Close to a Giffen good, depending on the therapeutic area, pharmaceutical products defy fundamental economic laws. Bad years are not so bad as they still exhibit an upward-sloping demand curve, while good years can be a bonanza. An overlooked obvious truth about the pharmaceutical industry is that it is a chimera of strong competing opposites—a 1980s Kombi van powered by jet engine thrusters, piloted by overly exuberant alternating with pessimistic MBA graduates. Through this lens, the stop/start progress and the multitudinous lurches toward both extreme success and extreme failure can be more clearly comprehended. On a deeper level still, pharmaceuticals are also a lifesaving endeavor inextricable to civilization’s forward march on one hand, while helping to extend and enrich each individual’s personal life on the other. In its inherently selfish human nature, the sector is thus well insulated from those deepest of natural financial troughs.

With this in mind, in gathering perspective on 2024’s industry performance, 

® asked industry workers for comments on challenges and opportunities. Responses were cross referenced with those from 

®’s annual employment survey (1) as a validation and quality control check.

The time and expense required to train and retain operators—and then to re-train them again when increasing molecular diversity for a new therapeutic requires the process to begin anew—is a commonly heard challenge. Christa Myers, senior fellow, Aseptic & Sterile Products, CRB, related that, “there is a lot to learn and be able to speak to within this highly technical side of the industry. Character and personality matter. An operator needs to be able to follow protocols for manufacturing of a quality product. Each also needs to be able to think in a quality improvement mindset in order to plan for corrective actions and preventative actions (CAPAs). Your operators are your most valuable asset and should be trained and treated as such” (2).

Many agree with training staff as a number one priority, as does Harry Benson, director of Human Performance Services at Commissioning Agents (CAI), who says, “finding, hiring, and retaining the workforce for many operational departments is a significant challenge—possibly the biggest barrier to growth opportunities and closing the drug shortage gaps. For this reason, the industry is giving much more attention to the availability of a skilled or trainable workforce in their site selection and expansion process.”

, this workforce challenge is commonly perceived as the biggest obstacle for industry progress, and is even mentioned, with variations, in the “others” category of 

, with frequent comments such as “I’d like to add that the onboarding and general information flow is horrible, and newcomers are completely left alone” or, “[there is a] lack of experience in the industry and common sense.”

Connie Leech, global director of Quality, Compliance, and Regulatory at CAI, puts an apostrophe on this, stating, “the post-pandemic workplace is uncertain, constantly changing, and ideal for workers with a breadth of knowledge and skills who thrive in ambiguity.”

Regulators asked to keep pace with innovation, shortages, and transparency

Ashleigh Dawley, director, Regulatory Affairs & Compliance, Integrated DNA Technologies, points out that, “The recent FDA approval of the first CRISPR [clustered regularly interspaced short palindromic repeats]-based gene therapy—which was just a vision a little over a decade ago—signals continued interest in the power of the CRISPR-Cas9 technology, as reflected in the more than 40 registered clinical trials globally to date. This rapidly evolving technology has the power to unlock innovation and future patient impact that we cannot even imagine today.” She continues, “An ongoing challenge remains in the ability of regulatory agencies to scale and grow with the pace of innovation. Of the current 40 active clinical trials globally, 23 of those are occurring in the [United States]. If successful, [FDA] could face challenges with regards to creating the infrastructure with the needed staffing resources and expertise to support regulatory reviews and approvals and enable the delivery of promising therapies to patients in need.”

Agne Vaitkeviciene, CEO of Memel Biotech and VP at LithuaniaBIO agrees. “As of today, [the] main obstacle in the pathway of new ATMPs [advanced therapy medicinal products] development is regulatory environment. Especially in [the] European Union. Changes in regulations comes slower than expected and leave Europe behind [the] US, Japan, and UK [United Kingdom]. Still, there are hundreds (543) of companies in Europe developing ATMPs and over [a] thousand (1235) in North America for a reason. Reaching patients, who could benefit for cell and gene therapeutics becomes significant, and improvements in regulatory landscape have to adjust faster to allow the access to the therapies. Together with regulatory approaches, reimbursement is one other obstacle that joint community efforts worldwide are trying to solve. Applying optimization in manufacturing processes and [a] fast marketing approval system might contribute to lowering ATMPs costs.”

