The biosimilars landscape is growing as market factors, such as pricing pressure, advanced bioprocessing and analytical technologies, and upcoming patent expirations for innovator biologics drive the development and commercialization of biosimilar products.

There are 45 biosimilars approved in the United States, which represents amazing progress over the past 10 years or so, remarks John Gabrielson, senior vice-president at JSR Life Sciences and head of Similis Bio.

Still, Gabrielson notes that the US lags roughly 10 years behind Europe in marketing biosimilars, with the first biosimilar, Zarxio (filgrastim-sndz), approved in the US in 2015 (1). “We’re looking at close to two decades now of experience worldwide with biosimilars,” Gabrielson says. “I think we’ve come a long way. I think the market is extremely strong right now, but there are still some challenges.”

Zara Bukhari, a PhD student in biochemistry at University of the Pacific, points out that bringing a biosimilar to market can be arduous, in particular because the developer has to have a large set of analytical similarity data when compared to the innovator biologic.

Another point that a biosimilar developer must contend with is interchangeability, Bukhari also points out. For example, with a small-molecule drug, it is simple to replace that drug at the pharmacy level with a generic because their similarity has been demonstrated. However, it is not the same when dealing with biologics because biologics are larger and more complex molecules. Thus, to show that a product is exactly similar, that it would have the same potency, the same kind of reaction in the body, any immunogenicity, or that it would have the same kind of impact that an innovator has, is quite challenging, Bukhari emphasizes.

“You need to show that they are interchangeable, say, if the innovative product is not available, then the doctor can feel comfortable prescribing a biosimilar,” Bukhari says, pointing to the fact that the comfort level of physicians is a significant factor in how well received a biosimilar is in the market. “Even though FDA has things in place, and there have been many talks that there is much money spent on a clinical trial to show that [a biosimilar and its innovator product are interchangeable], much of the time, the comfort level or the hesitancy is there among doctors—and patients—in prescribing the same medicine as that of the innovator. Even though [the biosimilar] is at a lower price, and you’ve established that it is pretty stable, will do the same thing in your body. That is a hindrance in biosimilars, I would say, the comfort level that doctors and patients feel in terms of getting an alternate medicine that would have the same impact,” Bukhari explains.

Noticeably, sales of biosimilars have not been as high compared to innovator biologics, despite being on the market for years, Bukhari remarks.This points to less-than-stellar uptake.

Gabrielson agrees, stating that market uptake has not met expectations in certain cases. This is partly due to the hesitancy among doctors and patients regarding the difference between biosimilarity and interchangeability, both Bukhari and Gabrielson specify.

“I think [this perception] is unique in the US market compared to other areas of the world,” Gabrielson says. “In the US, a biosimilar can be approved as a biosimilar without being interchangeable, or it can be approved as a biosimilar that is interchangeable, [which requires] switching studies as part of the clinical arms of the biosimilar approval, at least in some cases, to demonstrate that the product is, in fact, interchangeable.”

Gabrielson, however, considers that it may be a mistake to allow these two conditions of biosimilar approval in the US (i.e., biosimilar approved without interchangeability or biosimilar approved with interchangeability). “I feel like what [has been] created is two classes of biosimilars in the US market, where some are judged to be of ‘higher’ quality because they’re interchangeable, which wasn’t the intention. … A better model would be one where any biosimilar that’s approved, that goes through the rigorous approval process, by regulators in the US or elsewhere is automatically deemed to be interchangeable with its reference product, similar to the EU designation system. I think that would ease the burden,” Gabrielson says.

Easing the way for market uptake of biosimilars also requires discussion, Gabrielson notes. There is a need for discussion between patients and doctors and alignment among physicians with a goal toward raising the comfort level in prescribing biosimilars in lieu of the innovator product. The responsibility in changing the misperception that there exists a tier of “higher-quality” biosimilars falls upon multiple areas.

“It’s not a single organization or even a single sector that needs to take the lead in this. It’s about multiple groups working together to really change that mindset,” says Gabrielson. Certainly, the industry needs to take a role as well as regulators, he adds. Furthermore, he believes that Congress may also need to step in and change the underlying law that dictates interchangeability.

“[Interchangeability] is built into federal and state laws, which doesn’t provide FDA with as much flexibility as is needed,” Gabrielson states, going on to say, “but most of it really comes down to education.” Gabrielson specifies that education is needed in terms of: how to educate physicians on what biosimilars are, their quality, how rigorously they’re tested to ensure that they are safe and efficacious; and that, if a biosimilar doesn’t have the interchangeability designation, it is still a safe and effective treatment.

“Much of it really is just education materials that can be created by, for example, trade organizations or companies speaking to physicians, and, ultimately, even directly to patients,” Gabrielson says.

Other hurdles to bringing a biosimilar to market include the availability of the reference biologic and the batch-to-batch variability of that reference product; this is on top of the complexity of the molecule, notes Bukhari. “[Furthermore,] since you’re dealing with biologics, you cannot have exact true copies,” she adds. “The process and manufacturing aspect of it is patented, it’s not released. So, [a biosimilar developer] would need to figure it out [for themselves] and understand the process, and make it cost effective, all at the same time.”

Add to that the time it takes to bring a biosimilar to market and the challenge of navigating the regulatory pathways. “As mentioned earlier, [companies] have to provide much more CMC [chemistry, manufacturing, and controls] data for biosimilars compared to the innovator, and that takes time. [Companies] have to establish more steps, and need to have much more stability data available to show that [the biosimilar] product is actually doing what it is supposed to do,” Bukhari explains.

Another hindrance to progress is the ideology of adopting the innovation aspect and including it to justify one’s data to FDA, Bukhari adds. “Say [a company has] taken up an innovative method to produce a biosimilar, then that method also has to be validated. That validation data also has to be given to FDA, and that requires more time and cost,” she says.

“When we think about what it takes to bring a biosimilar to market compared to what it takes to bring an innovative product to market, of course, there’s a much lower clinical barrier, fewer clinical requirements. [It’s] abbreviated for a biosimilar,” says Gabrielson. However, Gabrielson clarifies, what most people don’t realize is that the amount of data from a CMC perspective, and particularly data demonstrating analytical similarity, is the same or higher than what it would take to bring an innovative product to market.

“There’s so much work that goes into demonstrating, analytically, that a product is comparable or similar to its reference product in order to achieve that type of regulatory approval,” Gabrielson states.

On the pricing pressure aspect in the biopharma market, Gabrielson asserts that pricing pressure is a central theme in biosimilars. “That is why these products exist, fundamentally,” he remarks, “to make life-changing medicines more available, more accessible, and more affordable to patients throughout the world. I think it makes sense that there is pricing pressure in this market, because I think everyone’s goal with this is to make medication more affordable to patients.” Thus, rather than a hindrance, and rather than existing in a mindset where developers need to find a way to overcome pricing pressure, Gabrielson points to pricing pressure on the market as the reality, and should be perceived as such.

“It’s a feature of the system, not a bug,” Gabrielson states, explaining that what biosimilar developers need to be doing is figuring out creative and innovative ways to build businesses that can be successful in an environment where there is going to be sustained, consistent pricing pressure. “Pricing pressure can come from lower priced, brand name biologics when they come off patent, it can come from competing biosimilars, but that’s the market. It’s not exactly like the generics market, but there are some parallels between what [is being seen] with biosimilars and what [has] already [been] seen with generics in terms of that pricing pressure. So, a good question that really needs to [be] asked [of] developers is, how is [a] particular product viable commercially, and even differentiated in the marketplace, in the face of this type of competition?”

