Feliza Mirasol is the science editor for 

Feliza Mirasol

The structure and activity of complex biotherapeutic molecules require sensitive analytical technologies that can characterize these complicated protein structures and observe their pharmacokinetic activity, elements fundamental to biotherapeutic drug development and production. Relevant technologies are needed to make detailed analytical assessments of structure and function, and further innovation is needed to address the continued challenge faced by the industry.

The problem with complex biological molecules

The structure of biological molecules poses interesting challenges to analytical assessment. Elucidating the purity or structure of a complex biological product often requires methods designed uniquely to the analyte, multiple analytical assays that leverage cross-modal technologies, and orthogonal analytics to differentiate the analyte from a biosimilar molecule, says William Bakewell, research fellow at PPD Laboratories’ GMP Lab.

Meeting Future Analytical Requirements for Novel Biotherapeutics

A fundamental challenge, says Shawn Fitzgibbons, manager, Catalent Biologics, is the large size of biologics materials, and, in turn, the micro-heterogeneity observed with respect to each attribute measured. “Every molecule will have unique characteristics, for example, although characterization of post-translational modifications is routinely addressed, there may be a specific challenge if the molecule has a high level of modifiable residues at, or near, important binding regions of the molecule, or if the molecule is particularly prone to these modifications because of its given formulation,” he says.

Challenges with respect to functional analyses often revolve around proper, timely, development of the necessary cells and antibodies, Fitzgibbons adds. Similarly, extractables and leachables assessments require a large panel of extraction and analytical techniques to be undertaken to assure detection, identification, and quantitation of a potential toxic, unknown material.

Meanwhile, structure–function assessments have been known to be challenging because the amount of material available to address all orthogonal methods is often limited, says Pedro Morales, director, Catalent Biologics. “For example, it becomes challenging to isolate and analyze variants that are present in only small amounts within the fractionated sample,” he says.

Morales also explains that the extraction and/or purification of variants needs to be done while avoiding the introduction of additional—or exacerbation of—post-translational modifications (PTMs) or impacting the material so as to create aggregates. He says that experimental design should include appropriate controls needed for analysis in parallel to the analyte(s) of interest. “It is always challenging to develop a robust functional assay that will ensure a solid structure–function correlation that can be unequivocally determined,” he states.

Jana Hersch, scientific consultant, Genedata, adds that correct prediction of how a complex biological will behave 

 remains the biggest challenge during the development of complex biotherapeutic molecules. She points out that antibody-drug conjugates (ADCs), bispecifics, and even engineered cell-surface receptors, such as chimeric antigen receptors (CARs), rely on the scientists’ ability to assess how these molecules will behave once inside the patient.

“It is important to predict not just whether they bind with the desired affinity and specificity to their target 

, but also that they bind in the right tissues, or that they have the appropriate level of clearance from the body to achieve the desired therapeutic effect and cause minimal harm,” Hersch says. She also explains that many different versions of a complex biological molecule are often tested in iterative pharmacokinetic and pharmacodynamic analyses before the preferred format as well as the final formulation are selected.

Campbell Bunce, chief scientific officer at Abzena, emphasizes the point that all drug developers should look to establish the right set of analytical methods and workflows to assess the developability of their individual drug candidate and ensure that those they take forward have a reduced risk of inherent liabilities at an early stage. He states a holistic approach that applies design of experiment principles is recommended to capture a wide range of parameters to focus across several characteristics, such as function, manufacturability, immunogenicity, and safety. “For example, through developability assessment, methods can be applied that will provide insights into the relative stability of the biological molecules and allow them to be ranked against comparators to help select the best lead candidate for development,” he says.

Bunce notes that often, novel biotherapeutic candidates are unique with little information that can be drawn on to help develop the right assays and comparative assessment parameters, as can be done with antibodies, where a significant amount of historical data exists. Certain aspects of novel biologic molecules can be pieced together, however, directed by experience-led trial and error to generate developability profiles that lead to better outcomes, he explains.

“There may be occasions where liabilities cannot be designed out using protein engineering techniques and thus, need to be managed through different approaches. Consequently, other mitigating options can be evaluated and applied,” Bunce states. Biologics, for example, may be affected by one or more degradation pathways that can be evaluated and addressed through the formulation development process to maximize the stability of the biologic drug candidate.

Because of the complexity of biological molecules, a panel of analytical methods is used to analyze the therapeutic proteins for lot release to ensure consistent product quality, safety, and efficacy, adds Gang Huang, senior vice-president, analytical sciences and clinical quality control, WuXi Biologics. Though enormous work is done in the clone selection and process development stages, it is still expected that a final drug product is composed of a mixture of hundreds to thousands of variants that differ in PTMs and higher order structure, he emphasizes. As a result, state-of-the-art analytical methods have been used to thoroughly characterize various PTMs and degradation pathways of these proteins.

Bakewell notes that mass spectrometry (MS)-based analyses are a critical component of determining primary structure for conjugated proteins, monoclonal antibodies (mAbs), and cell and gene therapy (CGT) products. He further explains that intact mass and peptide mapping-based MS methods allow for confirmation of protein sequence as well as identification of any disulfide bonds, oxidation/deamidation events, glycosylation patterns, and PTMs, all of which are key structural critical quality attributes (CQA). Meanwhile, information about secondary and tertiary structure can be determined through techniques such as circular dichroism, isothermal calorimetry, size exclusion chromatography, and multi-angle light scattering.

“Complementary to these protein-level analyses, sequencing of the products underlying DNA can be performed by Sanger sequencing, capillary electrophoresis with fragment analysis, and next-generation sequencing (NGS) approaches. The depth to which a product is sequenced—including a product’s expressing and non-expressing elements—and the degree to which sequencing results confirm a product’s identity has yet to be standardized,” Bakewell adds.

Fortunately, biopharmaceutical analysis methods have improved since the first mAbs were approved. The most significant change over the past 15 years has been the speed and resolution at which bioanalytical tools are employed, says Christopher Colangelo, biopharma business development leader, Agilent Technologies. The development of porous and/or sub-2-um chromatographic media coupled to ultra-high-performance liquid chromatography (UHPLC), for example, has enabled analytical method runtime to decrease 10-fold from 30–50 mins/sample to 3–5 mins/sample. Colangelo adds that a key success in many biopharma research labs today is the use of automation solutions in lab workflows. “For example, successful labs effectively use robotic sample preparation, liquid autosamplers, and automated data analysis tools,” he states.

Significant improvements have also been made in the sensitivity, resolution, accuracy, reproducibility, and specificity of analytical methods used to support the release and stability testing of biological drugs, Bakewell emphasizes. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and isoelectric focusing gel electrophoretic methods commonly used for release and stability in the 1980s have been largely replaced by capillary electrophoresis (CE) methods, for example, Bakewell says. “CE methods are deemed superior to traditional gel electrophoresis methods due to their improved resolution and their high degree of reproducibility and robustness,” he states.

Similarly, column chromatographic separation techniques have evolved and improved over time to where, for example, the operating pressures that analytical HPLC/ultra high (UH)PLC columns can tolerate has increased. This has resulted in a concomitant increase in analytical resolution, Bakewell explains. Furthermore, improvements in the quality of HPLC systems and batch-to-batch reproducibility of HPLC columns has led to better and more reproducible analytical methods, while data analysis for chromatographic separations has evolved from using paper integrators to 

) Title 21, Part 11-compliant analytical software, says Bakewell.