Communication, a key asset during pandemic vaccine development and deployment, is being called back into question. Thomas Hartman, president & CEO of the International Society for Pharmaceutical Engineering says, “Both the FDA and drug manufacturers will need to focus on earlier communication and collaboration to mitigate shortages and avoid facility shutdowns. This is especially true of generic manufacturers–many of which are either shifting from generic medicines because of a lack of profitability or closing down entirely. Alongside the FDA’s mobile app and web portal dedicated to reporting drug shortages, the introduction of new technologies in digital validation and data management will improve visibility within manufacturing and allow for early communication to the FDA regarding potential disruptions.”

Hanns-Christian Mahler, CEO of ten23 health, says he thinks of this line from Star Trek when approaching a challenge: “things are only impossible until they are not (Cpt Picard, Enterprise).” This certainly was borne out by recent Vertex and CRISPR Therapeutics approvals in the UK and US for Casgevy, alongside bluebird bio’s Lyfgenia, the first approved gene edited therapeutics, and Arcturus’ARCT-154 approval in Japan, the first self-amplifying messenger RNA (sa-mRNA) COVID-19 vaccine.

There is so much historical process habit in the bio/pharmaceutical industry that it can often be paralyzing to growth, most especially when confronting new modalities. Renee A. Hart, president and chief business officer, LumaCyte, thoughtfuly observes. “I have used the analogy before that the leaning Tower of Pisa is a great example of where process and technology sits in many verticals. Companies think that layering on new processes, people, and tools into already complex infrastructures will somehow check the box of driving innovation. Those organizations that choose not to invest in change management and continue to stack new processes on top of old will find themselves leaning so far away from growth that in time they will find their organizations are actually moving backwards. Process inefficiencies create unnecessary obstacles and bottlenecks to advancing the development and delivery of lifesaving treatments and cures, we can definitely do better.”

These therapeutics “herald new ways of thinking, new types of testing and validation, and new types of manufacture and end-point monitoring. Following on from the revolutionary COVID-19 vaccines, what comes next is more complicated, more difficult to price for reimbursement, and more effective in terms of cure rates. The toothpaste is out of the tube, and the industry must learn to adapt to take best advantage of what is becoming available, no matter how messy the process” (3). As can be seen in 

, cell and gene therapies are widely considered the biggest opportunity going forward.

When considering the evolving toolkit now available to pharmaceuticals, Michelle Fraser, head of Cell & Gene Therapy, Revvity, says, “notably, there are also technologies poised to edit mRNA rather than the genome, inducing transient changes, and addressing genetic alterations influenced by environmental factors through epigenetic modifications ... The trajectory points towards increased sophistication, efficacy, and safety in these therapeutic interventions … expect to see a surge in the adoption of more intricate gene editing approaches, signaling a year marked by significant advancements in the refinement and application of these transformative technologies.”

Personalization requires yet more systems convergence

While progress on advanced therapeutic medicinal products is now undeniable, a new type of effort begins in earnest. That consists of bringing still more technologies into alignment for these treatments to be deployed and accessed. Casgevy requires access to facilities specializing in bone marrow transplantation, for example, which necessitates knowledge transfer from hospital and medical staff being feedback to pharmaceutical scientists. Thermo Fisher Scientific and the University of California San Francisco (UCSF) established one of the early direct connections to hospital treatment, with a current good manufacturing practice facility adjacent to the UCSF medical center. About this movement toward distributed manufacturing (some term it near patient, or spoke and hub), Michel Lagarde, executive vice president and chief operating officer of Thermo Fisher, observed, “cell therapies represent a rapidly emerging field of biotechnology with tremendous promise for future therapeutic applications … with a record number of cell therapy approvals granted in the last two years, and CAR-T therapies becoming earlier treatment options, we’re in a golden age of biology, where new technologies and partnerships are evolving and transforming clinical care” (4).

This makes turning the page to the year 2024 a metaphorical, as well as literal, leap year. Pharmaceutical manufacturing is now (pun intended) an embedded part of the hospital-patient system.

2023 Pharmaceutical Technology/Pharmaceutical Technology Europe Employment Survey

Spivey, C. Hi-Fidelity Fill/Finish for Biologics. 