1. FDA. FDA Approves First Biosimilar Product Zarxio. Press Release, March 6, 2015.

Feliza Mirasol is the science editor for 


Volume 37, No.3
March 2024
Pages: 8–9,29

When referring to this article, please cite it as Mirasol, F. Moving Biosimilars Forward in a Hesitant Market. 

Articles

Cover Story

Despite a growing number of biosimilar approvals, market uptake remains a challenge.

Moving Biosimilars Forward in a Hesitant Market

Submitted: November 8, 2022.
Accepted: October 31, 2023.

The harvest step in a monoclonal antibody downstream purification process removes biomass, particulates, and other material from the cell culture to generate a product stream suitable for purification by liquid chromatography. In a widely employed, two-step harvest process, coarse solids and intact cells are first removed using a centrifuge, and the resulting centrate is further clarified by depth and membrane filtration in series. Conversely, direct depth filtration has the potential to greatly simplify the overall harvest process by directly loading the cell culture broth on high-capacity depth filters and consequently removing the need for centrifugation. The authors evaluated this potential of direct filtration for multiple biopharmaceutical candidates to understand the underlying mechanisms that define fouling and the resulting capacity limitations in these filters. Here, the authors present a methodology to assess the contribution of individual filters to overall capacity of the direct filtration train, and elucidate how capacity is affected by characteristics of the cell culture broth and process parameters, including for enhanced upstream processes such as those incorporating high cell density perfusion. The findings highlight the limitations of direct filtration when implemented in large-scale facilities for high cell density processes. Ultimately, increased understanding of the fundamental principles that govern depth and membrane filtration can aid in moving from a trial-and-error to a more predictive and methodology-based approach for harvest development. This article is Part 2 of the study.

In Part 1 of this study, previously published in the February 2024 issue of 

(1), the materials and methods, and study results for fouling Mechanisms of coarse depth filters were described. In this article—Part 2 of the study—the fouling mechanisms of fine depth filters, fouling mechanisms of sterile filters, relation between fouling and filter train capacity, and design strategy during harvest development will all be discussed.

robert.g.luo@gsk.com, is a scientific director, all at Downstream Process Development, BioPharma R&D, GlaxoSmithKline, King of Prussia, Pennsylvania 19406.


*To whom all correspondence should be addressed.


Vol. 37, No. 3
March 2024
Pages: 20–26

When referring to this article, please cite it as Koshari, S.H.S; Zhang, H.; Ubiera, A.; Luo, R.G. Fouling Mechanisms of Filters During the Harvest Development of Monoclonal Antibody Therapeutics with Intensified Upstream Processes—Part 2. 

Peer-Review Research

The authors evaluated the potential of direct filtration for multiple biopharmaceutical candidates. This article is Part 2 of the study.

Fouling Mechanisms of Filters During the Harvest Development of Monoclonal Antibody Therapeutics with Intensified Upstream Processes—Part 2

The growing demand for adeno-associated viral (AAV) vectors in therapeutic applications emphasizes the need for scalable and more efficient manufacturing methods. Establishing proprietary cell lines offers substantial opportunities for achieving high AAV titers. High titers allow shorter production times, rapid production cycles, higher throughput, simplified downstream processes, less likelihood of contamination, and ultimately can lead to greater flexibility in process scale-up, all of which can contribute to a reduction of costs and product variability, according to Ines do Carmo Goncalves, head of project management for cell-line and process development at Cytiva.

Proprietary cell lines may also lower the cost of input materials, improve cell growth properties, and/or improve the quality of the vector product. As such, it is possible for every AAV manufacturing process, regardless of the type of cell, to benefit from cell-line development, according to James Cody, associate director of technical sales and evaluations at Charles River.

Several approaches can be taken: isolation of high-producing natural clones for transient transfection or infection processes; engineering of cells for the same processes; and development of packaging/producer cell lines that eliminate the need for transfection/infection steps. The optimal technique depends on the nature of the AAV vector and intended therapeutic application.

The most widely used approach to cell-line development for AAV manufacturing is single-cell isolation and cloning. “This method offers the capacity to screen thousands of clones, allowing for the identification and isolation of highly productive and robust cell lines for further process development,” says Goncalves.

Notably, incorporating digital software for data management in the process enhances the ability to detect and identify genetic characteristics that were previously unknown, Goncalves adds. “Identifying these traits can help improve outcomes, such as increasing AAV titers or the percentage of full capsids. Once well characterized, these traits can then be introduced into other cell lines,” she comments.

Upon isolation, single cells are cultivated into clonal populations, ensuring genetic uniformity within the selected clone and contributing to the stability and consistency of the cell line, according to Silvia Ungari, upstream vector development manager for AGC Biologics.

Ideally, Ungari observes, selection and cloning are coupled with some type of imaging technology to provide documentation of single-cell sorting and growth, which allows for demonstration of final monoclonality in a single cloning run. Fluorescence-activated cell sorting (FACS) is an advanced method that enables the isolation of individual cells based on specific fluorescence markers that correlate in some way to higher vector titers, providing a means to select cells with particular characteristics, she notes.

SK pharmteco, according to Brian Tomkowicz, senior director of research and development, uses cell printing with high content imaging for selection and cloning. “With this efficient process, it is possible to plate one 96-well plate of single-cell clones in about four minutes. It is, in addition, a completely closed system that does not require a trained operator, as is necessary when using a flow cytometer capable of sorting,” he says.

One of the key advantages of single-cell selection and cloning is avoidance of the need for genetic engineering. “The optimization of a cell line for AAV production via the selection of clones from a bulk population with improved viral protein expression, or packaging efficiency, or other positive mechanisms resulting in an overall increased productivity, allows the identification of naturally occurring high-performing clones without genetic manipulation. These cells are more readily approved by regulatory agencies, potentially expediting the approval process for AAV production,” Ungari explains.

Consistency due to the uniform behavior of a specific clone rather than a heterogeneous bulk population is another advantage, according to Ungari. “Proprietary cell lines typically ensure batch-to-batch reproducibility and reduce variability in AAV titers during large-scale manufacturing,” she says.

To realize optimum results in cell-line development, Ahmed Youssef, senior manager of upstream process development at Ascend, emphasizes the importance of focusing on functional AAV titer as the primary read out. “High vector potency is key to improving both the quality and safety and reducing the cost of goods of a given product,” he comments.

Genetic engineering is used to add desired and/or remove undesired traits from cell lines for production of AAV vectors. As such, genetic modifications can enable not only increased titers, but increased AAV particle quality, according to Goncalves.

It is also possible, observes Bingnan Gu, head of viral-vector R&D with Lonza, to develop novel host-cell lines for AAV production from scratch. “It is essential with this approach to have full documentation of the process and materials used to achieve cell-line establishment/immortalization and to meet safety requirements, such as the absence of any adventitious agents.”

For a given host AAV cell line, one can either perform screening to identify clonal cell lines that can support relatively higher AAV productivity (compared to parental cells), or carry out rational cell-line engineering to upregulate or downregulate host-cell gene expression or introduce new ‘helper’ genes that could lead to increased AAV productivity, Gu explains.

The use of genetic modifications should, Ungari comments, be approached cautiously due to regulatory considerations.

