has emerged in recent years as an excellent excipient for stabilization. “The steric confinement of the API in nanosized pores after loading substantially reduces the mobility of the API in the formulation,” he explains.

Opportunities for instability still exist

Even though amorphous APIs are stabilized in ASDs, instability can remain an issue during storage and once the formulation has been administered to the patient. “The one downside to polymeric ASDs is the inherent mobility of both the polymer and the API in the formulations, which can lead to re-crystallization of poor glass-forming APIs,” Price states.

The mechanisms by which amorphous forms destabilize are phase separation and crystallization, according to Sis. “When an amorphous solid dispersion phase separates, distinct polymer-rich and API-rich amorphous phases form, which can make the amorphous API more prone to crystallization. Phase separation may or may not be a precursor to crystallization, with the rate-limiting step for crystallization being either diffusion or crystal nucleation,” he explains. Nucleation occurs when small seed crystals of the API form that then grow until a thermodynamically stable polymorph is obtained, according to Price.

Internal factors affecting the extent of stabilization of ASDs include the API loading, the polymer properties, and the drug-polymer interactions within the ASD, Sis outlines. For instance, hydroscopic excipients that absorb moisture will have negative consequences, as will formulations that contain plasticizers that increase polymer and API mobility, according to Johnson.

Environmental factors such as humidity and temperature can thus also affect the chemical and physical stability of the amorphous API within the formulation by increasing the kinetics of unwanted reaction pathways, such as degradation in the presence of water. Increased API mobility leads to a shift in the phase diagram, phase separation, and API recrystallization (3). “Smart formulation decisions and storage strategies are necessary to mitigate these risks,” Sis contends.

The behavior of the amorphous form upon dissolution in the body must also be considered, says Price. “When an amorphous API is dissolved, supersaturation generally occurs, and the API is in solution at concentrations higher than its thermodynamic solubility. Supersaturation is not a stable condition and is thermodynamically unfavorable. The API may precipitate out of solution and undergo nucleation and crystal growth, or API-rich liquid domains may separate out of solution,” he says.

Upon dissolution, observes Johnson, the stability of amorphous APIs is often segmented by their relative molecular and physiochemical properties, which will dictate which formulation approach is likely to be successful at stabilization. “In order to be amorphous and solubilized, any drug molecule needs to dissociate from its crystal lattice, and the dissolving media needs to solvate the drug molecule at the molecular level. Both of these processes require energy to either overcome drug-drug intermolecular forces or solvate the compound, respectively,” she explains.

Depending on which step is more solubility-limiting for a particular API, Johnson continues, the molecule can be referred to as a “brick dust” molecule or a “grease ball”. “Grease ball” and “brick-dust” APIs are associated with low and high melting points, respectively, and thus Tm and lipophilicity (log P) are often predictors of this categorization. In fact, Tm and log P can be used in combination with an API’s amorphous solubility to steer the selection of formulation type, according to Johnson.

“The supersaturation concentration achieved relative to that amorphous solubility is what dictates the destabilization process—whether direct crystallization that occurs below the amorphous solubility threshold or liquid-liquid phase separation that occurs above the amorphous solubility threshold,” Johnson concludes.

It is therefore important, stresses Price, to ensure not only the stability of the amorphous form in the solid state, but also stabilization of the supersaturated state upon dissolution.

Increasing interest in co-amorphous systems

Recent literature has expanded the interest in the field in co-amorphous systems, specifically ones that are not cocrystal forming. These co-amorphous systems include the API and a low-molecular-weight molecule that may be another pharmacologically relevant API or an excipient, Johnson observes.She adds that such formulations are commonly produced via mechanical milling, melt quenching, or solvent evaporation.

As an example, Price points to the spray drying of a mixture of an API with an amino acid, with the latter stabilizing the API in the amorphous form. “The two main benefits of this approach are processing and quality. For small-molecule coformers, it is easier to identify cosolvents for spray drying, and from a quality perspective, a small-molecule coformer is more well-defined and easier to control than a polymer,” he states.

Co-amorphous forms may also benefit from higher intermolecular interactions that prevent crystallization, higher conformational flexibility to prevent phase separation, and the anti-plasticizing effect of small molecules (5). They have also been shown to have increased solubility, dissolution rates, and physical stability compared to polymer ASDs with weak intermolecular interactions, Adam comments.

There are challenges to the formation of co-amorphous systems, however. Their typically higher molecular mobility, for instance, can lead to a greater tendency to crystallize during processing and storage, according to Johnson. She notes that significant performance variability can result, so co-amorphous formulations must be extensively studied in order to mitigate these impacts.

Recent advances in ASD technology are helping to overcome some of the limitations posed by the nature of poorly soluble APIs. High-shear mixing as an alternative to HME has the benefit of fast processing times combined with reduced exposure to high temperatures, according to Adam. “This technology has the potential to greatly impact the use of amorphous APIs by opening up the processing space where both SD and HME fall short, because it can be used for APIs with low organic solubility and high melt temperatures without degrading them,” she comments.