Spivey, C, New Medicines, Markets, and Manufacture: CRISPR for Sickle Cell Disease and β-thalassemia, 

Kurtzmen, L. Thermo Fisher Scientific Opens Cell Therapy Facility at UCSF to Accelerate Development of Breakthrough Therapies. UCSF press release, March 27, 2023.

Chris Spivey is the editorial director of BioPharm International.

When referring to this article, please cite it as Spivey, C. Leap Year. 

Articles

Cover Story

Revolutionary therapies restructure pharmaceutical manufacturing.

Leap Year

® survey audience members to see what has remained the same and what has shifted, regarding pharmaceutical workforce attitudes toward their jobs, their current employers, and the general direction the industry is heading. The industry’s response to COVID-19 has had the most significant measurable impact on the way both business and manufacturing processes were managed in the past two or three surveys. In 2023, the largest observable shift was brought about by the steady emergence of advanced therapeutic medicinal products (ATMPs). The last half of 2023 was a watershed moment for pharmaceuticals, given the industry saw the first set of approvals for gene edited therapies, as well as for a self-amplifying messenger RNA vaccine. But everyone experienced the year through the lens of the products and activities their own company sells. In this way, one can say of pharmaceutical manufacture, “All happy families are alike; each unhappy family is unhappy in its own way” (1). Let’s examine survey responses to see what these differences might be founded upon (2). There is a gulf between survey responses such as “struggling industry full of scientific brains lacking leadership, mentoring, and training” or “Party’s over–hunker down, folks,” to “Aside from a few ‘deplorables’, the industry is in good condition’’, “volatile, encouraging, innovative, nerve-wracking”, or “Incredible advances and treatment options.”

As the sugar rush of COVID-19 response funding wore off, so too did the number of hours workers put in on average. Hours worked returned to pre-pandemic levels, based on earlier survey results (see 

). This finding was somewhat surprising given conversations held during conferences, but there was perhaps some confirmation bias during conferences, versus survey results. It’s easier in a survey to say ‘I’m not working as hard’ compared with a network contact you are used to doing business with. The final question of the survey asks, “If you could describe the 2023 bio/pharma industry in five words or fewer, what would you say?” And this is where the quote from Tolstoy can most readily be explored.

Opinions about the post-pandemic industry were largely positively oriented, from “post-COVID readjustment” and “the 2023 bio/pharma industry has benefitted greatly from covid restrictions.”Responses ranged from “consolidation after covid pandemic rush” to “Great come back after covid.” But some were bleak in their appraisal, with “Pandemic stress, Supply chain collapse” and “Post-pandemic blues” being somewhat frequent responses.

Naturally, one can suppose the technologies and services most involved in the pandemic response, such as vaccine manufacture and distribution, were/are the respondents going through the deepest readjustment phase. One of the features of the pandemic was that it forced everyone to communicate more quickly, clearly, and completely. This seems to have remained.

Karolina Makovskyte, head of Business Development at Caszyme, said, “One positive aspect of CRISPR-Cas technology is the emergence of a more active dialogue about gene editing regulations, particularly in Europe. It is also somewhat linked to another positive aspect, which is increased exploration of the technology by new industries, seeking ways to integrate CRISPR-Cas into their activities.”

Satisfaction with pay in 2023 didn’t move to any great degree versus historical years (see 

). Perhaps the timing of the survey influenced a type of survival-driven satisfaction, as the last half of the year saw several companies markedly reduce workforce numbers as part of restructuring efforts. Pfizer axed approximately 900 staff, as their Covid vaccine reported $1.31 billion in sales, a 70% decline from the year before. “Wall Street had expected $1.53 in revenue. Paxlovid suffered an even sharper drop, with revenue of just $202 million—97% down from last year” (3). Not a big reduction but still significant for a company that sold $100 Billion in product for the first time in 2022. Grifols, a Spanish company specializing in blood plasma therapies, trimmed its operations in the United States by cutting approximately 2300 positions (which amounts to approximately 8.5% of its global workforce). Takeda braced for competitive threats with cuts of nearly 1400 staff, to prepare for generic competition to its diabetes drug Actos. Thermo Fisher Scientific adjusted to changing demands through a series of workforce cuts totally nearly 1300 staff, mainly due to decreased demand for COVID-19 testing products and “evolving economic conditions.” Genentech, Biogen, Catalent, and Amgen also witnessed significant workforce diminutions, while Novavax, Emergent BioSolutions, and Bristol Myers Squibb, round out the list (4).