In a packaging cell line, the helper genes and Rep-Cap genes are stably integrated into the cell genome and the AAV is produced upon induction of these genes and transfection of the gene of interest (GOI). In a complete AAV stable producer cell line (PCL), not only the helper and Rep-Cap genes but also the GOI are stably integrated in the cell. AAV production with stable cell lines is triggered upon induction of these viral genes without any helper virus infection or plasmid transfection. An AAV PCL, according to Gu, can significantly improve AAV titer and quality and enable robust large-scale production beyond 2000 L at much reduced cost and complexity.

Cost reduction is achieved because packaging/producer cell lines eliminate the need for costly plasmid scale-up and transfection reagents, according to Tomkowicz. Yields may also be improved without having to necessarily increase scale, he adds, as packaging/producer lines allow the end user to intensify production processes by taking advantage of high density in combination with perfusion culture.

“The key to successful development of packaging/producer cell lines is genetic manipulation of cellular machinery to mitigate host cellular immune responses against the AAV product, which to date has been investigated using a variety of approaches,” Tomkowicz says.

The challenge then becomes the time it takes to establish stable producer cell lines. “As a result, this approach to cell-line development is highly recommended for programs requiring high amounts of recombinant AAV (rAAV), such as preclinical studies performed in large animals, or gene therapies requiring large doses or involving large patient populations (e.g., cardiovascular diseases or diabetes),”Tomkowicz contends.

Both Youssef and Ungari agree that producer cell lines are more suitable for large-scale production and commercialization. “Stable cell lines prove to be better suited for advanced stages of clinical trials or commercial production, particularly due to the considerable time investment needed for their establishment and the requirement for a finalized process. An advantage is that in these phases, higher volumes of vector may be needed, which can be more easily produced in a stable manner,” Ungari notes. Packaging cell lines, meanwhile, are primarily used for small-scale rAAV production in research settings, according to Youssef.

Indeed, Cody believes that packaging and producer cell lines fit within a continuum of production cell options. “The choice of which cell line is best may come down to the timeline to the clinic and the overall goals of the program. A true producer cell line may take the longest time to develop, but may be the most cost-effective approach for commercial production of a gene therapy product,” he observes.

Proprietary packaging and producer cell lines play a crucial role in the advancement of the AAV gene therapy field, according to Goncalves. “They enable the production of high titers of AAV vectors with high quality while also ensuring scalability and reproducibility. In addition, the use of stable packaging and producer cell lines offers a massive reduction in the costs of goods for producing AAV vectors in quantities that meet the industry demand,” she states.

It is that cost reduction potential of producer cell lines that most interests Marguerite Campbell, associate director of research and development at SK pharmteco. “Million-dollar gene therapies are not sustainable for rare-disease individuals or large patient populations suffering from more prevalent diseases. Having off-the-shelf packaging lines with easy conversion to producer cells not only democratizes gene therapies, but provides benefit where it is needed most,” she comments.

While all cell types can benefit from cell-line development efforts such as enhancing packaging capacity or ensuring robust expression of AAV components, the specific actions required may differ based on the manufacturing process mechanisms involved.

Insect cell lines, for instance, suffer from heterogenous AAV vector packaging and thus require specific attention to vector integrity, says Youssef. Cell-line development for these cells also tends to focus on facilitating efficient viral entry or evading the anti-viral immune response, according to Ungari. Mammalian, and more specifically human cell lines, used for transient transfection processes, meanwhile, present process intensification challenges, Youssef notes. As a result, proprietary cell lines often are customized for improved transfection efficiency, Ungari remarks. “It is therefore crucial to tailor the optimization of each cell line according to its specific manufacturing process and specific product,” she concludes.

When it comes to developing packaging/producer cell lines, Tomkowicz notes that while transitioning from a triple transfection-based process is relatively straightforward because downstream considerations for purification and analytics are comparable, each cell line (HEK293, Sf9, etc.) presents its own challenges, and the characteristics of the selected cell line (origin/derivation, doubling time, and permissiveness for viral infection and replication) determine the efficiency of viral productivity. He adds that because the insect platform is completely distinct from mammalian HEK293 systems, it often requires more cell and viral engineers/scientists knowledgeable in the design and execution of the two separate platforms.

Developing proprietary cell lines can be a complex and challenging process. The main obstacles lie in the complexities of cellular biology, Goncalves notes. “Guaranteeing high productivity involves complex factors such as packaging efficiency and/or viral genome replication. Achieving maximized productivity demands a deep understanding of how to balance these factors. This level of optimization requires extensive genetic characterization and process development activities,” she observes.

Campbell highlights the need to have a validated cell line to start with as a barrier to beginning cell-line development. “While commercial cell lines exist, further engineering to include pHelper and/or Rep/Cap constructs is strictly prohibited [by regulators]. In addition, finding scientists who can engineer viral packaging constructs and who know how to single-clone and expand cells to GMP [good manufacturing practice]-banking is currently a challenge, as can be gaining access to advanced high-throughput technologies for accelerating cell isolation, screening, and selection. Once those issues are overcome, there is still a need to optimize cell-culture processes and customize the cell-culture media,” she explains.

Screening of cell lines, including selecting the best criteria for screening, is a significant challenge noted by Cody. In addition, he notes that cell lines can take significant amounts of time to develop, screen, and characterize, so unless the increased productivity is robust, it may not always be possible to recoup the cost of cell-line development. The need for comprehensive characterization of new cell lines can also be a hurdle. “Depending on the origin of the cell line and how it was generated, new characterization assays may need to be developed and will be dictated by the type of platform,” he says.

Other technical challenges pointed out by Tomkowicz include the need to assess anti-AAV immunity for understanding clinical data and product development, the need for a rapid assay to quantify infectious viruses and gene therapy vectors, and efficient harvest of AAV vectors from cell-culture media.

Furthermore, according to Youssef, cells affording high titers may not be easy to grow or maintain in culture; prevention of AAV particle degradation via proteases, nucleases, and shear forces is necessary by ensuring high cell viability despite harmful gene products expressed and required for AAV manufacturing; and the safety of AAV particles must be assured through testing for potential contaminants, pathogens, and host-cell genes.

For development of producer cell lines affording high AAV titers, the main challenge also gets back to biological mechanisms. “There is an incomplete understanding of the molecular mechanism that controls AAV production in host cells,” comments Gu. He adds that the necessity to demonstrate cell-line stability for any new cell line regardless of its derivation, which involves several months of testing, represents another impediment.

All these challenges, concludes Ungari, “underscore the need for a comprehensive approach, integrating growth efficiency, viability, stress-related cell resistance, aggregation profile, packaging efficiency, material costs, genetic stability, and regulatory compliance for the successful creation of high-titer AAV-producing proprietary cell lines.”

Ideally, observes Cody, an ideal cell line will improve not just overall productivity, but also product quality (lower residuals, higher percent-full capsids, better infectivity, etc.). Cell-line development, however, brings with it a number of new considerations, many of which play off one another, requiring the management of the benefits and risks. These trade-offs, Youssef says, can be complex and vary depending on the specific manufacturing platform and product being developed.

For one thing, introducing a new cell line into an existing process necessitates a revision of the timing and steps in the process and may impact downstream processing requirements, raw-material consumption and costs, and full/empty capsid ratios, which can potentially affect the overall potency, safety, and therapeutic effectiveness of the AAV product, according to Ungari. There can also be a threshold beyond which higher titers do not afford proportional therapeutics effects.

Changes in productivity can also change the density of cells needed during production, which could in turn impact residuals such as host-cell proteins and/or host-cell DNA (for better or for worse), adds Cody. “It is best to base tradeoff decisions on data, which is why it is important to test the critical quality attributes (CQAs) of AAV vectors made from a new cell line at a scale representative of GMP production,” he remarks.