“Over time, the variety of good manufacturing practice (GMP)-compliant analytical methods used to evaluate purity, potency, and stability of biopharmaceuticals has expanded and evolved. These now include multiplex and fluorescent plate reader assays, residual assays for process impurities such as host cell proteins and DNA, and 

 potency assays rather than animal models,” Bakewell adds. “Improvements in automation that remove analyst subjectivity for compendia methods such as color, clarity, particulate matter, sterility, and endotoxin analysis have increased the reliability of these methods.”

More recently, Hersch includes, advanced protein engineering has given rise to synthetic antibody-like molecules, including bispecifics, ADCs, or CARs with an antibody-derived targeting domain. “The analytical methods associated with modern protein design are now nearly always high-throughput and require automation, with researchers routinely screening large panels of molecules in different formats to select the very best candidates for further development. A large amount of data is generated at each step and using sophisticated data management solutions that can handle and structure all the associated data centrally across an organization have become the norm in the industry,” Hersch says.

Morales adds that technology, software, and biological knowledge have evolved in such a way that cell-based potency assays are expected to be more robust and precise by default. “Phase-appropriate validation enables the identification of critical reagents and steps, not only for analytical methods, but also from manufacturing process validations. Cell-based development reports are now an expectation to demonstrate that efforts were made to optimize assay precision,” he states.

Fitzgibbons further adds that advances in characterization have occurred within all areas, from improvements in cell-based functional analysis to detection mechanisms in MS-based solutions involving higher-resolution instrumentation. “In several instances, such as in mass spectrometry, data processing solutions have driven evolution where the large amount of data acquired has required powerful software to process the volume of information present in the analysis,” he says.

One hot analytical topic for biopharmaceuticals over the past few years has been sub-visible particle analysis, including particle counts using microflow imaging analysis and high accuracy products liquid particle counters, says Gary Watts, Analytics Manager at Abzena. “The understanding that ‘particles breed particles’ potentially leading to drug products failing specifications has led to emphasis on early detection to help de-risk the drug development pathway by identifying and removing the likelihood of any issues further down the line,” he explains.

Huang reinforces how analytical advancement has made significant strides for the sake of biological drug development since the 1980s, noting that advances in liquid chromatography (LC) and CE have had significant impact on biopharmaceutical development by improving method robustness and throughput.

“There was a time when scientists had to identify all peaks in a peptide map using Edman degradation—a tedious task with limited sensitivity and robustness—but now the peptide sequence can be easily elucidated via LC–MS. Beyond this, peptide-based multi-attribute method (MAM) as an emerging technology has the potential to offer enhanced sensitivity and selectivity for the simultaneous detection and quantification of multiple product attributes, even in the quality control (QC) laboratory,” Huang says.

For binding assays, Huang says, surface plasmon resonance and biolayer interferometry technologies have become increasingly promising compared with the conventional enzyme-linked immunosorbent assay binding method and have been used in both biologics development and quality control labs.

Moving forward, the increasing complexity of biologic modalities in drug development may also lead to analytical needs not yet met by current methods. “The field of complex biologicals is diverse and expanding rapidly,” says Hersch. “Standardization of analytical requirements for full characterization and quality control of these new modalities is, in some cases, still evolving and represents an important unmet need.”

According to Hersch, unmet need in the industry includes quality control analyses for the newest autologous cell therapy approaches, which must be performed quickly to ensure the shortest possible vein-to-vein time for critically ill patients. “To meet this need, researchers may have to rely more on faster NGS-based methods rather than more time-consuming traditional 

In addition, as biologic modalities become increasingly complex (e.g., bispecific antibodies, gene therapies, cell therapies), there will be a continued need to develop methods for characterization of the active biologics’ components, says Fitzgibbons. This development may involve analytical techniques that are only just now becoming widely used. “As new higher-resolution analytical techniques and more hybridization occurs within instrumentation, new opportunities for characterization present themselves,” he states.

One example of instrument hybridization involves the combination of CE techniques with MS, Fitzgibbons says. With hybridization such as this, identification of size or charge heterogeneity profiles becomes much more straightforward, he notes. He also says that analytical instrumentation that automates processes that were previously undertaken manually offers higher throughput and opens the way for new regulatory requirements for analytical characterization. “Additionally, as the development in viral vector manufacturing progresses, and as new gene therapy offerings are developed, new analytical challenges arise to assure CQAs are understood and controlled,” he adds.

Meanwhile, on-line/real-time monitoring of product attributes has gained increasing interest due to advancement in continuous processing for biologics manufacturing in recent years, says Huang. He also adds that a greater level of automation in sample preparation as well as applying MS techniques, such as peptide-based MAM, which has been used in quality control to simultaneously monitor different product attributes, can greatly enhance real-time monitoring and expedite biologics development.

“One gap that needs addressing is industry coalescence in developing standardized methods and reagents,” adds Colangelo. He points out that many manufacturers still develop fit-for-purpose methods for each biotherapeutic, which leads to a continual reinvention of the analytical methods for every new drug product.

Bunce points out that capacity is a major issue because all providers are looking to access platforms that can express more material in less volume, while maintaining the sensitivity of the process at scale. “Consequently, the industry needs to ensure that the analytical tools and methods are fit-for-purpose and will robustly inform the maintenance of the overall integrity and stability of the biopharmaceutical,” he says.

Bunce also stresses that it is imperative to get the analytical strategy and methodologies right in the early stages to support confidence in the results and mitigate issues with quality downstream. “Paying attention early on is crucial to long term success of the biologic and mitigates risk of correcting issues at later stages of development, which may have an impact both in terms of economics and safety to the patients. Getting things right up front is a mindset to be encouraged in analytical method development to characterize biopharmaceuticals and reduce risk in the drugs that we develop,” he asserts.

Marc Wolman, principal scientist at PPD Laboratories’ GMP Lab, meanwhile, explains that the biopharmaceutical industry has made significant strides in managing logistics and working within critical timelines, which has been especially and recently evidenced by the successful development, production, analyzation, and review of new pharmaceutical products in record time to battle COVID-19. “However, we also saw that drug sponsors, raw material and testing reagent suppliers, CDMOs [contract development and manufacturing organizations], CROs [contract research organizations], and regulatory agencies have the opportunity to implement faster, more-efficient means to develop, produce, analyze and review new pharmaceutical products, all while maintaining a critical eye on safety and quality,” he states.

For analysis of biotherapeutic product CQAs, the advent of rapid microbiological methods, rapid high-throughput screening methods, rapid release and stability methods—in particular rapid bioassay methods that mimic the 

 functionality of the product—are all areas where enhanced turnaround times will be critical to getting new products to market, Wolman emphasizes.

“The ability of the industry to meet rapid manufacturing and testing turnaround times will define the success and availability of promising autologous cell-based therapies,” Wolman adds. For example, to support an autologous cell therapy, a patient’s cells must be harvested and modified, and the modified cells must be rapidly tested for structure, function, identity, purity, and safety-related CQAs before the patient can be infused with the modified cells. “The analytical testing requires coordination of sample shipping and receipt with critical handling, execution of assays and data review, and quality assurance approval of the samples’ testing report,” Wolman explains. “This monumental effort may have a limited timeline of one to two weeks given the short shelf life of the 

 modified cells and the patient’s life-depending need to receive the therapeutic.”

When referring to this article, please cite it as F. Mirasol, “Complex Biomolecules Require Analytical Evolution” 

Articles

The ever-increasing complexity of biotherapeutic molecules presents unique analytical challenges for developers.