, job security hit its highest levels during the height of the COVID-19 pandemic. While the survey questions don’t allow enough depth to explored in detail, going back to the Tolstoy metaphor, the workers feeling most secure are likely the staff working on the most promising new advanced therapeutic medicinal products (ATMPs), as this is clearly where the excitement and major new investments are being ploughed into. For example, RoslinCT will manufacture Vertex Pharmaceuticals’ CRISPR-based gene therapy, Casgevy, recently approved for treating sickle cell disease and β thalassemia (5). Novo Nordisk is also planning a US$2.3 billion investment in new French facilities, partly to facilitate extra capacity for glucagon-like peptide-1 products (6). One of the survey respondents summed up the entire industry with the words “Sustained growth with new therapies” while another simply stated “Vaccines, proteins, blood, tissues.”

Similarly, optimism about future performance was also peaked during the pandemic.Somewhat alluding to the Tolstoy metaphor and also the COVID-19 sugar rush, Barbara Ryan, founder, Barbara Ryan Advisors commented, “It is interesting, however, because the market remains highly bifurcated—companies with good data are able to finance, and often the aftermarket performance is quite positive, as the deals are viewed as a clearing-event lifting the financing overhang. As of press time, there had only been 11 biotech IPOs this year, in contrast to more than 100 each in 2020 and 2021. Sadly, however, too many companies went public in 2020 and 2021 that were at a far-too-early stage in their development (i.e., preclinical, Phase I trials) to be public companies; they were not sufficiently funded to proof-of-concept data. This is where the real pain is” (7). Ryan went on to round out her assessment of the industry, ending on a highly positive note. “The innovation renaissance is alive and well, with record numbers of FDA new drug approvals in each of the past several years. M&A continues to be another bright spot. Beginning in 2025, large pharma will be losing exclusivity for products currently representing over $350 billion in revenue, leaving a huge growth gap that needs to be filled through M&As, licensing, and partnering. Dealmaking has always been a key pillar of the industry’s growth strategies. More good news: according to EY’s Annual Fire Power Report, large pharma has a record $1.4 trillion to fund deals. There have been a total of 30 public biotech acquisitions this year, and 15 of these were above $1 billion each, which surpasses the number in any year for the past 10. As we wind down 2023, the hope will be for rate relief in the year ahead—and substantially improved biotech performance.”And as if on que, the Federal Reserve just suggested three cuts to interest rates in 2024, sending the DOW into record territory by rising 1.4%, to close at 37,090.24 (8).

Q.ai Contributor Group, Pfizer Layoffs Confirmed as Vaccine Manufacturer Drives For Cost Savings. 

Buntz, B. 10 Major Pharma and Biotech Companies that Downsized in 2023. 

Mirasol, F. RoslinCT to Manufacture CRISPR-Based Gene Therapy Casgevy, 

Mirasol, F. Novo Nordisk to Make Multi-Billion Dollar Investment in Expansion of Production Facilities in France, 

Ryan, B. Will Calendar Change Bring an End to Run of Volatility in Biotech? 

Mena, B.; Wallace, A. Buchwald, E. Dow Surges to New Record as Fed Signals Three Rate Cuts in 2024. 

Chris Spivey is the editorial director of 

When referring to this article, please cite it as Spivey, C. New Therapies Cry Out for New Skills. 

Special Coverage

There are positive indications for future growth.

New Therapies Cry Out for New Skills

Cell culture process transfer requires a good understanding of the physicochemical conditions within a bioreactor. This includes the hydrodynamic environment induced by the agitator and the mass transfer capability of the spargers at different gas flow rates. Cell culture processes need to operate in a design space where the oxygen supply and carbon dioxide removal can match cellular metabolism. In addition, prohibitive high shear stress and high gas flow rates can create bubble damage or foaming issues and should be avoided. Identifying the boundaries of a design space for a robust process is difficult due to the extensive characterization data needed and the diversity of single use bioreactor design. The authors show that in-silico models can be used to determine ranges in the design space for agitation and gas flow rates that fulfill the requirements of oxygen transfer and carbon dioxide stripping. Furthermore, the design space can be explored by locking one or two input variables and using the model to calculate acceptable ranges for the remaining parameters. With this methodology, it is possible to navigate the design space, understand correlations of the operating parameters in silico, and build understanding about the limits for all operating parameters.