Potency can also potentially be reduced in high-titer AAV products due to increased aggregation, which can also increase the risk of side effects, according to Youssef. The introduction of new genetic sequences may also necessitate additional release testing.

“Aiming for higher AAV titers in the production process inevitably involves finding a delicate balance with other crucial factors. Striking the right balance between titers and potency, in particular, becomes essential in the optimization process,” Ungari contends. That requires a comprehensive understanding of the process and product and application of a quality-by-design approach during AAV process development, says Gu. Consequently, the availability of robust analytics that enable the definition and quantification of process CQAs is crucial, Ungari notes.

Today, most cell lines are developed to be effective at producing numerous different AAV serotypes and a broad array of genes of interest. Achieving this goal is an active area of research, according to Tomkowicz. He points to both upstream and downstream processes designed to enable the production and purification, respectively, of multiple AAV serotypes.

Such broad applicability, however, does not preclude the need for optimization. Indeed, Youssef notes that several factors can affect the versatility of a proprietary cell line. He highlights the fact that capsid proteins from different serotypes bind partially to different cellular proteins, which can result in sequestration of host cell factors required
for efficient AAV productivity or in increased levels of impurities; different serotypes may use different RNA splice sites; and serotype-specific leaky promotors may express toxic gene products, requiring specific gene-silencing approaches. “For these reasons, it is crucial to always test the cell line with the specific AAV vector intended for production to ensure high titers and to be prepared with mitigation strategies when specific products turn out to be challenging,” he concludes.

Optimization may also be needed for specific combinations of serotypes and genes of interest, according to Goncalves. The main reasons include the interplay between characteristics of the host cell, AAV serotype-specific capsid interactions, and the molecular properties of the gene of interest. It is also possible that different serotypes have specific characteristics that can interfere with the productivity. These issues may potentially be overcome through optimization of the cell-culture conditions, media formulation, supplements used, or transfection protocols, she says.

One approach to the issue of multiple serotypes posed by Ungari is to adopt “plug-and-play” platforms using the same cell line for all serotypes. “This approach facilitates a rapid and streamlined production process, allowing for efficient interchangeability between different AAV serotypes without the need for extensive modifications to the cell line or production process,” she states. Further optimization specific to a serotype or GOI may still be needed, however, through adjustment of various process parameters using a design-of-experiment approach.

The concept of proprietary technology often raises questions about whether its proprietary nature results in higher cost and/or limited access. In the context of cell-line development, there is no agreement on this matter.

Some believe the development of proprietary cell lines has positive implications for access. “A variety of commercially available cell lines offered through non-exclusive licensing provides AAV manufacturers the option to use validated, high-performing cell lines to support expedited pathways to pre-clinical and clinical studies,” Gu believes.

Ungari concurs. She notes that as a contract development and manufacturing organization (CDMO), AGC Biologics actively develops proprietary cell lines that seamlessly integrate with its production platforms, are meticulously designed to align with GMP standards, and are inclusively provided within the batch pricing structure. Simultaneously, the company uses cell lines developed by its clients, often with some fine-tuning, and offers clients the option of elevating their R&D cell banks to GMP status. “This flexibility underscores our commitment to providing comprehensive solutions that cater to the diverse needs and preferences of our partners within the biopharmaceutical industry,” she comments.

Others see the potential for restriction of access to AAV production. “These cell lines are typically owned by private companies, which may not make them available to other researchers or manufacturers,” notes Youssef. Intellectual property protection grants exclusive rights to the owners for commercial use and makes it impossible for others to use those cell lines without permission and/or payment. Smaller companies, meanwhile, may find cell-line development a cost-prohibitive activity, limiting their ability to pursue innovation that could provide significant productivity and quality benefits. Furthermore, Youssef comments that it becomes challenging for researchers to share and collaborate on their work if only a few companies have access to the best cell lines.

“The cell line is a critical component of the manufacturing process, so at the very least, any proprietary cell line is likely to have restrictions on use and distribution,” Cody states. Some proprietary cell lines also come with licensing fees or milestone payments. “For a gene therapy product developer, the key is to decide if the cost of those fees is offset by potential savings on manufacturing cost due to higher productivity and/or some kind of benefit to product quality,” he observes.

The potential result, Tomkowicz summarizes, is the possible reduction in openness and collaboration in the field of AAV manufacturing, with the focus more on individual proprietary advancements rather than communal development.

On the other hand, Tomkowicz acknowledges that proprietary cell lines can “massively decrease” the cost of AAV production. In particular, providing access to packaging/producer cell line technologies is a perfect symbiosis for a CDMO like SK pharmteco that offers AAV and lentiviral vector manufacturing services. “While the use of these cell lines might involve licensing fees or other costs associated with using patented technology, combining the use of proprietary technology along with scalable GMP-processes as a platform technology is immensely cost-effective for the investigator as well as the manufacturer,” he explains.

At the same time, if certain higher performing cell lines are proprietary, those without access to them could be at a competitive disadvantage, potentially stifling innovation and limiting the diversity of approaches in AAV vector production, according to Tomkowicz.

Therefore, Tomkowicz concludes that “it is a delicate balancing act between providing for the patient, yet generating revenue in the form of IP [intellectual property] to support the business and further drive innovation.”

Despite these issues, ongoing development and innovation in cell-line development for AAV manufacturing is expected to continue, with advances having overall positive impacts across the sector. “There will be increasing interest in proprietary cell line development in the future, as there is always pressure to reduce the cost of production, and increasing productivity is one way to make manufacturing more efficient,” Cody observes.

“The development of proprietary cell lines is an ongoing and continuous process that never stops,” Ungari adds. She also notes that the competitive nature of the biopharmaceutical industry motivates companies to stay at the forefront of innovation, and continuous development allows companies to maintain a competitive edge, ensuring that their proprietary cell lines offer superior performance and efficiency compared to existing alternatives. “Moreover,” she says, “advancements in reagents, technologies and AAV knowledge offer new tools for optimizing cell lines, enhancing AAV production efficiency, stability, and consistency.”

There are, adds Goncalves, several positive aspects in the development of proprietary cell lines. In addition to the investment in new technologies and innovation, she points to an increase in the development of specialized applications that expand the use of AAV therapies. “Developing cell lines tailored for specific therapeutic targets helps to enhance the accuracy and effectiveness of gene therapies, ultimately accelerating the path to clinical applications,” she explains. “New proprietary cell lines,” Goncalves continues, “also help in reducing production costs and contributing to strategic collaborations that foster exchange of knowledge and provide access to proprietary technologies.”

Ascend, in fact, considers proprietary cell-line development the most promising approach to increase cell-specific and volumetric productivity of AAV manufacturing platforms in a disruptive manner, according to Youssef. “By adopting a balanced approach to proprietary cell-line development, we can help ensure that AAV gene therapy remains a promising and accessible treatment option for patients with various diseases. Furthermore, we believe that cell-line development will be an enabler for bringing AAV gene therapy to patients suffering from more prevalent diseases at affordable costs of goods,” he comments.

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

®
Vol. 37, No. 3
March 2024
Pages: 10-15

When referring to this article, please cite it as Challener, C. Proprietary Cell-Line Development for High-Titer AAV Manufacturing. 

Development

Proprietary cell lines offer opportunities for achieving high AAV titers.