Complex Biomolecules Require Analytical Evolution

Novel biotherapeutics are expected to have higher heterogeneity (variants); learning and understanding what these variants are will increase assurance of product consistency, says Pedro Morales, director, Catalent Biologics. He explains that massive genomics and proteomics data will need to be correlated with functionality data to identify what is truly critical to set specifications for release. “The establishment of proteomics and genomics databases are also a challenge as one needs to consider not only the product, but the genetic makeup of the patient as part of the correlation. Next-generation sequencing will be crucial to the implementation of cell and gene therapies (CGTs) because of the nature of personalized medical approaches,” Morales says.

As the industry moves gradually towards more personalized biotherapeutics, the ability to craft a biotherapeutic for each patient in real-time becomes more of a reality, asserts Christopher Colangelo, biopharma business development leader, Agilent Technologies. He states that there is a need, however, to scale and commoditize lab analytical workflows so that the same robust purification and critical quality attributes characterization can be performed at a lower cost per sample, without compromising sample or data quality.

Decreasing the cost of developing immunotherapeutic modalities, especially those based on CGT approaches, will be essential for their broad adoption, says Jana Hersch, scientific consultant, Genedata. “Innovative digitalization and new manufacturing practices will provide key solutions. New and enhanced digital solutions are already becoming available to support CGTs. Since cell and gene therapies often serve relatively small patient populations, miniaturization with patient-scale manufacturing represents another tangible step towards lowering costs,” she says.

“While these technologies predated the therapeutics, mechanisms to manage the data generated from these analytical approaches and to validate the instruments’ software for GMP [good manufacturing practice] testing is lacking. For nucleic acid sequencing, whole genome and single cell-based sequencing platforms continue to evolve with new, powerful software tools to support data analysis,” Marc Wolman, principal scientist at PPD Laboratories’ GMP Lab says. These tools, however, require substantial manual intervention and analyst interpretation when performing the analysis or retrieving sequencing data from tangential databases, he specifies.

“If techniques like AUC [analytical ultracentrifugation] and NGS [next-generation sequencing] are to become mainstream GMP methodologies, then innovations in data management and analytics regulations must be developed and implemented to meet 

The methods used to characterize, inform, and select the best biologic drug candidates are becoming more complex, requiring different analytical parameters to be assessed, observes Campbell Bunce, chief scientific officer at Abzena, who reinforces the need for speedy analysis with emerging drug platforms. For example, in the context of personalized cell therapies, a fast turnaround time from cell collection, modification, scale up, and treatment are critical to the patient outcome. “Innovative means of applying sophisticated analytics to these complex therapeutics in a fast and efficient way is required to help streamline the manufacturing process to increase the chances of success and pre-empt or reduce safety risks” he says.

When referring to this article, please cite it as F. Mirasol, “Meeting Future Analytical Requirements for Novel Biotherapeutics” 

The future pipeline of complex novel biotherapeutics will require new approaches to analytical assessment.

The emergence of new biotherapeutics has prompted the advancement of innovative technologies that enable or support unique approaches to biotherapeutic drug development, including vaccines. Some of these new technologies include intranasal formulation technology, new cell reprogramming technology, and virus-like particle technology, among others. The progress of drug development technologies will also have implications for the future direction of new biotherapeutic modalities.

The challenges to creating new biotherapeutic modalities stems from technical, scientific, and clinical advances that are resulting in a wider range of these modalities. This wider range of biologic modalities has led to a frequent requirement for bespoke development and characterization on a product-by-product basis to ensure that all possible interests, concerns, and compliance requirements of the international regulatory authorities have been considered and addressed, points out Margaret Temple, business development director of SGS.

“For example,” Temple explains, “Novel cell therapies have very limited or no downstream purification, which leads to a requirement to conduct very detailed safety testing and characterization of the cell bank and clinical product.”

“The development of COVID-19 vaccines in 2020 required an enhanced focus on documentation and study timelines to ensure lot-by-lot data [were] available in a coordinated and timely manner, whilst ensuring full compliance with the product release criteria,” she adds.

Joe Foster, chief operating officer of Mogrify, a UK-based cell therapy company, adds that all medicinal products have strict manufacturing and quality standards to follow to deliver safe and efficacious treatments to patients. In situations where the active pharmaceutical ingredient is biological, variation in the raw material and manufacturing process is difficult to eliminate entirely, which challenges the scalability of manufacturing biological modalities.

“In cell therapy,” Foster notes, “the most clinically successful cell type, T cells, currently follows a highly standardized ad-hoc manufacturing process where the quality of the final product is subject to the quality of the source cells initially isolated from the patient.”

“While ad-hoc protocols are associated with high costs and limited scalability, a challenge more broadly encountered in the translation of cells into safe and efficacious cell therapies is the need to modify, expand, and maintain cells in 

 conditions, evident in the scant number of cell types that have been delivered to the clinic,” Foster adds.

Challenges such as these are what have driven the innovation being seen in drug development and manufacturing technologies. For example, it is expected that the rapid development and regulatory authority approval of several COVID-19 vaccines will lead to a shorter timeframe to develop and approve new biologics, says Temple. This shortened timeframe will be aided by developments in the availability of more rapid testing and techniques, such as 

 toxicology and next-generation sequencing, she adds.

Meanwhile, translating new technologies to commercial-scale production poses its own challenges. “Scaling up of cell culture is a continued challenge in the application of stem and primary cells as therapeutics when compared to the scale up of, for example, stable cell lines such as [those derived from] CHO [Chinese hamster ovary] cells (1),” says Temple.

“In scaling up new technologies, manufacturers are expected to account for the requirements to generate sufficient clinical material and additional material for testing and stability study purposes,” Temple emphasizes.

Another potential challenge, Temple says, is clarifying the chemistry, manufacturing, and controls (CMC)-related studies that must be performed if a novel technology (e.g., novel equipment, novel manufacturing process, and/or novel biological substrates) is used, as there is little or no precedence to follow. However, on the positive side, innovations in the use of synthetic raw materials and disposable equipment are expected to enhance manufacturing process development, she says.

 The furious pace with which COVID-19 vaccines were developed has shone a spotlight on the need for more agile responses to health emergencies, including the need for quicker and easier administration of vaccines. While not necessarily a new technological idea, intranasal vaccines is one potential answer to addressing the need for global administration. US-based Meissa Vaccines is working to develop an intranasal live-attenuated vaccine (LAV) for COVID-19, with Phase I trials already underway (2). Meissa’s vaccine candidate can be manufactured cost-effectively with single-dose immunity to meet global demand, according to Marty Moore, founder and CEO of Meissa Vaccines.

Meissa is overcoming traditional challenges in attenuating a live virus for use in an intranasal vaccine. “The attenuation of a virus to produce a vaccine must balance reducing its virulence while retaining its ability to stimulate an effective immune response, or immunogenicity,” says Moore. The company started out to develop a pediatric vaccine for respiratory syncytial virus (RSV). “Natural RSV infection does not stimulate a robust immune response, and immunity following SARS-CoV-2 infection wanes, so vaccine technologies, such as our AttenuBlock platform, that enhance immunogenicity are required,” Moore explains.

The traditional processes for making live attenuated vaccines involve serial passage, gene deletions, or other mutation strategies, he notes. “These strategies, however, typically affect the viral replication machinery, which can compromise immunogenicity of the vaccine candidate.”

 In Meissa’s case, the development of its intranasal live attenuated recombinant vaccine also carries important opportunities as an end game vaccine for COVID-19. Moore uses the example of the live oral polio vaccine, which was successful in its ability to block transmission. Compared to the inactivated polio vaccine, the oral live attenuated (replicating) polio vaccine provided strong immunity and blocked transmission well. This example motivated the company in its development of an intranasal vaccine candidate.