Submitted: July 5, 2023
Accepted: Nov. 16, 2023

William Tran is a senior scientist, Jack Bowers is a senior principal scientist, and T. Craig Seamans is a senior principal scientist; all at Biologics Process R&D, Merck and Co. (MSD outside of the United States and Canada). Helena Öhrvik is senior product manager, and Andreas Castan is strategic technology leader; both at In Silico Process Development, Cytiva.


Volume 37, No. 1
January 2024
Pages: 26–30,33

When referring to this article, please cite it as Tran, W.; Bowers, J.; Seamans, T. C.; Öhrvik, H.; Castan, A. Right the First Time: Bioreactor Scale and Design Translation. 

Peer-Reviewed Research

The authors give an overview of scaling approaches and present good scaling practices for the biopharma industry.

Right the First Time: Bioreactor Scale and Design Translation

With the success of messenger RNA (mRNA) in the COVID-19 vaccines, the biopharma industry is responding with enthusiasm and intense focus on the development of mRNA-based therapeutics for other disease states. A primary area of focus is oncology, where developers see much promise in their use for treating cancers.

mRNA has a number of properties that can be exploited in oncology therapeutics, says Tim Van Assche, PhD, director, Business Development, Etherna, a Belgium-based mRNA/lipid nanoparticle (LNP) technologies company. Van Assche lists the following mRNA properties:

Induction of first-in-class immune responses, which makes mRNA the most optimal platform to develop cancer vaccines as evidenced by the recent success of the recent mRNA-based cancer vaccines.

Induction of local protein expression, in which mRNA is the most suitable platform to express multiple targets intratumorally. It can alter the tumor and tumor microenvironment to make the tumor more susceptible to the immune system.

mRNA is translated into protein in the cytosol, making mRNA an ideal platform to express factors intracellularly. Certain factors with antitumor activity that have intracellular targets, such as antibodies, can be challenging to deliver into the cell, but by encoding the factors into mRNA, this delivery challenge can be circumvented.

mRNA-based therapeutics offer a personalized cancer therapy and have the potential to revolutionize cancer treatment with a highly tailored approach, emphasizes Andreas Dreps, PhD, senior vice-president, Drug Development Services, ICON. “Cancer antigens, or the molecules that immune cells target, can vary greatly from person to person. This means that no one treatment is going to be effective for all patients, even within the same cancer type. However, mRNA cancer vaccines can be tailored for specific antigens based on the unique expression of an individual’s tumor,” Dreps states.

Dreps further explains that the ability to specifically tailor mRNA cancer vaccines allows these vaccines to harness the patient’s own immune system to specifically target and eliminate tumor cells. This results in a highly personalized approach to oncology that primes the immune system to target an individual patient’s specific cancer.

There are currently several methods in play to treat cancer, notes Yasser Kehail, senior product manager—mRNA CDMO, Aldevron. These methods include chemotherapy, stem cell treatment, monoclonal antibodies (e.g., human epidermal growth factor receptor positive, anti-programmed cell death protein 1), and now chimeric antigen T cell (CAR-T) therapy, he lists. “However, treating a solid tumor remains a challenge, and is not being addressed with CAR-T,” Kehail says.

The biggest advantage of using mRNA for oncology therapeutics, therefore, is targeted delivery. “We have seen success recently with hepatocarcinoma, where the mRNA–LNP [lipid nanoparticle] complex is delivered directly to the liver and inhibits the cancer from spreading. In addition, we have also seen success with mRNA neoantigens for pancreatic cancer, as seen in a study published by Memorial Sloan Kettering (1),” Kehail states.

An mRNA-based cancer vaccine functions similarly to mRNA vaccines for pathogens, such as COVID-19, by indirectly provoking an immune response, explains Martin Lachs, PhD, vice-president, Project Management, Oncology, ICON. “The mRNA contained in the vaccine encodes the instructions for healthy cells to temporarily produce cancer-specific antigens. Immune cells will recognize these antigens as foreign and become trained in targeting and destroying cells that express them. This provides the immune system with an improved capacity to recognize and fight cancer cells,” Lachs says.