Proprietary Cell-Line Development for High-Titer AAV Manufacturing

Sudden demand for large-scale manufacturing of messenger RNA (mRNA) drug substances during the COVID-19 pandemic has few parallels in the history of pharmaceuticals. Some might posit penicillin production during World War II as an analog, or perhaps early insulin availability. Pandemics and epidemics such as smallpox, typhus, cholera, influenza, HIV/AIDS, or the bubonic plague, all relied more on infection control for resolution than on manufacturing capacity. New therapeutic uses for mRNA are now ramping up good manufacturing practice (GMP) demand, especially for clinical trial supply. Moderna collaborated with CARsgen Therapeutics to combine an mRNA therapeutic cancer vaccine with a solid tumor chimeric antigen receptor (CAR)-T cell therapy, aiming to treat multiple cancers (1). Pfizer partnered with Beam therapeutics to co-develop mRNA and base editing approaches (2), while BioNTech and DualityBio have a partnership to develop differentiated antibody-drug conjugates, for solid tumors (3). In terms of upstream manufacturing for novel mRNA drug products, this technical complexity forms a tidal wave of question marks.

A prominent paper (4) examined reducing complexity by evaluating what common process could be standardized through extrapolations of clinical trial complexity, based on four published papers Phase I-II dose ranging studies. While not the amounts of product required during the COVID-19 pandemic, clinical trial doses quickly mounted up in terms of both volume and also manufacturing challenges related to techniques pursued, but also formulation delivery requirements. The authors state, “from the four studies, the average mRNA usage was 5500 μg and for the 75 trials in 2021, 412 500 μg of RNA would need to be formulated, purified, sterile filtered, and fill-finished. At concentrations of 100 to 200 μg of mRNA per mL, this would amount to 2063 to 4125 mL of drug product” (4). As an interesting foray into possible future manufacturing, the authors went on to contrast traditional mRNA with a self-amplifying method. Often, this is cited as potentially a 100 to 1 decrease in required finished product due to higher potency performances, but this study chose a picturesque perspective, saying, “assuming the entire population consists of 7.7 billion people, and each person receives two doses, a total of 15.4 billion doses or 4.5 million L of the vaccine is required. In perspective, 4.5 million L would fill two Olympic-sized swimming pools (2500 m

), whereas saRNA [self-amplifying RNA] vaccines, which we assume are 100-fold more potent, would only require 0.02 Olympic-sized swimming pools. Assuming that production costs, which account for materials, RNA, and LNP [lipid nanoparticles] production, scale linearly, vaccinating the entire population with Pfizer/BioNTech’s mRNA vaccine would cost approximately 150 billion USD. In contrast, with saRNA, it would only cost approximately 1.5 billion USD” (4).

Commercial-scale mRNA is produced through chemical, recombinant, or enzyme-based methods. For chains under 100 nucleotides, the standard chemical technique “utilizes phosphoramidite chemistry and solid-phase support promoting chain elongation from the 3′ end to the 5′ end” (2). It is amenable to automation, as is recombinant production through host cells such as 

), which has some cost advantages as it can utilize legacy protein manufacturing lines, already in GMP compliance. Enzymatic processes are newer to arrive and have tradeoffs still being worked through. A study conducted by the Duke Human Vaccine Institute explored creating standardized robust manufacturing processing steps, saying they confronted the lack of established quality standards by, in part, surveying vendor operations “through questionnaire and/or audit relating to the vendors’ quality systems and operations as they pertained to manufacturing and/or testing of the materials” (5). The study adopted the Weismann–Karikó method of production, the upstream process requiring three enzymatic steps of plasmid linearization, mRNA transcription, and DNA template digestion. In the starting out phase, fundamentals the authors emphasized were that “for optimal 

 transcription yields, it is critical to start with high quality template DNA. Some key quality attributes that are required before use include the presence of only a single band of linearized DNA, a supercoiled ratio of at least 70%, and a sequencing-verified correct poly(A) tail length” (5). They also noted that “endotoxin control was the primary raw material risk due to its variability in raw material lots and the difficulty of its removal in subsequent downstream purification steps … reaction agitation, temperature, incubation time, and reactant concentrations were also carefully examined for each reaction step in order to yield a robust upstream platform process across multiple mRNA constructs” (5).

To help guarantee the safety, quality, and regulatory compliance of the final product, Dale Patterson, vice president and general manager, Thermo Fisher Scientific, says, “First, raw materials should be of high quality and meet established specifications. This includes the use of pharmaceutical-grade chemicals, enzymes, and reagents that comply with relevant pharmacopeial standards. Suppliers should provide appropriate documentation, including certificates of analysis, transmissible spongiform encephalopathy agents (TSE) and Bovine Spongiform Encephalopathy (BSE) statements, etc. to demonstrate compliance with GMP requirements.” Patterson also talks about transparency, traceability, and documentation being crucial for ensuring quality control and for meeting regulatory requirements.

Melanie Cerullo, chief quality and regulatory officer, ReciBioPharm agrees and points to FDA’s new guidance on the Advanced Manufacturing Technologies Designation Program. “This is a great program where sponsor companies who are using novel manufacturing technologies can request to be considered,” Cerullo says. “The benefits include early interactions with FDA to help navigate the challenges associated with CMC development of novel technologies … With this program, FDA is saying that they are here to help support innovation and want to interact with companies early on.”

Optimizing synthesis “involves customizing the synthesis parameters, such as nucleotide or amidite ratios, modifications, and purification methods, to enhance DNA or RNA stability, translational efficiency, and therapeutic efficacy,” says Patterson. He goes on to emphasize that “process scalability is crucial for personalized cancer treatments or rare diseases, where patient-specific DNA or RNA therapeutics may be required in small quantities. “Implementing flexible and modular manufacturing systems,” says Patterson, “such as bead-based solid phase mRNA synthesis or biosynthesis of oligonucleotides that can be easily scaled up or down to accommodate varying production volumes is essential. This ensures efficient and cost-effective manufacturing while maintaining product quality and regulatory compliance,” he avers.

Cerullo draws attention to infectious disease, where “you need to scale up the manufacturing process to very high levels as we saw with [COVID-19]. So, the challenge becomes how to make a lot of material most efficiently. We are currently working with [FDA] and MIT [the Massachusetts Institute of Technology] on a grant to develop a continuous mRNA-LNP manufacturing process to enable rapid scale up and manufacturing of large quantities of doses for this very reason.”

Patterson continues, “another strategy is to leverage automation and process optimization technologies. This includes the use of robotics, machine learning, and artificial intelligence to automate production tasks and improve process control. Automation can reduce labor costs and minimize the risk of human error, resulting in higher efficiency, consistency, and product quality.” One can refer to an exploration by FDA to pursue an mRNA potential “platform pathway” for mRNA in the regulatory space. The concept gets complicated whether one is dealing with saRNA or circular RNA (circRNA), as handling and manufacturing aspects vary significantly. As they also do in terms of quantities, potency profiles, or absorption, distribution, metabolism, and excretion (ADME) considerations, or when discussing a generalized vaccine versus personalized oncology vaccines, gene editing, CAR-T, antibodies, or protein replacement therapies.

However, Cerullo contends, “the most efficient way to make process improvements is to have good, standard workflows for process development and manufacturing, evaluate and apply learnings from each time you run the process (like a continuous improvement loop), and be mindful that some constructs will behave differently and need individualized solutions.”

For its part, Pfizer found end-to-end automation for in vitro transcription reactions increases process throughput of mRNA vaccines and therapeutics. Because of this, Pfizer is creating a spin-off team to further develop assays capable of automation. Karena Smith, senior associate scientist at Pfizer, said this “allows template-directed synthesis of mRNA molecule. Therefore, developing a method to understand how each component affects the process is incredibly valuable” (5). By using a high-performance liquid handler for automation, “Pfizer increased the reaction throughput eightfold whilst achieving a tenfold decrease in reaction scale.” Smith continued, saying, “the automated process saw less than a 10% difference in final concentration samples, and no differences in mRNA attributes when compared to a manual process.”

