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

Cynthia A. Challener

Treatment of bacterial infections with bacteriophages (phages)—viruses that enter bacterial cells and rapidly multiply, quickly rupturing the cells and destroying the bacteria—has been known to be effective since the early 1920s. The advent of broad-spectrum antibiotics led to a halt in phage therapy research, however, except in Eastern Europe, particularly Georgia, and to some extent France (1).

Bacteria, meanwhile, have developed resistance to many antibiotics, and new “superbugs” that cannot be treated with even the most powerful antibiotics are increasingly common. There is real potential that the world could enter a post-antibiotic era in which giving birth or getting a simple
cut could present a serious risk of death. The situation is exacerbated by the fact that antibiotics have long since been commoditized, and most major pharmaceutical companies halted their antibiotic development programs in favor of investments in more lucrative areas such as oncology. Few new antibiotics drugs have been approved as a result.

Consequently, the need for alternatives to traditional antibiotics has become urgent. In addition to leading to the development of novel chemical approaches, the current threat of antimicrobial resistance is driving renewed interest in phage therapy. A lack of appropriate infrastructure and specific regulatory framework are two main challenges to the realization of marketed products. Fortunately, developers of phage-based treatments expect these hurdles to be surmountable in the medium term, with several candidates in or soon to enter clinical trials.

Fighting bacteria that are resistant to current antibiotics is what most people see as the main driver of interest in the development of phage-based therapies. For Alexander “Sandro” Sulakvelidze, president and CEO of bacteriophage developer Intralytix, there is another important factor: growing understanding of the human microbiome and its role in health and disease.

“The microbiome field is exploding, with a lot of bright minds working in the area. People are establishing links between microbiome composition and diseases ranging from those having obvious bacterial etiology (e.g., infectious and gastrointestinal diseases like bacterial dysentery) to disorders that are not typically associated with bacterial infections, such as obesity, cancer, and others,” Sulakvelidze explains. “That means that with bacteriophages we not only have the potential to manage drug-resistant infections, but also a tool that may be able to beneficially modulate the microbiome to address both infectious and noninfectious diseases,” he states.

Indeed, the human microbiome comprises hundreds of bacterial species, and maintaining the right balance of “good” and “bad” bacteria in the gut microbiome helps maintain health through multiple mechanisms including modulation of inflammation and regulation of protective gastrointestinal functions, according to Sulakvelidze. “Lytic bacteriophages are superbly suited for gentle and targeted fine-tuning of the microbiome by killing their specific targeted bacterial pathogens without disturbing the normal microflora—a unique biological property that is increasingly explored for developing novel tools for microbiome modulation and research,” he observes.

While broad-spectrum antibiotics are attractive because they can be used to treat a wide range of bacterial infections, they also do not discriminate between “good” and “bad” bacteria. Dysbiosis and secondary infections (e.g., fungal infections, inflammatory bowel disease, reactive arthritis, etc.) can occur as a result, complications that have not been observed with phages (1). Phages could also be beneficial for patients with allergies to the commonly used penicillins, sulfonamides, and tetracyclines.

At the simplistic level, notes Sulakvelidze, phages infect bacteria by first attaching to their cell membranes. They then inject DNA into the host cells. Within 60 seconds, the phage DNA takes over and shuts down the cellular machinery of the bacteria, which allows for phage DNA replication. Within 20–40 minutes, approximately 40–200 bacteriophages exist in each of the bacterial cells, causing the cells to burst. The bacteria die, and the phages are released to seek out and infect additional targets.

This mechanism of action is different from that of traditional antibiotics. As a result, while bacteria can become resistant to phages, the mechanisms involved are very different from the one against antibiotics. “The resistance that bacteria develop to antibiotics does not affect their resistance to phages, making these two approaches complementary,” Sulakvelidze observes. Indeed, often phages can be effective against bacteria that have developed resistance to antibiotics. There have been several case reports of the effective emergency treatment of patients for which conventional antibiotics have failed (1).

The specificity of phages is advantageous because it does not result in the death of desirable bacteria. It is, however, one of the reasons that phages fell out of favor after the introduction of antibiotics. For phage therapy to be effective, it is necessary to know which bacterium is causing the infection, which is not the case for broad-spectrum antibiotics, according to Sulakvelidze. It can take time to identify the specific phage or cocktail of phages that will be effective, time that some patients may not have, particularly in cases where phages are used as a treatment of last resort (1).