The treatment resemblesnatural processes because cellular immunity within a body works the same way, says Kaido Kurrikoff, CEO, Vectiopep. “The dendritic cells ‘become aware’ of a new pathogen and forward the molecular signature of the pathogen to the killer T cells. These, in turn, eliminate (kill) the unwanted cells. In the case of mRNA therapeutics, we just have to insert the molecular signature of the cancer in mRNA, making the dendritic cells ‘aware’ of cancer cells, which results in the killer T cells eliminating malignant cells,” he explains.

“The beauty of this approach is that our immune system is able to continuously survey and detect potential recurrence or metastases, whereas the traditional therapies last only as long as the drug is present in the circulation,” Kurrikoff adds.

The core of an mRNA therapeutic for oncology treatment lays in its ability to induce a tumor-antigen specific cytotoxic T cell response and induce intratumoral expression of mRNA-encoded immune-modulatory factors. “The induction of a systemic anti-tumor immune response has the potential to eradicate all present tumors and prevent tumor recurrence,” says Van Assche.

In addition to their therapeutic advantages, mRNA products have an easier manufacturing process, according to Van Assche. Manufacturing of mRNA is also faster and cheaper compared to other biologicals, he emphasizes. Furthermore, in-vivo drug delivery is facilitated via lipid nanoparticles (LNPs), and significant efforts are being made to develop LNPs targeted to specific organs and cell types. “In time, LNPs will be developed that can be targeted to specific cell types in the tumor,” Van Assche predicts.

From Kehail’s perspective, however, new challenges arise even as the industry explores the advantages of mRNA therapy in cancer. For example, he explains that, for neoantigens and personalized medicine, manufacturing challenges arise because of the cost of producing good manufacturing practice (GMP)-grade material.

“Essentially, a batch is manufactured for only one patient at a time. This one batch needs the same amount of analytics, development, and quality control oversight as the batch process for thousands of patients. In addition, the output volume required for a patient-specific manufacturing process is extremely small,” Kehail explains.

Kehail points out that current GMP manufacturing processes are designed for larger batches, which will require contract development and manufacturing organizations to either retrofit systems or create purpose-built systems for personalized medicine. “Industry innovations are needed to overcome the limitations for GMP-grade, small-scale manufacturing,” he adds.

However, the overall attitude is optimistic toward further development of mRNA therapeutics for cancer indications. “The first-generation immunotherapies (immune checkpoint inhibitors) have already demonstrated unprecedented therapeutic effects, never observed before with traditional therapies such as chemotherapy. The hope is that, with the addition of an mRNA approach, such effects can be extended to even higher numbers of patients and tumor types,” says Kurrikoff.

Also, mRNA-based drug delivery typically utilizes nanoparticles—often LNPs—to transport the therapeutic to the desired location; this delivery has been found to be more stable and biocompatible than traditional drugs, notes Dreps. LNPs are known for efficient intracellular delivery, and they provide a mechanism for targeted delivery, he adds.

Meanwhile, Lachs points out that, because of their targeted nature, mRNA vaccines direct immune response solely to cancer cells, rather than damaging healthy cells and tissue the way many chemotherapies do. “Additionally, when used in combination with other cancer treatments, mRNA cancer vaccines enable the treatment of a broader range of patients than other types of cell and gene therapies (CGTs). For example, early clinical trials of one combination therapy that utilized mRNA vaccines showed efficacy in treating patients with solid tumors, whereas the vast majority of successful CGTs have only been able to treat liquid tumors,” Lachs states.

As the mRNA therapeutic field is relatively new in terms of having marketed products, there are still hurdles to overcome in getting to market. In the field of oncology, for instance, precisely targeted mRNA vaccines may not be effective on their own. “Cancer is highly adaptable and adept at developing mechanisms to continue proliferation and survival,” says Dreps. “This includes evading the immune system, such as by using immune checkpoints to prevent attacks from T cells. As a result, some cancers may develop ways to resist the targeted immune response resulting from an mRNA vaccine.”

“To increase effectiveness,” Lachs adds, “mRNA cancer vaccines currently in development are primarily administered in combination with other cancer therapies, including immune checkpoint inhibitors or chemotherapies. This introduces challenges related to the complexity of therapeutic development and cost inflation for therapeutic development.”