“That’s our goal for our COVID-19 recombinant live attenuated vaccine: provide strong immunity with a single dose that’s intranasal with a high potential to effectively block transmission,” Moore states.

Meissa’s proprietary platform, known as AttenuBlock, uses synthetic biology to generate live attenuated RSV vaccine candidates designed to increase antigen expression and decrease or eliminate the expression genes that counteract the immune response. “The AttenuBlock platform incorporates 10 years of research and development at Emory University, where researchers employed rational and precise codon deoptimization and other genetic strategies to produce hundreds of targeted mutations into the RSV genome, providing exquisite control over viral protein expression,” Moore explains.

“We used codon deoptimization to reduce the efficiency of translating viral mRNA [messenger RNA] into proteins. By carefully selecting and replacing commonly used codons with nonpreferred codons in viral genes that inhibit the immune response, the translation of these viral mRNAs into proteins becomes inefficient. This approach results in heavy attenuation, optimized immunity, and genetic stability,” Moore further states.

For COVID-19, Moore points out that it is important to note that Meissa’s vaccine candidate is not a whole SARS-CoV-2. “Ours is designed as a live attenuated RSV backbone that expresses a fully functional SARS-CoV-2 spike protein, in place of the RSV antigen. The reason we did that is because coronaviruses, as a family of viruses, have a high rate of recombination, and viruses like RSV do not. We viewed that genetic instability of coronaviruses as a major hurdle to the development of a traditional live attenuated SARS-CoV-2,” he explains.

Live attenuated vaccines are typically manufactured on a cell line with relatively high productivity of doses/liter of capacity, resulting in a significantly lower footprint and cost of goods to manufacture. In addition, the dose of a live attenuated vaccine is typically much lower than all the other non-replicating vaccine types, including genetic (RNA/DNA), viral vectors, protein subunit, inactivated, and virus-like particles (VLPs). Thus, to meet global supply and demand, Meissa is implementing straightforward, economical, and scalable vaccine manufacturing technologies, Moore says.

“Meissa’s vaccine candidates grow well in cell culture to support manufacturing. Furthermore, Meissa’s single, low-dose vaccine candidates allow for a smaller manufacturing footprint and fewer batches to support production,” Moore states.

 When the pandemic began, Icosavax, a US-based biotechnology company pursuing VLP technology applied its technology platform to develop a vaccine candidate against COVID-19. The company’s vaccine program is supported by a $10 million grant from the Bill & Melinda Gates Foundation and $6.5 million from Open Philanthropy, a US-based research and grantmaking foundation (3).

“Although the application of VLP technology to the prevention of respiratory pathogens is fairly new, VLPs have been around for a long time,” says Adam Simpson, CEO of Icosavax. “In fact, licensed VLP vaccines are extremely effective. For example, naturally occurring VLPs have delivered effective licensed vaccines, including against human papillomavirus and hepatitis B.”

Simpson explains that VLP-based vaccines present key parts of the pathogen in a symmetrical and repetitive way, similar to how a virus would present itself. Meanwhile, the immune system has evolved to detect things that are presented thus as a danger signal and to react strongly to them. “However, reworking naturally occurring VLPs has been difficult to do for the display of proteins from certain pathogens, including those with complex heterologous antigens, such as RSV and SARS-CoV-2,” he states.

In Icosavax’s case, the company’s technology is designed to enable the use of VLP vaccine technology for a broader array of pathogen targets. Naturally occurring VLPs have historically induced strong, broad, and durable immunogenicity and protection, Simpson asserts. “However, naturally occurring VLP vaccines have limitations in displaying complex heterologous antigens. Our technology is designed to overcome this limitation to enable the incorporation of a broad array of complex heterologous antigens into VLP structures,” he says.

Virus-like particles: call to innovation.

 The idea behind Icosavax’s technology is based, not on reliance on molecules that naturally form VLPs, but rather on starting from scratch and using the power of computational protein design to create fully self-assembling proteins, Simpson explains. “We do not have to rely on the limits of what we see in nature, and we can optimize the particles accordingly via our technology,” he says.

Icosavax’s computationally designed VLP technology is designed to solve the problem of constructing and manufacturing VLPs displaying complex antigens. “The technology generates computationally designed proteins that separate the folding of individual protein subunits from the assembly of the final macromolecular structure. The individual proteins are expressed and purified using traditional recombinant technologies and then self-assemble into VLPs when mixed,” Simpson illustrates.

“We often describe our VLP technology as a soccer ball. The black parts are at the base of the antigens (e.g., the prefusion structure of the RSV F glycoprotein or the SARS-Co-V receptor binding domain) and the white parts are there as the second piece to help create an icosahedral particle in the middle of the vaccine. When mixed, the black and white parts self-assemble into the soccer ball, displaying the antigens in a repetitive fashion, much like a virus,” Simpson says.

However, the translation from lab-scale to commercial-scale production can be challenging if the technology was not designed with large-scale manufacturing processes and purification methods in mind, Simpson cautions. In addition, he adds, new technologies often have stability challenges at temperatures above sub-zero, which can lead to commercial distribution challenges.

“Our technology utilizes self-assembly of two protein components that can be manufactured using traditional recombinant protein manufacturing techniques. The technology is designed to be highly scalable and distributable,” Simpson says. Icosavax can use off-the-shelf technologies available at many recombinant protein contract manufacturers, he assures. “Both key intermediate components for IVX-411 [the company’s VLP COVID-19 vaccine candidate] have high manufacturing yield, and all data gathered to date support a competitive cost-of-goods. In addition, our final vaccine product is expected to be stable at 2–8 °C,” he adds.

Cell therapy development: the challenges.

 Mogrify, meanwhile, has developed a proprietary suite of platform technologies that utilize a systematic big-data approach to drive the speed, efficiency, and maintenance of cellular reprogramming. Its platform technologies have applications in generating the scalable source of functional cell types required to underpin the development of 

 cell therapies and also the potential to pioneer a new class of 

 reprogramming therapies for indications of high unmet clinical need in immuno-oncology, ophthalmology and other disease areas.

The main hurdle for a new technology such as this in its translation into viable biotherapies is proving clinical benefit and safety 

, Foster says. “In cases of innovative treatment

modalities, especially, there is limited experience and historic data to refer to,” he emphasizes. In terms of the development of allogeneic cell

therapies, for example, clinical efficacy has been proven through autologous cell therapies, but the use of pluripotent cells as a starting material and the integration of genetic modification requires thorough risk assessment for tumorigenicity and genetic instability, he explains.

Cell therapy development: a call to innovation.

 “To deliver safer, more efficacious, and scalable cell therapy to patients, particularly in immunotherapy, developers are aiming to reduce toxicity-inducing factors, shift toward allogeneic cell therapies, and expand the range of clinically viable cell types,” says Foster.

He points out some current developmental trends, such as the use of gene-editing tools to remove immunogenic components, incorporate safety switches, and reduce the expression of cytokines associated with severe adverse events. He explains that the expansion of therapeutic cell types has seen progress in the increase of alternative cell types reaching clinical trials, such as natural killer cells and macrophages. This progress is accompanied by continuous efforts into the characterization of rare subtypes with high levels of desired functionality, such as gamma delta T cells, and the discovery of new cell types through single-cell innovations.

“Taking inspiration from [K. Takahashi et al.’s] discovery of the OKSM [OCT4, SOX2, KLF4 and MYC] pluripotency-inducing factors (4), the field has been working to generate a universal induced pluripotent stem cell (iPSC) source for the derivation of any cell type of interest,” Foster says.