Antibiotics creating problem and inhibiting alternative solutions

Another difficulty facing the advance of phage-based therapies is the fact that the medical infrastructure that exists for the treatment of bacterial infections in most of the world has been designed around antibiotics (2). From susceptibility testing kits to the sophisticated machinery required for high-throughput testing and the training of hospital and clinical personnel, everything is geared toward antibiotics, Sulakvelidze comments.

“The business model for phages is very different from that for antibiotics, which creates a whole range of challenges from manufacturing and distribution to diagnosis and identification of the right phage treatment for each patient,” says Sulakvelidze.

For Grégory Resch, head of the laboratory of bacteriophages at the Center for Research and Innovation in Clinical Pharmaceutical Sciences at Lausanne University Hospital, Switzerland, one of the biggest challenges is the complexity around producing many different specific phages under good manufacturing practice (GMP) conditions, which contrasts with the existing access to broad-spectrum antibiotics. He also notes that patenting phages is not as obvious, because many of them can easily be isolated from the environment.

Phages are manufactured via fermentation using the bacteria that serve as the natural host of the phage in question. The phage reproduces, killing the bacteria. The phage must then be separated from the dead bacteria and purified, as the dead bacteria and other contaminants can cause undesired immune responses. Fortunately, advances in fermentation technology and downstream processing have enabled successful, large-scale production of high-quality phage-based products, according to Sulakvelidze.

Production challenges, although they are being addressed, are further complicated by a lack of an established regulatory framework for the approval of phage-based therapies. The absence of a clear definition for bacteriophages, common and validated dosage protocols, and the length of phage treatments, for instance, creates uncertainty with respect to the development of clinical programs (2). On the positive side, however, many of the disadvantages of phage-based therapy relate to infrastructure and knowledge gaps that can be resolved as the field progresses.

Even so, Sulakvelidze comments that the advances Intralytix and other bacteriophage developers, such as Adaptive Phage Therapeutics and Armata Pharmaceuticals, have made have taken longer than he anticipated. “Even with the many years of work and growing number of companies involved in the field, it will still be many years before phage-based therapy becomes a mainstream treatment or at least somewhat commonly used,” he contends.

Intralytix was formed in 1998 by Sulakvelidze and his post-doctoral advisor Glenn Morris, an infectious disease specialist who had a patient die after a sophisticated surgical procedure due to a drug-resistant infection of the intestine—and who was not familiar with phage-based therapy. The initial goal of the company, says Sulakvelidze, was to develop phages to treat infections caused by vancomycin-resistant bacteria called enterococci or VRE. It became quickly apparent to the founders that the time was not right for targeting clinical applications given the long list of challenges for human medical applications.

Instead, Intralytix shifted its focus to food safety, which has given the company time to establish greater knowledge, build a manufacturing facility and develop effective production processes, work with regulators, and establish some of the infrastructure needed to support phage-based products. Its first commercial product was ListShield, an all-natural, non-chemical antimicrobial preparation for controlling the foodborne bacterial pathogen Listeria monocytogenes.

Today, Intralytix is using the sales revenues from its portfolio of phage-based food protection products to support the development of nutraceuticals and drug candidates. The company will launch its first dietary supplement/probiotic pill to enhance natural gut resistance against certain pathogenic bacteria in March 2022, with a second to be introduced in 2023.

Some phage-based companies focused on the development of drug candidates are also leveraging advanced genome sequencing and metagenomics technologies to engineer genetically modified phages with improved patentability, according to Resch. He adds that the hope is that these advanced phages will also have enhanced activity without impairing safety, but sufficient evidence of this benefit has yet to be gathered.

Academic centers and government bodies are also getting involved in the field of phage-based therapies, helping startup companies with funding and other means of support. One example highlighted by Resch is IPATH, the Center for Innovative Phage Applications and Therapeutics, which is located at the University of California, San Diego School of Medicine and the first dedicated phage therapy center in North America.

Over the past one hundred-plus years, phages have been administered to humans orally (tablets and liquids), rectally, topically (skin, eye, ear, etc.) as rinses and creams, intravenously, and via other
delivery approaches.

Today, phage-based therapies are in preclinical development for the treatment of a wide variety of diseases, including lung, wound and gut infections, as well as endocarditis and many other diseases that are difficult-to-treat because of the potential for complications due to antibiotic resistance.