Yet, the greatest hurdle is still the delivery, states Kurrikoff. “Current lipid-based technologies are very efficient in targeting cells in the liver, but not so good at any other tissues,” he explains. However, he believes that certain peptides may offer an efficient alternative, as these peptides have shown exceptional selectivity towards target cells, in this case, dendritic cells.

Van Assche points out that the current furthest advanced mRNA-based cancer therapies are cancer vaccines. “The development of mRNA-based cancer vaccines suffer from previous failures in the field with suboptimal cancer vaccine platforms, such as peptide-based vaccines. Due to these failures, there is a general cautiousness to invest in cancer vaccines from both the investment world and pharma. This restraint hampers the fast and thorough development of mRNA-based cancer vaccines,” he states.

“This is unfortunate,” Van Assche continues, “as they have the potential to bring significant and sorely needed added value to the treatment of numerous patients as evidenced by the recent success with mRNA-based cancer vaccines.”

Looking ahead, “With the success of mRNA as a vaccine modality, we are now seeing mRNA being developed for enzyme replacement therapies such as ornithine transcarbamylase (OTC) deficiency and phenylketonuria,” says Kehail. “In addition, we are also seeing increased development activities in gene editing for hematological diseases, as well as protein replacement. Of course, the main challenges compared to vaccine development is high dose requirements, targeted delivery, and expression levels.”

Van Assche further elucidates by pointing out that the potential of mRNA-based therapies is only limited by the delivery methods for the mRNA. “Once a solution is found to deliver mRNA to any cell type and tissue, mRNA-based therapies can be used for any pathology,” he asserts.

Van Assche lists the following therapeutic applications that are within reach of mRNA-based therapies with the delivery solutions currently at play. These include:

Kurrikoff echoes the sentiments, also noting that protein replacement is a potential therapy area, provided that mRNA delivery is efficient. “We could, in theory, introduce any desired protein to our cells. This way, we could replace any dysfunctional protein with a new one and cure basically any disease. Obviously, we are far from such efficacy, and some tissues pose a considerable barrier, such as the brain,” he states.

“While the field of mRNA vaccine research is still in its early stages, the technology’s versatility, scalability, and speed of development make it especially promising for numerous disease applications,” Dreps foresees. He explains that the production process for mRNA vaccines is especially advantageous for developing vaccines against emerging infectious diseases.

“Successful mRNA vaccines for COVID-19 have paved the way for the treatment of numerous infectious diseases, such as HIV, tuberculosis and Zika. Recent research with mRNA technology shows promise for developing a universal influenza vaccine that could immunize against all major influenza subtypes,” Lachs adds.

1. Rojas, L. A.; Sethna, Z.; Soares, K. C.; et al. Personalized RNA Neoantigen Vaccines Stimulate T Cells in Pancreatic Cancer. 

, 144–150. DOI: 10.1038/s41586-023-06063-y

Feliza Mirasol is the science editor for 


Volume 37, No. 1
January 2024
Pages: 14–17

When referring to this article, please cite it as Mirasol, F. From Vaccines to Oncology and Beyond: Tracking mRNA’s Progress. 

Development

The success of mRNA-based vaccines paves the way for mRNA in oncology and beyond.

From Vaccines to Oncology and Beyond: Tracking mRNA’s Progress

Cell culture is fundamental to biomedical research and can be categorized into two major platforms: adherent and suspension. Both approaches are suitable for a range of diverse research applications, including basic cellular biology and disease modeling as well as for biopharmaceutical manufacturing, including biologics production, cell and gene therapies, and vaccine production. Each approach has advantages and limitations to be considered along with other factors, such as scalability, resource requirements, facility space, and process monitoring. This article provides an overview of these considerations.

Adherent cell culture describes the method for propagation of anchorage-dependent cell types on a growth substrate. Anchorage-dependent cell types require physical attachment to a substrate or extracellular matrix (ECM) for proliferation and survival (1–3). Many common primary and continuous cell lines are adherent, including mesenchymal stem cells (MSC), induced pluripotent stem cells (iPSC), human embryonic kidney 293 (HEK293), Vero, and Chinese hamster ovary (CHO). Adherent cell types fall into several groups based on their origin and morphological features such as nuclei size, cytoplasm ratio, and shape (

). For example, fibroblast-like cells are elongated with a spindle-shaped morphology (4), epithelial cells exhibit a polygonal shape (5), and endothelial cells elongate and assemble in a ductal fashion (6), whereas neuronal cells spread with projecting processes (7). Many adherent platforms enable visualization of cells to monitor cultures, which is valuable because cell morphology is an important phenotypic indicator of cellular biology and function.