“Considerable effort has been invested into the development of off-the-shelf cell therapies derived from iPSCs; however, progress has been limited by the field’s ability to identify and recapitulate developmental pathways to freely differentiate target cell types from iPSCs whilst also acquiring and maintaining their required functional maturity for therapeutic purposes,” Foster adds.

In the meantime, with the increasing availability of high-throughput bioinformatics data, researchers have developed innovative computational approaches to systematically tackle the challenges posed by cellular reprogramming.

Platform technologies, such as MOGRIFY, utilize transcriptomic and regulatory network data to predict the key regulatory factors and small molecules that direct cellular reprogramming, Foster explains. Using the data in this way enables the transdifferentiation of any target cell type from any source cell type. Other complementary technologies, such as epiMOGRIFY, deploy epigenetics to predict optimal xenogen-free culture conditions for cell maintenance and to support cell reprogramming.

“When combined, such techniques provide a unique opportunity to enhance existing stem-cell-forward reprogramming methods or bypass development pathways altogether, allowing the direct reprogramming of the scalable source of functional cell types required to transform the development of 

 cell therapies, and furthermore, the exploration of new classes of 

 reprogramming therapies, which offer the potential to introduce cell reprogramming 

The future direction of drug development technologies may largely follow the near-term results of biotherapies currently in development and how they are handled. For example, the long-term prospects for mRNA vaccines are contingent on improving on the current cold temperature requirements for storage and shipment.

“If this is feasible, it would be expected that development of nucleic acid-based technologies progresses to develop products with other applications for healthcare, such as protein-based therapeutics. The international regulatory authorities have been supportive of advances in molecular biology, both with regard to product analysis and testing and with regard to product application, and this would be expected to continue,” Temple says. She further adds that it is not unreasonable to suggest that more product and clinical data and data analysis may be required to answer the question of what direction RNA-based therapeutics may take in the future, and to compare the application of nucleic acid technology with, for example, the application of cell-culture derived viral vaccines, which have been developed and approved for a significantly longer time.

Intranasal, vaccines generate both mucosal (IgA) antibodies in the nasal cavity and antibodies that circulate in the blood (serum), providing an alternative to injected vaccines, which typically do not do a sufficient job in blocking transmission, points out Moore.

“In contrast, injected vaccines typically induce circulating but not mucosal antibodies. While circulating antibodies are important for preventing serious lung disease, mucosal antibodies are important for blocking infection and transmission of respiratory viruses,” he states.

“We believe that end-game vaccines for COVID-19 need to be able to do both: prevent disease and block transmission,” Moore adds.

The preclinical data for Meissa’s COVID-19 recombinant LAV candidate shows that the vaccine induces a SARS-CoV-2-specific mucosal IgA response, causes the generation of serum-neutralizing antibodies, and provides efficacy against challenge, Moore emphasizes.

“With the current injected vaccines, we are going to need boosters for both durability and protection against variants. A real mucosal transmission-blocking vaccine could put a tight lid on SARS-CoV-2 and be an important end-game strategy to really put this to bed and get back to normal. That’s why we need intranasal vaccines,” Moore concludes.

Moore says that Meissa’s vaccine candidate has the potential to deliver a single-dose of 104–106 plaque-forming units (PFU)/dose compared with multiple doses of 109–1011 PFU/dose of non-replicating vaccines or multiple milligrams for subunit vaccines. “The footprint for manufacturing live attenuated vaccines is significantly smaller and does not necessitate single use technologies,” he also adds.

Meanwhile, an aging population also poses a challenge for traditional vaccine technology because the older a person gets, the more difficult it is to induce a robust immune response. “We believe that a technology that induces higher neutralizing antibody titers will have the best chance of optimal and lasting protection in older adults,” says Simpson

“Our vaccine candidates have shown a strong immune response in preclinical models, and we believe that our candidates could become important for older adults, where immunosenescence plays a role in the effectiveness and durability of other vaccine technologies,” Simpson states.

“Furthermore,” he continues, “from a global health and access perspective, the high yield and stability of the assembled VLPs suggest that manufacture of VLP vaccines will be highly scalable, and our final vaccine product is not expected to require subzero storage.”

The advantage of Icosavax’ technology is that it was designed to be highly scalable and distributable, according to Simpson. “That said, COVID-19-related efforts have resulted in near-term shortages in materials, and, with the number and scale of existing vaccine manufacturing efforts worldwide, there are limited openings and manufacturing facilities. Right now, the biggest challenge we face is similar to many vaccines and therapeutic manufacturers in the current environment: access to facilities and materials needed for manufacturing and fill and finish at a large scale,” Simpson says.

“Currently, there are a small number of cell types that have been successfully delivered as therapies to the clinic, due to the limited capacity in directing cell fate and cell conversion,” says Foster, speaking from a company translating cell reprogramming into viable cell and gene therapies.

Mogrify is currently in the preclinical proof-of-concept stages of development, so discussion of commercial-scale aspects is early at this point; however, with the broad opportunities offered by the company’s proprietary technology platforms, its biggest challenge to date has been the identification, consolidation, and prioritization of the disease areas of focus, says Foster.

“We have chosen to focus on immuno-oncology and ophthalmology because they are both areas with clear regulatory pathways, and building on known therapeutic potential, will allow us to deploy our novel science and progress our lead assets within a well-defined clinical roadmap,” Foster states.

1. C. Du and C. Webb, “Cellular Systems,” in 

, pp. 11–23 (Elsevier, Amsterdam, Netherlands, Second Edition, 2011).
2. Meissa Vaccines, “Meissa Announces IND Clearance for Phase 1 Study of COVID-19 Intranasal Live Attenuated Vaccine,” Press Release, March 16, 2021.
3. Icosavax, “Icosavax Launches COVID-19 Vaccine Program with Preclinical Data and $16.5 Million in New Funding,” Press Release, Oct. 30, 2020.
4. K. Takahashi, et al., 

When referring to this article, please cite it as F. Mirasol, “New Biotherapies Push Technological Innovation Forward,” 

The emergence of new biotherapeutics is both the driver and result of innovative drug development technologies.

New Biotherapies Push Technological Innovation Forward

Dynamic light scattering (DLS) is a well-known technique for determining sample interactions, particle size, and aggregation of molecules dispersed or dissolved in solution. The technique can also be a reliable method for characterizing protein stability (1).

Among the most challenging aspects of conducting analytical stability studies on protein therapeutics is protein instability, which can be caused by many different mechanisms, says Hanna Jankevics-Jones, PhD, segment manager, pharmaceuticals, at Malvern Panalytical. A change in the local environment around the protein, such as change in pH, ionic strength, or temperature, or an external factor; for example, agitation, can cause stress that affects the non-covalent bonds in the protein’s structure. These stresses can reduce or increase the strength of these non-covalent bonds (depending on the magnitude and direction of change) and thereby affect the likelihood of the protein maintaining its structure and stability, she explains.

“If the stresses are significant enough to cause unfolding, this often manifests itself in secondary effects, such as protein aggregation,” she adds.

Hanna Jankevics-Jones also notes that many analytical techniques used in stability studies measure the secondary effects of instability, such as aggregation, rather than the primary cause of instability, such as reduced repulsion due to increased ionic concentration in the buffer. “A combination of analytical techniques is often needed to better understand or pinpoint the likely cause of instability and identify potential remedies,” she states.

Another challenge with protein therapeutics is that they are quite complex molecules, remarks John Bak, principal scientist, at PPD Laboratories GMP Lab. Proteins are more fragile than typical small-molecule therapeutics, and their function depends not only on their chemical identity (primary structure), but on the shape of the molecule (secondary and tertiary structures), and even multi-subunit proteins (quaternary structure). Proteins are, in addition, particularly sensitive to temperature changes, pH, and conductivity, he says. It is thus imperative that proteins be reconstituted and/or diluted with a diluent in an ionic environment in which the protein is stable. “Ensuring that the proteins are produced in the proper form, and that the form remains stable all the way from production to the patient, is required,” he states.