When it comes to clinical trials, only a few have been conducted and/or are ongoing in Western medicine, according to Sulakvelidze. Phase I and Phase II studies involving phage-based therapies have been completed in chronic otitis media, respiratory infections, infected burn wounds, gastrointestinal disorders, urinary tract infections, and venous leg ulcers (1). The most common targets, says Sulakvelidze, are gastrointestinal and wound/skin infections.

One of Intralytix’s prototype phage preparations for treating infected wounds was successfully used during a Phase I human clinical trial in Lubbock, Texas—the first Phase I trial in wound healing in the United States. The company’s EcoActive bacteriophage therapy targeting adherent-invasive Escherichia coli(AIEC) in Crohn’s disease patients entered a Phase I/IIa clinical trial in 2019 and is currently enrolling participants at the Icahn School of Medicine at the Mount Sinai Hospital in New York, NY.

In October 2021, Intralytix received clearance from FDA for an investigational new drug (IND) application for ShigActive, a bacteriophage preparation to manage Shigella infections in humans, of which there are approximately 125 million cases and 14,000 deaths worldwide, the majority of the latter in children under the age of five. The company is also in the pre-IND stage with a candidate to treat VRE infections. Altogether, Sulakvelidze says Intralytix will be starting three additional clinical trials in the next 18 to 24 months.

The increasing number of developmental drug products and clinical trials is a hopeful sign for the future of phage-based therapy and a first step in advancing the field. During the next few years, Sulakvelidze expects to see even more clinical trials and greater numbers of people involved in generating an ever-broadening base of scientific knowledge and establishing the infrastructure needed to enable a phage-based approach to the treatment of disease.

“Eventually,” Sulakvelidze remarks, “there will be more reference centers and clinics—maybe a handful—where people can go to get treated with bacteriophages by properly trained experts with access to the right equipment. When enough centers are established, phage-based therapy will become more commonplace nationwide, but progress toward that goal will be incremental.”

Resch adds that greater capacity to produce phages under GMP conditions will need to be constructed to support the products that advance to late-stage clinical trials and commercialization.

Part of Sulakvelidze’s reason for having high expectations for bacteriophages in medical applications is the experience Intralytix has had with FDA regarding its food safety products. “The first product took four years to get approved, with a lot of back-and-forth between the company and FDA that largely involved educating one another. The agency has required considerably less time (9 to 12 months) to reach decisions on subsequent products,” he observes.

Sulakvelidze expects that experience to translate to the company’s human health products. “FDA’s Center for Biologic Evaluation and Research is very much open and willing to work with companies developing phage-based therapies to establish a regulatory framework for ascertaining their safety and effectiveness,” he comments.

“With the potential for bacteriophages when used in combination with antibiotics as first-line therapies to provide personalized treatment solutions for challenging patient infections, there is the real [opportunity] for this new approach to be democratizing,” insists Resch. That potential in conjunction with the urgent need for solutions to the rapidly growing threat of antibiotic resistance point to a bright future for phage-based therapies.

1. J-P Pirnay, T. Ferry, and G. Resch, “Recent Progress Toward the Implementation of Phage Therapy in Western Medicine,” 

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


Vol. 46, No. 3 
March 2022 
Pages: 21–23

When referring to this article, please cite it as C. Challener, “The State of Phage Therapy,” 

Articles

Bacteriophages could be crucial weapons in the fight against antibiotic-resistant bacteria.

The State of Phage Therapy 

The success of the messenger RNA (mRNA) vaccines against the SARS-CoV-2 virus brought attention to a field that has steadily advanced for many years. Investment in this space has ramped up dramatically, and many applications of mRNA technology are rapidly advancing through pre-clinical development as well as clinical trials.

As with all drug or vaccine candidates, proposed mRNA products must meet regulatory requirements for analytical characterization as outlined by the International Conference on Harmonisation (ICH), the World Health Organization (WHO), FDA, the European Medicines Agency (EMA), and other health authorities around the globe.

Unlike most other drug substances, mRNA must be formulated within specialized nanoparticles, most commonly consisting of lipids, both to protect it from degradation and to facilitate its transit across cell membranes. Additional analytics are therefore required to ensure the quality and safety of the nanoparticle formulation as well as the mRNA molecule itself.