For APIs with poor solubility in common SD solvents, Adam points to temperature-shift spray drying as another important manufacturing advance. In this process, a slurry of the API is rapidly heated using an inline heat exchanger before atomization to increase solubility and throughput around 10-fold (6).

Another development noted by Adam involves increasing the drug loading for ASDs while improving stability and maintaining performance. High loaded dosage form (HLDF) architecture takes advantage of a high Tg polymethacrylate-based copolymer to significantly improve the stability of the ASD and HPMCAS to help sustain supersaturation upon dissolution. “This technology is applicable in formulations where the API tends to crystallize in gastrointestinal fluids and a polymethacrylate copolymer alone cannot effectively stabilize the amorphous API under these conditions,” she explains. “HLDF not only allows for higher ASD drug loadings, but also decreases tablet size and tablet burden,” she adds.

With traditional ASDs, the API is already in the amorphous state before it is processed into tablet form. Induced amorphization is the process of amorphizing crystalline API within a dosage form, thus converting from crystalline to amorphous phase during tableting.Deliberately utilizing this technique on a final dosage form can be described as in situ amorphization (ISA) and can be achieved using microwave radiation and other methods (7,8).

Price points to several examples in which APIs have been combined with small-molecule coformers such as amino acids, compressed into a tablet, and irradiated with microwaves to trigger ISA. One of the benefits of this approach is the ability to avoid using large weight fractions of polymer excipients, which dilute the concentration of API in the product, according to Sis. Physical stabilization of ASD formulations typically involves employing large weight fractions of polymers or other excipients in the final dosage form, which in some cases causes increased tablet burden, possibly lowering patient compliance (8). “Decreasing the need for long-term amorphous API stabilization results in final dosage forms containing only crystalline API and having dramatically reduced excipient fractions,” Sis explains.

In addition, eliminating the need to manufacture SD or HME formulations could avoid flowability issues associated with ASDs and drive down manufacturing costs (7). “Other important advantages to this approach relate to the stability and shelf-life of the product,” Price comments. “If the API is converted to the amorphous form directly at the point of care (pharmacy or hospital), the shelf-life relating to the stability of the amorphous form is less important,” he posits.

There are significant hurdles that must be overcome before such a dosage form is realized, however, particularly with respect to regulatory and quality concerns, Price temporizes. The technology is currently in its infancy, however, Sis notes.

In at least one example, some seed crystals were still present following microwaving of a tablet formulation (8). For some drug products, that may not affect the dissolution rate, but for others it could have a negative impact.

Overall, Sis believes ISA technology appears promising and warrants further development and investigation, with efforts focused on complete crystalline to amorphous conversion. He observes that considerations should also be taken to develop and understand the landscape in which FDA approval could occur. “ISA process technology must first be proven safe, effective, and scalable,” he concludes.

Rapidly evolving characteristics for candidate drug substances are driving the need for continued advances in technologies for the stabilization of amorphous APIs. Already formulators are challenged by the poor solubility of Biopharmaceutics Classification System II and IV APIs in the development pipeline.

“The ideal scenario for each of these drug substances would be to achieve a kinetic (beyond physio-relevant timelines) or thermodynamic increase in solubilization that allows for effective bioavailability,” contends Johnson. To achieve that goal, she believes continued investment in novel excipients that balance the need for increasing solubility while not hindering absorption due to competing thermodynamic or partitioning mechanisms will be necessary.

Adam points to the increasing number of “brick dust” candidates in the pipeline that are poorly soluble in both water and typically used organic solvents. That makes SD infeasible due to poor throughput or high emissions of harsh solvents. Because they often have higher Tm values, HME is also often challenging or not possible. “Strategies that can overcome the manufacturing complications with brick dust compounds will drive future formulation opportunities for needed therapeutics,” she concludes.

Drug loading also remains an issue for amorphous API dosage forms because they sometimes require high amounts of polymers to help with stabilization. In addition to HLDF architecture, Adam remarks that further improvements in stabilization technologies that enable decreased polymer amounts are needed to address this issue.

For Price, growing numbers of proteolysis targeting chimeras (PROTACs) and beyond rule-of-five (bRo5) compounds with molecular weights greater than 500 Daltons making their way through the clinic will be an important driver of innovation in amorphous technologies. “These larger molecules have the potential to totally transform the treatment of disease, in much the same way that mRNA is currently doing. The big difference is that PROTACs and bRo5 molecules have a high likelihood of being orally bioavailable and thus can be delivered orally. Development efforts must therefore be directed at identifying amorphous technologies for the formulation and stabilization of these novel and exciting modalities to ensure acceptable absorption from the GI tract,” he says.

1. J.M. Baumann, M.S. Adam, J.D. Wood,Annual Review of Chemical and Biomolecular Engineering 2021.

2. S.V. Bhujbal, et al., Acta Pharmaceutica Sinica B 11, 2505-2536 (2021).

Polymeric amorphous solid dispersions are the most commonly used technology, but amorphous APIs remain inherently unstable.