Volumes are small compared to traditional bioprocessing, being on the order of less than 100 liters for commercial products (compared with 2000 liters for proteins), and according to Cerullo, while “most mRNA manufacturing platforms for the drug substance are aqueous based and don’t have organic solvent requirements … However, organic solvent is widely used for lipid nanoparticle formulation, so advances in new delivery technologies, which would eliminate the use of large volumes of organic solvent, would have a positive impact on reduction of waste. This is the part of the process that presents the most challenges from a waste and safety infrastructure perspective,” she concludes.

Patterson points again to process optimization. “By fine-tuning reaction conditions, such as temperature, pressure, and reaction time, it is possible to minimize the need for excessive solvent volumes. Employing innovative techniques, such as continuous flow processing or solid-phase synthesis, can also help limit solvent requirements.” Patterson goes on further to point out that the ““design and implementation of closed-loop systems and solvent recovery processes can further reduce solvent waste. These systems enable the capture, purification, and reuse of solvents, minimizing both environmental impact and production costs.” Patterson also states, “another alternative is to take a biochemical, rather than purely chemical approach to synthesizing oligonucleotides. The chemistries that are currently used to produce oligonucleotides using phosphoramidites require significant quantities of organic solvents. Instead, several groups have been researching the possibility of synthesizing oligonucleotides with various enzymes. Initial studies have shown there may be a possibility that at least some oligonucleotides could be synthesized with enzymes in aqueous, non-toxic solutions. This is an alternative oligonucleotide synthesis technology that certainly has promise in reducing solvent consumption if it can be put into practice.”

CARsgen. CARsgen Collaborates with Moderna to Evaluate CT041 in Combination with an mRNA Cancer Vaccine. Press Release. Aug. 21, 2023.

Pfizer. Pfizer and Beam Enter Exclusive Multi-Target Research Collaboration to Advance Novel In Vivo Base Editing Programs for a Range of Rare Diseases. Press Release. Jan. 10, 2022.

BioNTech. BioNTech and DualityBio Form Global Strategic Partnership to Accelerate Development of Differentiated Antibody-Drug Conjugate Therapeutics for Solid Tumors, BioNTech. Press Release. April 3, 2023.

Blakney, A.; Webb, C.; Bathula, N. Current Status and Future Perspectives on MRNA Drug Manufacturing. 

 19(4). doi: 10.1021/acs.molpharmaceut.2c00010.

Johnson, M.; Denny, T.; Sempowski, G.; Haynes, B. Development of mRNA Manufacturing for Vaccines and Therapeutics: mRNA Platform Requirements and Development of a Scalable Production Process to Support Early Phase Clinical Trials. April 2022, Translational Research.

Smith, K. Presentation. Bio Process International US West, Feb. 27-March 2, 2023.

 This is an extended version of the article that appeared in the March 2024 print issue of 

When referring to this article, please cite it as Spivey, C. The Making of mRNA. 

Upstream Processing

A tidal wave of questions floats the need for more upstream automation.

The Making of mRNA

All drugs are subjected to regulatory scrutiny during the full development lifecycle because, whichever way a drug is administered, it should always be effective and, most importantly, safe for the end-user. The requirement for safety, however, is ever more pressing for parenteral products, as these drugs are administered either directly into the bloodstream or into other delicate mucus membranes.

Focusing on parenteral drug manufacturing in particular, a critical step that ensures sterility and contaminant-free results is fill/finish. “Following drug development and production, fill/finish is the next step in the process—and crucial for getting the drug into the required container and making it ready for distribution,” confirms Andreas Mattern, vice-president Strategy and Global Product Management Pharma at Syntegon.

“Fill/finish manufacturing entails the process of aseptically filling and sealing products, in this case pharmaceutical products, ensuring that precision, sterility, and compliance with regulatory standards are meticulously followed,” concurs Carlos Quintero, director, Industry Services—PMMI (the Association for Packaging and Processing Technologies). “This is critical to ensure safety, efficacy, and shelf life.”

It is imperative for both bio/pharma manufacturers and any contract service providers, such as contract manufacturing organizations, to pay close attention to fill/finish processes, making sure there is the utmost sterility while also being robust and reliable, notes Mattern. “Filling and closing are especially demanding [because] the drug and its container come into contact with many different parts of equipment,” he adds. “To date, some of these processes still require manual intervention from operators, which makes them even more prone to contamination.”

In 2022, a key regulatory revision was released—the European Union’s Good Manufacturing Practice Annex 1 revision (1)—that has far-reaching consequences for sterile filling operations, not just within the EU but for the global bio/pharma industry, Mattern reveals. “The specifications affect the manufacturing processes of all drugs imported into the EU,” he says.

“Among the major challenges of the new Annex 1 is the separation of the aseptic process area from the operator environment,” Mattern continues. “Pharmaceutical manufacturers therefore require at least restricted access barrier systems (RABS) for the approval of new products.”

As a result of these new regulatory requirements, there is now high demand for new machines and barrier technology upgrades, Mattern explains. “In the case of older machines, the question is whether to upgrade or invest in a completely new line with pre-installed barrier technology,” he says.

The revised Annex 1 also clearly sets out the requirements for air flow, Mattern specifies. “Unidirectional air flow is mandatory for open isolators and RABS in all class A areas and should not be interrupted between the air inlet and the open product. Yet this uninterrupted flow is not possible everywhere,” he says.

“A typical exception is the filling needle together with the filling needle holder, which must be positioned exactly above the open, sterilized packaging material in order to achieve loss-free filling,” Mattern states. “It is expected that these components are aerodynamically designed to minimize turbulences and that these items can be sterilized separately and installed aseptically.”

As alluded to earlier, the revised regulatory requirements are leading to the need for new technologies to be used in fill/finish processes. A further elaboration on this matter is highlighted in the second chapter of Annex 1 (1), where automation and robotic systems are penned as appropriate technologies, confirms Mattern. “By using these technologies, gloves and human intervention in barrier systems can be reduced to an absolute minimum or even eliminated,” he says.

“The issue of gloves presents many challenges, which are acknowledged in separate paragraphs,” Mattern continues. “For example, gloves are to be additionally tested in operation during longer campaigns—which is not possible with the glove testing equipment used to date without risking the integrity of the sterile room. A switch to gloveless systems with robotics that handle these manipulations is already possible.”

Additionally, in conjunction with robotic filling lines, artificial intelligence (AI)-influenced line controls and automation will be able to offer faster scalability and increased flexibility for manufacturers, remarks Quintero. “Automation and AI are always accelerating the rate of precision, quality control, and efficiency of the fill and finish-seal process in manufacturing,” he says. “This has a huge impact on reducing contamination risks and the overall effectiveness and performance of the lines.”

Another pressing issue for the bio/pharma industry and its stakeholders is sustainability. More sustainable equipment and processes are needed across the board in bio/pharma manufacturing to ensure various reporting requirements can be met, which is something that technology providers, such as Syntegon, can help with, notes Mattern.

In terms of the sustainability of fill/finish manufacturing, Quintero anticipates a marked and quick change in the coming years. “Fill/finish manufacturing most likely will rapidly evolve into a zero-waste process and will incorporate more and more, sustainable renewable energy sources,” he states.