A key activity in the early development phase of mRNA therapeutics is extensive characterization of mRNA and nanoparticle quality attributes as these are known to strongly influence biological efficacy. Given the shrinking timelines for drug development, access to faster and higher-throughput analytical methods has become vital. Indeed, it is essential that analytical approaches for use during mRNA product development and quality-control (QC) testing must keep pace to ensure the identity, safety, and efficacy of these new prophylactic and therapeutic modalities.

Although analytical requirements for mRNA candidates are more complex than those for traditional biologics, some testing needs are similar while others are quite different. “There is a common need to adequately characterize both protein therapeutics and mRNA therapeutics to confidently identify and quantify any quality attributes that will impact their efficacy and safety,” says Todd Stawicki, global marketing manager, mass spectrometry, for the biopharmaceutical industry at SCIEX. He points to the need to identify process-related impurities—host-cell proteins (HCPs) in the case of protein therapeutics and a variety of genetic impurities like truncated transcripts or incorrect DNA template in the case of mRNA.

The differences are notable, however. Rather than focusing on post-translational modifications as is the case for protein therapeutics, there is a lot of emphasis on characterization of mRNA, the delivery systems, and cellular uptake at the discovery phase of drug development, because unlike engineered proteins and monoclonal antibodies (mAbs) that are ready to start functioning upon delivery, mRNA once delivered to cells provides instructions for producing the proteins that do the work, according to Brian Liau, a solution development scientist at Agilent Technologies, Singapore.

Manufacturing for mRNA products, furthermore, requires unique process steps such as in vitro transcription (IVT) and lipid nanoparticle formulation, which naturally creates different analytical needs, adds Ryan Hanko, manager of quality control for Catalent. IVT is a chemical rather than a cell-based process, expounds Joe Fredette, senior business development manager of biopharmaceuticals at Waters. It also involves the use of an enzyme called an RNA polymerase that transcribes DNA into mRNA.

As an example of different analytical needs, Hanko notes that mRNA products require QC assays to be developed that measure residual enzyme (such as polymerase or endonuclease), which may be present after the IVT step, to ensure that it is not present in the final product. Confirmation of the RNA sequence and determination of the efficiency of capping at the 5’ end and poly(A) tail distribution at the 3’ end are also unique to mRNA, notes Ashleigh Wake, business development director for Intertek Pharmaceutical Services. The potential impurity profile is also quite different to other molecules, and methods to quantify any residual plasmid or double-stranded RNA are also required.

In addition, lipid analysis workflows are necessary for characterization and chemistry, manufacturing, and controls (CMC) testing of mRNA products, including liquid nanoparticle (LNP) composition analysis to ensure the right mix ratio of lipids within the formulation, lipid ID confirmation, and impurities analysis and screening, according to Fredette.

In essence, summarizes Wake, there is a similarity in the overall requirements for product characterization in that the intent is to assess or confirm identity, purity/impurities, stability, activity, etc. “However,” she underscores, “to fully accomplish this, there is a need to include mRNA-specific analytics that allow assessment of these attributes.”

Evolving analytical needs from early to late-phase development

Another important difference between the development of protein- and mRNA-based products is the analytical burden during the early discovery phase. As traditional drug development typically involves mAbs, says Dr. Liau, bioanalytical requirements tend to be relatively modest because even sub-optimal molecules may be used to demonstrate target engagement and a therapeutic effect. In contrast, mRNA products require several biological processes, including cellular uptake, endosomal escape, and protein translation to take place efficiently before any effect may be observed. Hence, analytical characterization of mRNA and delivery vehicle quality attributes are important from the outset.

“For process scale-up and GMP [good manufacturing practice] manufacturing/product release, chemical characterization of mRNA remain important. However, the focus tends to shift towards ensuring consistent size polydispersity and mRNA encapsulation in the formulated product, as well as identifying process-related impurities,” Dr. Liau comments.

With mRNA candidates, Stawicki adds that scale up of the enzymatic process requires significant and difficult scale up for all upstream raw materials—nucleoside triphosphate (NTPs), enzymes, capping reagents, cofactors, and multiple classes of lipids. “The majority of these components are large, complicated biomolecules with their own associated scale up, purification, and analytical challenges,” he explains.

There are also some differences in the attributes that are monitored during production, according to Hanko. A260 concentration checks and osmolality testing are the primary monitoring assays in early development and process scaleup for in-process observations, while during GMP manufacture RNase testing is important to ensure the final product will not be compromised by incidental contamination from non-product materials.