Because a stability study involves setting aside quantities of candidate formulations for long periods of time to evaluate shelf life, it becomes essential to minimize the number of candidates and pre-optimize the selection as much as possible, adds Dan Some, principal scientist at Wyatt. “This process reduces the risk of not finding a suitably stable formulation and consequently delaying commercialization. Effectively screening a wide range of formulations in order to select the best candidates for long-term stability studies is a major challenge.”

DLS, also known as photon correlation spectroscopy (PCS) or quasi-elastic light scattering (QELS), is just one analytical technique that can offer a solution to the challenges of studying protein therapeutics to determine their stability, says Bak. For ideal samples, DLS has sensitivity starting at a few nanometers. For instance, he points out, one can see sodium dodecyl sulfate (SDS) micelles at about 4 nm. The upper range of detection is about 1000 nm or more, depending on how quickly particles settle. In comparison, laser diffraction methods tend to lose sensitivity under 100 nm, and electron microscopy or X-ray methods tend to have more difficult sample preparation and cannot always be performed in solution. DLS, however, can provide measurements in a few minutes using under 100 µL of solution, Bak explains.

“A protein is far from an ideal scatterer,” remarks Eric Olson, senior research scientist at PPD Laboratories GMP Lab. “Proteins are not hard spheres, but instead deformable and porous, so many of the assumptions of DLS are broken. For instance, DLS assumes particles are moving by Brownian motion, but, since proteins are not hard spheres, the collisions with solvent molecules could be considered inelastic rather than elastic,” he states. Olson uses the example of punching a pillow instead of a basketball to illustrate the difference between protein and small-molecule detection using DLS. “Some of the energy is lost in the collision as it deforms the protein instead of sending it flying in a different direction,” he says.

Proteins may not be ideal scatterers, Bak chimes in, but DLS can still be used to measure them, although it will not have the same sensitivity as with more ideal particles. “Typically, a protein will need to have a minimum diameter of several tens of nanometers to be detected. The sensitivity of a protein will depend on several factors: the relative refractive index; optical density; and the relative degree of hydration inside the structure of the protein to name a few. A very rough estimate of the radius (not diameter) of a globular protein is the cube root of its molecular weight in kilodaltons,” he emphasizes.

Meanwhile, sample preparation is also a significant issue in that the structure of the protein cannot be allowed to change in response to preparing the test solution, Olson points out. “Ideally, the sample should be measured neat. If it needs to be diluted, the formulation buffer should be used to keep the environment the same. Temperature is also an issue, so it must be controlled as well, though most modern instruments will do that,” he says.

In stability studies, DLS plays two major roles, says Some. One is to detect large aggregates in the course of long-term stability studies—aggregates that are too large for size-exclusion chromatography (SEC). The second role is the most important, he notes, which is to screen a matrix of formulations to identify those that are most likely to succeed in the course of long-term stability and other stability stress tests.

Some points to several different measurements that can be made with DLS to quantify stability predictors and help optimize the candidate selection process: aggregation, colloidal stability, thermal stability, and conformational stability:

Size distributions measured by DLS—these measurements are useful in determining the average protein size and detecting the presence of small and large aggregates. A relatively large average size may be indicative of physical instability, and the presence of aggregates is a definitive sign of poor formulation.

Measurement of kD (diffusion interaction parameter)—this parameter is derived from the change in diffusion coefficient (determined directly by DLS) with increasing protein concentration and provides a measure of colloidal stability. Formulations with poor colloidal stability cannot be expected to do well over the long term.

Accelerated stability tests–these tests hold the formulation at an elevated temperature, typically 40 °C, while monitoring aggregation rate. DLS provides rapid feedback on aggregate content of the solution, and the measurement cell may be held at temperature for extended periods, making this method a core predictor of stability, one that is fully enabled by DLS.

Temperature ramp experiment—here, DLS monitors the change in size and molar mass of a protein solution as a function of temperature. The temperatures that experience the onset of unfolding (Tonset) and aggregation (Tagg) are also considered key predictors of stability.

Isothermal chemical denaturation—DLS can also be used to monitor conformational stability via isothermal chemical denaturation. In this technique, the size of the protein is measured as a function of the amount of a denaturant, such as urea or guanidine hydrochloric acid, added to the formulation, which causes the protein to unfold. A higher resistance to chemical denaturation is a sign of higher conformational stability.

Other DLS-based techniques—other techniques are being developed to provide more robust formulation ranking. A technique published in 2018 (2) monitors the protein size upon denaturation and subsequent dilution back to near-formulation conditions. According to the study, proteins that are more stable tend to refold more compactly as the degree of denaturation is reduced.

DLS’ sensitivity is based on the impact of particle size change on the amount of light that particle scatters, adds Jankevics-Jones. The light scattered by particles in the typical size range for proteins “scales as the sixth power of the particle diameter, meaning that even a small change, such as the formation of dimers from monomers, can be detected by an increase in light scattering,” she states.

DLS alone, however, is not sufficient as a standalone analytical technique to evaluate protein stability studies, Jankevics-Jones further states. “As previously mentioned, DLS reports on aggregation, which is really an indicator for many types of instability. It should therefore be applied as part of a portfolio of techniques whose data can be used collectively to identify degradation pathways or compare stability under different environmental conditions,” she says. An example would be the use of DLS in combination with differential scanning calorimetry (DSC) to cross-correlate any aggregation reported by DLS with changes in the higher-order structure and content of folded protein material, she adds.

The level of complexity in protein molecules requires the use of many other technologies in addition to those used in the development of small-molecule therapeutics, Olson says. Including other analytical detection technologies can help ensure that the different levels of protein structure are maintained for the highest therapeutic efficacy, safety, and quality. “One property of proteins in solution that can be monitored is the particle size.Particle size is difficult because proteins can be small. They range in size from a few nanometers to tens of nanometers for individual molecules and up to hundreds of nanometers for multi-subunit structures,” Olson explains.

“Full characterization of a protein therapeutic requires a large battery of tests,” Bak concurs. “Focusing only on particle size, an excellent way to improve the performance of DLS is to use it as a detector on a separation technique such as SEC or field flow fractionation,” he says. Bak explains that the limitations on the minimum size that can be detected will still be there and will be exacerbated with the sample going through a separation; the concentration will be lower, so there will be less signal generated for the DLS detector. “When it does work though, we can use the particle size as a rough check on the molecular weights reported by SEC,” he says.

In long-term storage studies, DLS is primarily useful for its ability to quickly detect large aggregates, adds Some. Some also agrees that DLS needs to be combined with other analytical techniques, including SEC coupled to multi-angle light scattering (SEC–MALS), which identifies small aggregates and fragments; field-flow fractionation coupled to multi-angle light scattering (FFF–MALS), which provides detailed information on both small and large aggregates; various types of high-performance liquid chromatography (HPLC) coupled to mass spectrometry to identify chemical degradation; and other techniques that can identify modifications such as charge variants.

“In the preliminary candidate selection process, DLS should be combined with SEC for fragment/aggregate quantification and techniques mentioned above that evaluate chemical degradation, especially with respect to the different stress tests. It may also be valuable to add dye-based extrinsic fluorescence assays that can detect changes in hydrophobicity and significantly small amounts of aggregates,” Some says. He further adds that another commonly used analytical technique during this stage is differential scanning calorimetry, which is considered the “gold standard” for measuring the protein melting onset temperature of each domain in a multi-domain protein.