Furthermore, the drive towards automation of fill/finish processes will continue into the future as humans remain to be the main source of contamination, Mattern adds. “While drugs are becoming more and more complex, and as such expensive, separating pharmaceuticals from operators has been gaining attention throughout the industry for some years—a topic that is still becoming more pressing when considering small and even micro batch filling of highly potent substances,” he says. “Hence, automation and digitalization will play an increasingly crucial role.”

“While it is not easy to integrate what is known as ‘Industry 4.0’ into complete pharmaceutical manufacturing operations, many individual solutions are already available,” he summarizes. “The main question here is how to make use of the enormous amounts of data generated by fill/finish (and other pharmaceutical) processes every day.”

1. EC. Annex 1 Manufacture of Sterile Medicinal Products, 

Felicity Thomas is the European/senior editor for 


Vol. 37, No. 3
March 2024
Pages: 28–29

When referring to this article, please cite it as Thomas, F. Automating the Future of Fill/Finish. 

Manufacturing

Given the criticality of fill/finish processes, it is clear that automation is the next technological step.

Automating the Future of Fill/Finish

It’s no secret that proteins dominate the world of biology. Human cells have more than 20,000 proteins and over one million proteoforms, orchestrating countless mechanisms within the body (1). Looking beyond a natural physiological role, biologics are a mainstay in the biopharma industry’s repertoire of therapeutic modalities. Monoclonal antibodies (mAbs) and other protein-based biologics can be found in most modern therapeutic development pipelines. The industry’s collective knowledge of these biomolecules and their interactions is vast, yet there is still much to learn. Small variations can have significant consequences in the clinic. An accurate understanding, down to each amino acid and small variation, is key to the discovery and development process, requiring broad access to single-molecule protein sequencing technology.

Remarkable advancements have been seen in molecular biology and engineering in the past three decades, resulting in the modern revolution in medicine today (2). Protein-based therapeutics offer a host of benefits when compared to traditional small molecules, including the potential for greater target specificity and improved pharmacokinetics (PK) (3).

The prevailing role for proteomics in drug discovery and development is from an analytical and quality control (QC) perspective. Working with biologics requires a sequence-level understanding of the molecule at each stage of development in order to assess and instill the desired properties in the investigational drug. Their molecular complexity, however, poses a challenge.

With complexity comes opportunity for variation, which can be introduced during transcription and/or translation, by misincorporation at various levels including the DNA, RNA, and amino acid level, and by the introduction of co-translational modifications (CTMs) or post-translational modifications (PTMs) (4). Post-translational modifications such as methylation, acetylation, glycosylation, ubiquitination, phosphorylation, or other modifications can change the way proteins stabilize, fold, function, and localize (4). For example, among the long list of possibilities that can alter the behavior of a biologic in clinically significant ways is errant replacement of an amino acid in amino acid misincorporation. Variations such as these may alter a biologic’s charge and size, reduce or alter target affinity, or lead to unintended aggregation (4–7).

Given the significant opportunity for deviation from the intended product that can occur within the synthesis and larger manufacturing process, these molecules and their variants must be closely monitored and tested in the preclinical setting as they are characterized and optimized as biological drug candidates and, later, in the clinical setting as their PK and therapeutic concentrations are closely scrutinized.

In early drug discovery, fundamental sequence understanding is key, particularly as multiple variants of the biologic are intentionally produced. The overarching goal is to find the optimal biologic drug candidate or variant, including considerations such as function, specificity, stability, and safety, among others. Understanding of the investigational asset at single molecule amino acid resolution provides information regarding how specific changes within the sequence of that biologic drug impacts the function of that molecule and provides a detailed mechanistic understanding of what is driving biological interactions. Proteomics workflows and biophysical techniques are used together to characterize sequence and function, which are often directly related to one another.

As molecules move into clinical trials in humans, they are exposed to entirely new parameters. Protein activity can be affected by interactions with small molecules, cofactors, metals, nucleic acids, and other proteins, which may change with variations in protein sequence. Variations can also impact target-binding affinity or promote off-target binding. These factors make a detailed understanding of structure important (8–9). Having confidence that the biologic drug is being made consistently with low batch variation may allow more confidence and insight in the interpretation of clinical trial data and ensure continuity in effect when molecule production is scaled up for commercial availability.

In manufacturing, lot purity must be maintained as cell-line mutations and amino acid misincorporations can cause major supply chain problems. Significant effort is put into maintaining the integrity of cell lines with regard to biologics that are recombinantly expressed; however, there are still opportunities for deviation. Being able to look at a manufacturing product at amino-acid-level resolution provides insight into the degree of consistency within the product over time and batches.

Taken together, these considerations make proteomics tools indispensable to scientists and technical personnel at each stage of the therapeutic lifecycle.

Current sequencing and mapping techniques

Biologic drugs are complex molecules produced by living cells through sophisticated manufacturing processes. As one of the fastest growing segments in the bio/pharmaceutical industry, biologics have the potential to provide targeted therapy and treat diseases that were once considered untreatable (10). However, successful biologic discovery comes with many challenges, such as high costs, workflow complexities, and end-product safety considerations (11).

Biologics are more complex due to their larger structures and complicated processes in their analysis, development, and manufacturing. When developing a biologic, critical quality attributes such as structural elucidation, physiochemical, immunological, biological properties, as well as impurities and contaminants (12), must be defined and well documented to ensure successful therapy and delivery. Characterization of these attributes are performed with bioanalytical tools that include chromatographic, electrophoretic, spectroscopic, and electrochemical techniques (12).

Rising to the top of these methods is liquid chromatography–mass spectrometry (LC–MS). LC–MS is a key tool in a scientist’s toolbox and often the available method of choice for protein identification at varying levels of detail (13). The sample of interest is digested into protein fragments that will be in-range for the instrument’s sensitivity and applied to a liquid chromatography column where particles are separated by size and other biophysical properties. Reverse-phase liquid chromatography is the most common choice and uses a hydrophobic stationary phase with separation via hydrophobicity with a polar mobile base. The sample is then ionized and sent over a detector, yielding a molecular mass to charge ratio (

While LC–MS is an extremely powerful technique, it is equally as complex, with a multitude of possible workflows and expertise required in sample preparation, instrument operation, and output analysis. The resultant spectrum must be deconvoluted into the appropriate peptide fragments. Available automated tools make this process easier; however, subtle changes in 

 and deviations from what may be expected within the workflow can result in misinterpretation of the data. Further, results depend on the existence of a 

 difference between the samples and the ability to separate those samples on the LC column, which is not always possible. Some peptides may not even elute from the column due to strong hydrophobicity. In instances of PTMs, where there may not be a mass change or a mass change on the order of Daltons, LC–MS may not offer the sensitivity required. In instances where there are multiple PTM possibilities within a sequence, the results may indicate those PTMs exist but not their location, obscuring insight into the frequency and heterogeneity of PTMs that can be critical for the function of biological drugs.

Immunoassays, such as Western blot and enzyme-linked immunoassay (ELISA), are also within the scientist’s toolbox, owing to their ability to quickly identify proteins in a sample. Immunoassays could confirm the presence of PTMs or that the biologic drug is interacting with the target of interest, although they don’t provide sequence-level information. Western blot separates proteins by molecular weight through gel electrophoresis, which are then moved to a membrane and analyzed via antibody binding. ELISA also uses antibodies to detect a protein sample; it is easy to use with the potential for higher throughput as compared to Western blot. Conversely, Western blot can analyze a more complex protein mixture.