Core release tests include identity determinations, final concentration, impurity content (e.g., residual protein, dsRNA, rDNA), capping efficiency, and poly-A tail, in addition to typical compendial evaluations for safety and quality attributes.

There is an extensive list of critical quality attributes (CQAs) that must be monitored to meet regulatory requirements regarding the assessment of the identity, functionality, and safety of mRNA therapeutic/vaccine products (see 

). “Confirmation that the specific mRNA sequence has been generated, that the mRNA solution is at target pH, and that any potential process impurities (solvent, protein, DNA-related) have been controlled will be required,” says Matthew Howard, a senior specialist in quality assurance at Catalent.

Table I. Key critical quality attributes (CQAs) that must be monitored for

messenger RNA (mRNA) therapeutic/vaccine products.

Assurance will also be needed that the translation-promoting and
degradation-preventing characteristics of the 5’ cap and poly-A tail are sufficient, says Howard. The efficiency of capping at the 5’ end can influence both target protein production and overall immunogenicity, while the length/distribution of the poly(A) tail is critical for translation and overall protection of the mRNA, Wake explains.

Adds Maxwell Meller, senior scientist, quality control, with Catalent: “The 5’-capping of the mRNA molecule is critical to its integrity upon delivery to the target site and is a unique requirement for mRNA production compared to other kinds of drugs. Characterization and monitoring of capped material, however, have proven to be time- and resource-intensive.”

For the lipid components, confirmation of the identity of each lipid in the LNP mix and that they are present in the correct ratio is essential as these CQAs can directly impact LNP formation and efficacy, according to Fredette. It is also important, he emphasizes, to ensure that lipid impurities are well controlled for and below critical thresholds, as both lipid and mRNA impurities can adversely impact process outcomes and in some cases lower the potency of the mRNA.

The sensitivity of mRNA to degradation by enzymes, particularly RNase, creates additional unique requirements when working with mRNA-based therapeutics and vaccines. For instance, Hanko observes that the use of RNase-eliminating preparations is vital and laboratories should consider physical space segregation in addition to personnel training and awareness regarding the maintenance of RNAse-free zones, equipment, and personal protective equipment (PPE).

These actions are important, Hanko says, because a compromised mRNA sample will show lower-than-expected purity in those assays. Some analyses, as those for residual impurities, are arguably not impacted by compromised mRNA and others may only require heightened RNase sensitivity depending on solution preparations and material manipulations. “Even so,” Hanko states, “the core method performance expectations must be understood for all methods regardless of the level of mitigation required.”

One of the biggest issues facing mRNA analysis is experienced across the board in the biopharmaceutical industry. “The great enemy here is time,” Stawicki asserts. “The unfortunate reality is that many of the current functional and biophysical tests are still too slow to generate actionable answers in a timeframe that enables intervention in the process,” he explains.

For instance, Stawicki highlights cell-based functional assays, which are the gold standard today. “This test is the best early indication that you’ve got everything right,” he says. “You dose your formulated mRNA LNP to cells and see if you get sufficient yield of the right protein or antigen. The problem is that it can take days to get a read out. Therefore, often times developers have to proceed at risk in their processes without having the analytical data to back up their process parameters.”

Use of numerous complementary techniques during early development

Bioanalysis of mRNA products requires a combination of familiar and more novel analytical techniques and technologies. It also necessitates, Hanko reiterates, the control and elimination of RNase enzymes, which may compromise product integrity.

A wide variety of analytical techniques are employed to evaluate these properties. What is interesting to Stawicki is that they have come from a variety of different backgrounds. High-performance liquid chromatography (HPLC) and mass spectrometry (MS) have migrated from protein-therapeutic characterization. Electrophoretic techniques (most notably capillary electrophoresis) have come from classic molecular biology use. Particle sizing techniques that have come from polymer analysis or materials lab use.

“Since mRNA therapeutics are such large, heterogenous complexes, it really comes down to which part or parts of the candidate require characterization,” Stawicki remarks. For the mRNA component, he notes that capillary electrophoresis or even slab-gel electrophoresis methods are preferred. For characterization of lipids and LNPs, HPLC with charged aerosol detection (CAD) or LC-MS are generally the methods of choice. For complete mRNA-LNP complexes, techniques such as particle sizing or cryo-electron microscopy are preferred.