An important area of improvement for DLS is that the technique can use multi-angle detectors. Olson says that most DLS instruments only have one angle of detection; however, by using multiple angles one can more accurately perform molecular weight measurements and measure the radius of gyration. These measurements would help account for particles that are not spherical, he states. In addition, using a green or violet laser will increase light scatter and improve sensitivity as well as resolution. “The one drawback of green and violet lasers is that any fluorescence caused by them would interfere with the DLS measurement,” Olson cautions.

“Other techniques that can access this size range also include analytical ultra-centrifugation and resonant mass measurement. X-ray (SAXS) and neutron scattering (SANS) are used for micelle work and can be applied to proteins. Each of these techniques has its strengths and weaknesses, but they can be considered for use,” says Olson.

Meanwhile, another technique that uses Brownian motion is nanoparticle tracking analysis, adds Bak. Rather than monitoring light scatter intensity, a camera monitors the locations where particles are emitting scattered light and analyzes their Brownian motion, allowing for the sizing of individual particles. “Unfortunately, it is much less sensitive than DLS, so it only will detect larger proteins, starting at about 75 nm to 100 nm. For larger aggregates in the micron size range, flow microscopy can be used,” he states.

“DLS can often be a bit of a black box in that it’s easy to get a quantity out, but much harder to confirm that the value is correct and not subject to experimental artifacts, so as to filter out any poor data,” states Some, who points out that one of the main challenges for DLS users sifting through high-throughput experiments is to receive robust indications from the software that the results are valid, when they have many measurements yet to review.

Furthermore, DLS is extremely sensitive to the presence of small amounts of aggregates forming in the sample because of the light scattering’s dependency on size, which means that even tiny changes are magnified in the millions, Jankevics-Jones says. This extreme sensitivity can, however, also be a drawback for DLS as a technique, as low levels of contamination can influence DLS measurements. “This has, up to recently, meant that sample handling has had to be meticulous in order to avoid any extraneous contamination,” she says.

“Recent developments have meant that the analysis algorithms applied to collect and analyze DLS data, such as adaptive correlation (3), have become ‘cleverer’, scanning the profile of a sample at all times and identifying those particle populations that can be detected only occasionally. This reduces the likelihood of results being skewed by extraneous contaminants. It also extends the application of the method to novel, challenging modalities, such as gene therapy vectors and next-generation vaccines,” Jankevics-Jones explains.

Biochemistry and Molecular Biology Education

 40 (6) 372–382 (2012).
2. H. Svilenov, et al., 

 107 (12) 3007–3013 (2018).
3. A.V Malm and J.C.W. Corbett, 


Vol. 34, No. 4
April 2021
Pages: 41–43, 46

When referring to this article, please cite it as F. Mirasol, “Stability Testing of Protein Therapeutics Using DLS,” 

Dynamic light scattering presents a good analytical technique for testing protein stability.

Stability Testing of Protein Therapeutics Using DLS

With curtailed on-site inspection activities for manufacturing facilities in the past year as a result of the COVID-19 pandemic, regulatory authorities such as FDA have had a challenging time. However, maintenance of quality and safety for regularly produced pharmaceuticals remains a priority. The past year especially saw the challenge of a slew of drug recalls caused by the detection of nitrosamine contamination. A roundup of voluntarily initiated recalls due to this contamination is summarized below.

Metformin had the largest number of recalls throughout 2020 and at the start of 2021 with FDA citing nitrosamine contamination as the primary cause of the recalls. Nearly every month, starting in May 2020, drugmakers were announcing/reporting product recalls. In May 2020, Amneal Pharmaceuticals issued a voluntary recall of its metformin hydrochloride (HCl) extended release tablets, USP, in 500 mg and 750 mg strengths because of N-nitrosodimethylamine (NDMA) impurities detected in the products (1). FDA had notified Amneal of the impurity detection after seven lots of the products showed the presence of NDMA in amounts above acceptable levels and recommended the recall. Also in May 2020, Apotex issued a voluntary recall of its metformin hydrochloride extended-release tablets, USP 500 mg, when FDA tested one lot and found NDMA levels above the acceptable limits (2).

June 2020 saw several other companies issuing recalls for their metformin products. Marksans Pharma, Teva Pharmaceuticals USA, and Lupin Pharmaceuticals all issued voluntary recalls for their respective metformin products based on NDMA contamination. Marksans Pharma recalled metformin HCl extended-release tablets, USP 500 mg, lot no. XP9004 (3); Teva recalled 14 lots of metformin HCl extended-release tablets, USP 500 mg and 750 mg, in 100- and 1000-count bottles (4); and Lupin Pharmaceuticals recalled metformin HCl extended-release tablets, USP (generic equivalent of Fortamet), 500 mg, lot G901203 (5).

In July 2020, Lupin Pharmaceuticals issued another recall, this time for all batches of metformin HCl extended-release tablets, USP 500 mg and 1000 mg as a part of FDA’s “ongoing assessment” and the continuation of a dialog between company and agency under which additional analysis showed that certain tested batches exceed acceptable limits of NDMA. Lupin Pharmaceuticals decided to issue the July recall “out of an abundance of caution,” the company stated in its announcement at the time (6). In the same month, Granules Pharmaceuticals voluntarily recalled 12 lots of metformin HCl extended-release tablets, USP 750 mg in 100- and 500-count bottles (7).

In August and September of 2020, Bayshore Pharmaceuticals and Sun Pharmaceutical Industries, respectively, issued voluntary recalls for their metformin products. Bayshore recalled one lot, lot no. 18641, of metformin HCl extended-release tablets, USP 500 mg, in 1000-count bottles and one lot, lot no. 18657, of metformin HCl extended-release tablets, USP 750 mg, in 100-count bottles after testing found NDMA levels in excess of acceptable limits (8). Sun Industries recalled one lot, lot no. AB06381, of its Riomet ER (metformin HCl for extended-release oral suspension), 500 mg per 5 mL (9). Meanwhile, later in October 2020, Marksans Pharma expanded its earlier recall to include an additional 76 unexpired lots of metformin HCl extended-release tablets, USP 500 mg and 750 mg, out of an abundance of caution after testing unexpired identified marketed lots (10).

Nostrum Laboratories issued multiple voluntary recalls towards the end of 2020 and in early 2021. In November 2020, the company voluntarily recalled two lots of metformin HCl extended-release tablets, USP 750 mg (11), and two lots of metformin HCl extended release tablets, USP 500 mg (12). Following these announcements, the company issued further recalls in January 2021 for two more lots of metformin HCl extended-release tablets, USP 750 mg (13,14).

Ranitidine was another drug plagued with NDMA impurities last year. In January 2020, Appco Pharma and Denton Pharma, Inc. dba Northwind Pharmaceuticals, an affiliate of Northwind Pharmaceuticals, issued voluntary recalls for ranitidine products. Appco recalled all quantities and lots, within expiry, of ranitidine HCl capsules in 150 mg and 300 mg strengths (15). Denton Pharma also recalled all unexpired lots of ranitidine tablets, 150 mg and 300mg, in response to the manufacturer’s recall of all unexpired lots of ranitidine tablets, 150 mg and 300 mg, which Denton Pharma had repackaged (16).

Later in February 2020, American Health Packaging recalled 11 lots of ranitidine tablets, USP 150 mg, 100-count unit dose blisters. The company initiated its recall in response to the manufacturer’s (Amneal Pharmaceuticals) recall, which included affected lots that were repackaged by American Health Packaging (17).