Both of these methods are easily accessible tools that offer high degrees of sensitivity and low sample input. However, they require customized reagents, such as high-quality antibodies, for each and every target of interest and additional analysis in order to get protein sequence or individual amino acid information. Further, challenges can arise in the reproduction of results due to biological variation (i.e., polyclonal vs. monoclonal antibodies) leaving a need for enhanced QC (14).

Each of these methods involve trade-offs between ease of use and level of detail in protein identification data, highlighting the need for technological advancements that make detailed proteomics more accessible and targeted toward the field’s evolving needs.

Next-generation sequencing in genomics catapulted science forward. It enabled cost-effective parallel sequencing of millions to billions of DNA fragments and has been applied in investigating molecular-level variation in cancer, rare genetic diseases, and complex microbiomes (15). Single-molecule protein sequencing has the potential to be just as revolutionary. Understanding of biologics on a single amino acid level can provide deeper insights into what may be driving biologic function against a target, allowing optimization on a level that otherwise is unattainable. In larger-scale production, single-molecule protein sequencing may allow more precise quantification of changes occurring from batch to batch, enabling close monitoring of the types of events that are impacting product quality.

Overall, single-molecule protein sequencing technology has the potential to decrease cycle time while facilitating improved quality of biologic drugs available through improved stability and less batch-to-batch variation. Having tools available that enable deeper insights can reduce development time and ultimately get new drugs to patients faster.

The industry is facing a critical challenge within the field: elevating analytical techniques to match the sensitivity that is now required within the drug discovery and development process. There is a great need for sensitive and unambiguous amino acid detection at a scale and ease accessible to broader populations of scientists. If more researchers can “see” proteins with this level of clarity, then there is a tremendous opportunity to transform the way they apply proteins in research and medicine.

1. Aebersold, R.; Agar, J.N.; Amster, I. J.; et al. How Many Human Proteoforms are There? 

 (3), 206–214. DOI:10.1038/nchembio.2576
2. Chen, Z.; Wang, X.; Chen, X.; et al. Accelerating Therapeutic Protein Design with Computational Approaches Toward the Clinical Stage. 

, 2909–2926. DOI:10.1016/j.csbj.2023.04.027
3. Oo, C.; Kalbag, S. S. Leveraging the Attributes of Biologics and Small Molecules, and Releasing the Bottlenecks: A New Wave of Revolution in Drug Development. 

 (6), 747–749. DOI:10.1586/17512433.2016.1160778
4. Jefferis, R. Posttranslational Modifications and the Immunogenicity of Biotherapeutics. 

, 5358272. DOI:10.1155/2016/5358272
5. Samodova, D.; Hosfield, C. M.; Cramer, C. N.; et al. ProAlanase is an Effective Alternative to Trypsin for Proteomics Applications and Disulfide Bond Mapping. 

 (12), 2139–2157. DOI:10.1074/mcp.TIR120.002129
6. Wong, H. E.; Huang, C. J.; Zhang, Z. Amino Acid Misincorporation in Recombinant Proteins. 

 (1), 168–181. DOI:10.1016/j.biotechadv.2017.10.006
7. Bradbury, A. R. M.; Trinklein, N. D.; Thie, H.; et al. When Monoclonal Antibodies are Not Monospecific: Hybridomas Frequently Express Additional Functional Variable Regions. 

 (4), 539–546. DOI:10.1080/19420862.2018.1445456
8. Po, A.; Eyers, C. E. Top-Down Proteomics and the Challenges of True Proteoform Characterization. 

 (12), 3663–3675. DOI: 10.1021/acs.jproteome.3c00416
9. Mateus, A.; Savitski, M. M.; Piazza, I. The Rise of Proteome-Wide Biophysics. 

 (7), e10442. DOI:10.15252/msb.202110442
10. Pharmaceutical Technology. High Biologics Demand Spurs Need for Greater Contract Manufacturing. 

pharmaceutical-technology.com/sponsored/biologics

, April 24, 2023.
11. Olsen, C. Challenges in Biologics R&D. 

, Nov. 7, 2022.
12. Alhazmi, H. A.; Albratty, M. Analytical Techniques for the Characterization and Quantification of Monoclonal Antibodies. 

 (2), 291. DOI:10.3390/ph16020291
13. Song, J. G.; Baral, K. C.; Kim, G. L.; et al. Quantitative Analysis of Therapeutic Proteins in Biological Fluids: Recent Advancement in Analytical Techniques. 

 (1), 2183816. DOI:10.1080/10717544.2023.2183816
14. Baker, M. Reproducibility Crisis: Blame It on the Antibodies. 

, 274–276. DOI.org/10.1038/521274a
15. Satam, H.; Joshi, K.; Mangrolia, U.; et al. Next-Generation Sequencing Technology: Current Trends and Advancements. 

Ben Moree is principal scientist at Quantum-Si.


Volume 37, No.3
March 2024
Pages: 30–33

When referring to this article, please cite it as Moree, B. Biologics Quality Control: The Growing Need for Accessible Proteomics. 

Analytics

There is a great need for sensitive, precise, and easily accessible analytical detection techniques for protein sequencing.

Biologics Quality Control: The Growing Need for Accessible Proteomics

Novo Holdings’ acquisition of Catalent, in retrospect, was entirely logical (1). We are tempted to see echoes of Thermo Fisher’s buyout of Patheon, or Lonza’s swallowing of capsule maker Capsugel. But this situation is different. Catalent makes significant production of Novo’s Ozempic and Wegovy, but also Lilly’s Mounjaro and Zepbound. And while Catalent’s acquisition is definitely about making drugs, it’s also very much about making money.

Making money takes various forms—taking away a competitor’s capacity to do so is doubtless one of the more intriguing variants. David Ricks, Lilly’s chief executive, was quoted as saying, “the reality is there just isn’t built capacity that’s available. These are technically complex facilities, there’s not an infinite number of people who know how to set them up. And the supply chain for the machines that make the products is also constrained” (2).

An interesting ironic side note is that Catalent has had a checkered compliance past. FDA visited the company’s Brussels plant in October 2021 and August 2022. Reuters reported that “inspectors said the lapses at the plant … represented the most serious form of violations, according to the reports, which show Catalent shut the facility down twice between the two inspections” (3). Reuters goes on to say, “at a meeting with Novo executives in May, two investors out of a group of about five expressed concerns about the choice of Catalent as manufacturing partner, citing the contractor’s history of compliance issues with FDA, according to one of the shareholders who was present. The investor, who declined to be named due to the sensitivity of the matter, said Novo’s Chief Financial Officer Karsten Munk Knudsen had responded by saying that, in hindsight, the company may have made a mistake in choosing Catalent and was now tightly overseeing the firm’s filling operations of Wegovy in Brussels” (3).

Novo Nordisk and Eli Lilly are spending heavily to boost their manufacturing capacity. “That could help them fend off competition from specialized pharmacies that are making generic versions of semaglutide and tirzepatide, an option created by the FDA designating the drugs in shortage” (2). Nevertheless, Anat Ashkenazi, Lilly’s chief financial officer, said, “we intend on holding Catalent accountable to their contract with us” (2). Given new ownership, its uncertain if Lilly will be first in line, on the production line.

Catalent. Novo Holdings to Acquire Catalent. Press Release. Feb. 5, 2024.

Gilbert, D. Makers of Ozempic, Mounjaro Race to Meet Demand for Weight-loss Drugs. 

While Catalent’s acquisition is definitely about making drugs, it’s also very much about making money.

BioPharm International, March 2024