Some common activities during early development are mRNA codon optimization and selection of optimal 5’ and 3’ untranslated regions (UTRs), says Dr. Liau. As this entails cloning coding sequences out of libraries and combining them in multiple plasmids with different UTRs, Sanger sequencing, capillary or slab-gel electrophoresis, and polymerase chain reaction (PCR) remain mainstay analytical methods during this phase of development.

Characterization and CMC testing for the mRNA component of the drug, according to Fredette, includes a range of nucleic acid (DNA/RNA) analytical workflows to confirm the identity and purity of the mRNA, including 5’ Cap analysis, PolyA tail analysis, oligo mapping (similar to peptide mapping, but for nucleic acids), and MS-based sequencing.

MS, meanwhile, is also used to establish key quality attributes such as capping efficiency and poly(A) tail distribution, notes Wake. She adds that all of these analyses are performed in addition to more traditional QC requirements, such as assay [ultraviolet (UV)/HPLC)] analytics, residual solvents, metals, etc.

To confirm the biological activity of mRNA candidates, well-studied lipid delivery systems using cationic or ionizable lipids may be used to transfect standard cell lines in vitro. “For such early-stage experiments,” Liau observes, “it is often unnecessary to use highly purified mRNA or monodisperse preparations of lipid nanoparticles. Instead, bulk dynamic light-scattering and zeta potential measurements are often used to ensure the nanoparticles have approximately the correct size and surface-charge properties to permit efficient cell transfection. Biological activity may then be determined by fluorescence microscopy, enzymatic assays, or immunostaining as appropriate.”

Looking for simpler, more robust solutions during late-stage development

Similar analytical techniques are generally used from early in development through the GMP environment. For instance, LC and LC-MS workflows bring value across all stages of development and for process monitoring in manufacturing, Fredette observes.

Because the goals of process optimization and scaleup are often quite different from those of early-stage development studies, however, Liau comments that different analytical techniques are required in some cases. “During process optimization and scaleup, the mRNA sequence is fixed, and its biological activity known, and the focus is on monitoring and/or improving the process yield and mRNA quality attributes,” he says.

LC-MS is very useful both for monitoring the progress of in vitro transcription reactions and determining the status of critical quality attributes such as 5’ capping, Liau adds. Separation-based techniques such as size-exclusion chromatography-multiangle light scattering (SEC-MALS) and field-flow fractionation (FFF)-MALS may, meanwhile, be useful for monitoring the properties of LNPs and optimizing the complexation process.

One important trend noted by Stawicki that is also seen with protein therapeutics is the preference for increasingly robust and simpler techniques the further down the pipeline programs get, Meller agrees. For instance, he notes that while LC-MS is a promising tool for characterizing and quantitating capped species, a more simplistic approach lends itself more appropriately to routine use. “In the QC environment,” says Meller, “the chromatography analysis must be condensed to an HPLC assay to prioritize speed and efficient data analysis.”

In general, Wake believes that many of the methods or approaches used to fully understand mRNA are novel in the QC area, when compared to those used for other modalities. “To fully support all regulatory requirements, greater use of technologies such as next-generation sequencing, which have not been typical technologies operated in a GMP environment, has been necessary,” she explains.

For other considerations such as capping, however, Wake says that the technologies used are more familiar and tend to be heavily MS-based. Even in these cases, though, she remarks that the overall sample preparation approach is often novel and challenging. “A lot of work is being done in our and other laboratories to develop platform approaches to overcome these challenges considering best science, quality data, and regulatory compliance,” she concludes.

There is, in fact, tremendous effort being made to advance analytics for mRNA vaccines and therapeutics. “Scientists have been working with mRNA for decades, but a lot of that effort has focused optimizing analytical methods to enable production-grade techniques. With the explosion of genetic therapies and the now intense interest in mRNA, you are seeing a tremendous amount of investment and innovation by scientists and instrument companies to develop the best analytical solutions to support that work,” Stawicki contends.

SCIEX, for instance, has introduced electron activated dissociation (EAD) as a new MS fragmentation technology in the ZenoTOF 7600. Initially launched with small-molecule and protein therapeutics in mind, the company has since learned, according to Stawicki, that EAD technology is also powerful for analyzing mRNA products. “EAD has amazing utility for both oligonucleotide and LNP characterization, allowing, for example, the sensitive and complete structural characterization of lipids including double bond positions and isomer configurations in a single experiment.”