Besides metformin and ranitidine, some other solid-dose products were also affected by NDMA contamination. In January 2020, Mylan, via its US-based business, Mylan Pharmaceuticals, issued a recall of three lots of nizatidine capsules, USP 150 mg and 300 mg strengths. The recall was based on the detection of trace amounts of NDMA in the API, nizatidine, USP, which was manufactured by Solara Active Pharma Sciences (18).

In addition to NDMA contamination, the pharmaceutical industry has had to contend with other contamination-based recalls in the past year. Taro recalled one lot, lot no. 331771, of lamotrigine 100 mg tablets in 100-count bottles in January 2020. The lot was found to have been cross contaminated with a small amount of enalapril maleate, another drug substance that is used to manufacture a different product at the same facility (19).

In May 2020, MasterPharm recalled one lot of Finasteride Plus 1.25mg capsules when an independent testing laboratory found undeclared minoxidil, an antihypertensive drug, in the product at levels that exceed FDA limits. According to a company statement, consumption of undeclared minoxidil would likely result in low blood pressure, rapid heartbeat, and salt and water retention, which leads to swelling, and could put patients at risk for developing heart failure or other heart damage (20).

Aside from contamination, some solid-dose drugs were recalled because of failure to meet certain specifications. In April 2020, Avet Pharmaceuticals recalled eight lots of tetracycline HCl capsules, USP 250 mg and 500 mg, in 100-count bottles because of low out-of-specification dissolution test results (21).

Similarly, Torrent Pharmaceuticals recalled one lot, lot no. BFD1G001, of anagrelide capsules, USP 1 mg in 100-count bottles also due to dissolution test failure that was detected during routine quality testing. Failed dissolution would likely result in a slower rate and extent of drug release, meaning that less anagrelide would be available in the body (22).

In a separate situation, certain thyroid medications were recalled due to potency issues. In May 2020, Acella Pharmaceuticals recalled 13 lots of 30-mg, 60-mg, and 90-mg NP Thyroid (thyroid tablets, USP, composed of levothyroxine and liothyronine) when testing found those lots to be superpotent, containing potentially up to 115% of the labeled amount of liothyronine, one of the APIs (23).

In September 2020, RLC Labs recalled 483 lots of Nature-Throid and WP Thyroid (thyroid tablets, USP, composed of liothyronine and levothyroxine) in all strengths and all counts of product, including 30-, 60-, 90-, 100-, and 1000-count bottles. Potency was also an issue in this recall; FDA tested samples from six lots and found them to be sub potent (24). Similarly, Acella also recalled one lot, lot no. M327E19-1, of 15-mg and one lot, lot no. M328F19-3, of 120-mg NP Thyroid in September 2020 also because testing found these lots to be sub potent (25).

Amneal Pharmaceuticals LLC Issues Voluntary Nationwide Recall of Metformin Hydrochloride Extended Release Tablets, USP, 500 mg and 750 mg, Due to Detection of N-Nitrosodimethylamine (NDMA) Impurity

Apotex Corp. Issues Voluntary Nationwide Recall of Metformin Hydrochloride Extended-Release Tablets 500mg Due to the Detection of N-nitrosodimethylamine (NDMA)

Marksans Pharma Limited Issues Voluntary Nationwide Recall of Metformin Hydrochloride Extended-Release Tablets, USP 500mg, Due to the Detection of N-Nitrosodimethylamine (NDMA)

Teva Pharmaceuticals USA, Inc. Initiates Voluntary Nationwide Recall of Metformin Hydrochloride Extended-Release Tablets USP 500 mg and 750 mg Due to Detection of N-Nitrosodimethylamine (NDMA)

Lupin Pharmaceuticals, Inc. Issues Voluntarily Nationwide Recall of One Lot of Metformin Hydrochloride Extended-Release Tablets USP, 500mg Due to the Detection of N-Nitrosodimethylamine (NDMA)

Lupin Pharmaceuticals, Inc. Issues Voluntarily Nationwide Recall of Metformin Hydrochloride Extended-Release Tablets, 500mg and 1000mg Due to the Detection of N-Nitrosodimethylamine (NDMA) Impurity,

Granules Pharmaceuticals, Inc. Issues Voluntary Nationwide Recall of Metformin Hydrochloride Extended-Release Tablets USP, 750 mg Due to the Detection of N-Nitrosodimethylamine (NDMA) Impurity

Bayshore Pharmaceuticals, LLC Issues Voluntary Nationwide Recall of Metformin Hydrochloride Extended-Release Tablets USP, 500 mg and 750 mg Due to the Detection of N-Nitrosodimethylamine (NDMA) Impurity

Sun Pharmaceutical Industries, Inc. Issues Voluntary Nationwide Recall of RIOMET ER (Metformin Hydrochloride for Extended-Release Oral Suspension) Due to N-Nitrosodimethylamine (NDMA) Content Above the Acceptable Daily Intake (ADI) Limit

Marksans Pharma Limited Issues Expansion of Voluntary Nationwide Recall of Metformin Hydrochloride Extended-Release Tablets, USP 500mg & 750mg, Due to the Detection of N-Nitrosodimethylamine (NDMA)

Nostrum Laboratories, Inc. Issues Voluntary Nationwide Recall of Metformin HCl Extended Release Tablets, USP 750 mg, Due to N-Nitrosodimethylamine (NDMA) Content Above the Acceptable Daily Intake (ADI) Limit

Nostrum Laboratories, Inc. Issues Voluntary Nationwide Recall of Metformin HCl Extended Release Tablets, USP 500 mg, Due to N-Nitrosodimethylamine (NDMA) Content Above the Acceptable Daily Intake (ADI) Limit

Nostrum Laboratories, Inc. Expands Voluntary Nationwide Recall of Metformin HCl Extended Release Tablets, USP 750 mg, Due to N-Nitrosodimethylamine (NDMA) Content Above the Acceptable Daily Intake (ADI) Limit

Appco Pharma LLC Issues Voluntary Nationwide Recall of Ranitidine Hydrochloride Capsules 150 mg and 300 mg Due to an Elevated Amount of Unexpected Impurity, N-Nitrosodimethylamine (NDMA)

Denton Pharma, Inc. dba Northwind Pharmaceuticals Voluntarily Recalls All Unexpired Lots of its Ranitidine Tablets and Ceases Distribution, Due to Possible Presence of N-nitrosodimethylamine (NDMA) Impurity

American Health Packaging Issues Voluntary Nationwide Recall of Ranitidine Tablets, USP 150 mg, 100 Count Unit Dose Blisters Due to the Detection of N-nitrosodimethylamine (NDMA) Impurity,” Company Announcement

Mylan Initiates Voluntary Nationwide Recall of Three Lots of Nizatidine Capsules, USP, Due to the Detection of Trace Amounts of NDMA (N-Nitrosodimethylamine) Impurity Found in the Active Pharmaceutical Ingredient Manufactured by Solara Active Pharma Sciences Limited

Taro Pharmaceuticals U.S.A., Inc. Issues Voluntary Nationwide Recall of Lamotrigine Tablets USP, 100 mg, 100 Count Bottles

MasterPharm, LLC. Issues Voluntary Nationwide Recall of Finasteride Plus 1.25mg Due to the Presence of an Undeclared Antihypertensive Drug,” Company Announcement

Avet Pharmaceuticals Inc. Issues Voluntary Nationwide Recall of Tetracycline HCl Capsules USP, 250 mg and 500 mg Due to Failed Dissolution Specifications

A slew of solid-dose drug recalls were issued over the course of 2020 and in early 2021 after nitrosamine contamination was found in products.