Stawicki also points to SCIEX’s new BioPhase 8800 multi-capillary CE instrument, which allows up to eight analyses to be performed simultaneously on the same or different samples. “We are exploring how to leverage the high throughput capabilities of the BioPhase for a whole range of different mRNA applications,” he says.

Agilent recently developed an improved method for rapid analysis of mRNA 5’ capping by LC-MS, according to Liau. Enzymatic cleavage is required for this analysis because mRNA is such a large molecule. The established method required two challenging sample preparation steps: affinity capture using biotinylated oligonucleotide probes, and site-directed cleavage of affinity-captured mRNA using RNase-H.

This method failed to work reliably with some mRNA sequences due to inefficient affinity purification, so Agilent developed an improved sample preparation method comprising site-directed cleavage in solution without affinity capture followed by purification of the cleaved oligos and mRNA sample matrix using silica-based columns. Using a thermostable version of RNase-H enzyme allowed the sample preparation time to be reduced as well.

The total process—sample prep, analysis, and data processing—requires just 75 minutes and allows separation of the oligos of interest from the mRNA sample matrix without further sample cleanup.“When used in conjunction with a specially-designed flushing protocol to mitigate sample matrix buildup on the column, this method yielded superior linearity, sensitivity, and reproducibility,” Liau says.

Howard highlights several promising techniques currently being refined for the evaluation of protein expression in cells, such as flow-cytometry for the detection of intracellular dsRNA levels and antigen-specific fluorescent detection of expressed proteins, both of which provide rapid results for cell uptake, RNA amplification, and protein production. “Such direct measurements could reduce the turn-around time for in vivo antigen studies. The new techniques certainly look promising and could be practical in the next few years,” he believes.

Wake returns her attention to NGS technology; even though it isn’t new in general, its use in the GMP environment is, and for mRNA products the introduction of NGS is vital to achieving the required quality of control. Similarly, she says that electron microscopy is an essential technique, along with approaches for establishing in vitro release performance, for evaluating encapsulated products. Doing so, however, has required the adaptation of a kit that has mainly been associated with US Pharmacopeia type 4 dissolution testing.

The importance of developing platform analytical technologies is also reiterated by Wake. The continual improvement of platform approaches suited for establishing if mRNA is truly encapsulated within the nanoparticle system at the right internal liposome pH to support early-phase products without the need for extensive development is a promising example, she says. As the product moves into later phases, these same methods are optimized and eventually validated for each specific product. “However,” Wake argues, “understanding these critical aspects as early as possible in the development cycle necessitates the need for a general approach.”

It is important to remember that mRNA technology itself is advancing rapidly and evolving on a continual basis. Developers of analytical methods for mRNA therapeutics and vaccines are therefore challenged not only by the complexity of these biomolecules and their delivery vehicles, but also by the need to develop flexible methods that can accommodate the ongoing changes in mRNA structures and final formulations.

As an example, Wake notes that while to date mRNA products have been developed as injectables, inhalation and nasal delivery present viable alternatives. “These routes of delivery can not only aid in achieving improved bioavailability, but also lead to products that can be delivered in a less evasive manner, even in the absence of a healthcare lead administration system,” she explains.

Scientists, Fredette recommends, should think about the analytical platforms and workflows they need to deploy across their organizations as they grow and how best to harmonize everything, from method development and method/data transfer; to ensuring data integrity, traceability, and compliance; to the level of service and support they want and need.

“I don’t even think we have started to approach the steady state for what analytics look like for mRNA therapeutics,” states Stawicki.“Just the space of what we consider mRNA therapeutics continues to grow rapidly since elasomeran (Spikevax) from Moderna and tozinameran (Comirnaty) from Pfizer-BioNTech became world-changing vaccines. Many companies are now developing variations on mRNA such as self-amplifying mRNA and circular mRNA.In addition, scientists are developing new types of vehicles and targeting strategies for mRNA therapies.So yes, we are still at the beginning stages of this technology revolution,” he concludes.

Cynthia A. Challener, PhD, is contributing editor to 


Vol. 46, No. 2
February 2022
Pages: 16–21

When referring to this article, please cite it as C. Challener, “Analysis of mRNA Therapeutics and Vaccines,” 

Analytical approaches must keep pace to ensure the identity, safety, and efficacy of evolving mRNA candidates.

Analysis of mRNA Therapeutics and Vaccines

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,” 

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.

Dosage form and patient needs drive excipient choice.