The intracellular signaling cascades that regulate many cellular functions, including cell cycle progression, cell differentiation, and responses to growth factors are governed by cell–cell and cell–matrix interactions (3). Adherent cell culture provides a more natural environment because it can recapitulate these interactions. Vessel surfaces can be modified with various treatments and biologically relevant coatings to optimize cell adhesion for specific cell types and experimental conditions. In this way, adherent cell culture vessels can be tailored to specific applications to achieve or maintain a particular cellular phenotype. In particular, iPSCs often exhibit changes in morphology depending upon the surface they are cultured upon. Gelatin-coated surfaces promote endothelial differentiation (8), whereas natural ECM-based hydrogels provide the signaling cues to maintain pluripotency (9).

Adherent culture is essential in applications requiring closely controlled cell physiology and function (1,2). For regenerative medicine and tissue engineering applications, structural scaffolds are employed to support cell growth and differentiation. Examples include MSCs expanded on ECM hydrogels for bone and cartilage repair (10–12) and hiPSCs cultured on adherent substrates to produce cardiomyocytes for heart tissue repair (13). Additionally, skin grafts for wounds are created by culturing fibroblasts and keratinocytes on collagen matrices (14), highlighting the necessity of adherent culture for tissue architecture fidelity.

Adherent platforms offer versatility not only in simply surface modifications, but also in size range, making them adaptable to diverse experimental needs. Traditional dishes, flasks, and microplates are widely used for a broad spectrum of research applications and drug screening. For larger-scale production, roller bottles continue to be one of the major platforms for vaccine production. Stacked platforms (e.g., CellSTACK Culture Chambers, Corning) and more advanced technologies amplify conventional planar adherent culture by packing multiple layers into a standard footprint. Fixed-bed bioreactors combine an adherent substrate in a bioreactor vessel with media conditioning and process automation to deliver high yield and larger scale. In short, there are a breadth of offerings at different scales, from R&D through to clinical production, for a range of budgets and accounting for different spatial footprints.

However, adherent platforms are limited in scalability. Because adherent cells are attached to the growth substrate, culture expansion is limited by surface area. When cells have covered the growth surface, the cells must be subcultured by either chemical (e.g., ethylenediaminetetraacetic acid), enzymatic (e.g., trypsin), or mechanical dissociation (e.g., scraping) to disrupt the cell–cell and cell–substrate bridges (15,16). Many cell types tolerate dissociation well. However, care must be taken to minimize degradation to surface proteins, which could disrupt adherence to subsequent culture vessels and/or negatively impact downstream applications, such as surface marker identification (17). There are also added process steps and labor involved in subculturing adherent cells—wash steps, incubation, quenching, and/or centrifugation to remove the dissociation reagent (15,16).

Labor also becomes a factor in the scalability of adherent platforms. At some point with some vessel types (e.g., multilayer stacked vessels), greater surface area can only be achieved by scaling out the process by adding multiple units of the same vessel versus scaling up through progressively larger vessels. Scaled-out processes generally require more manual labor and have a much larger footprint per growth area compared to suspension platforms (2).

When considering scaling adherent cultures versus suspension, there are some additional limitations to factor in: media usage and open versus closed systems. Some adherent culture vessels may have fixed or more restrictive media volume requirements. Nevertheless, incorporating circulation with in-process monitoring can help to get media usage per growth area to a more favorable level for specific dynamic adherent platforms. Finally, many traditional adherent vessels are handled as open systems. While open system handling of dishes and flasks on a small scale might not be problematic, there is risk of contamination associated with manual open handling of roller bottles and larger stacked vessels (2). Closed system configurations and accessories are becoming increasingly available for some adherent vessels to mitigate contamination risk for large scaled-out processes.

Suspension is a method for cultivation of cells suspended in growth medium (Figure 1). Suspension cells do not require attachment to a substrate; they grow free-floating as single cells or as multicell clumps or clusters (16). This growth paradigm is the native state for hematopoietic stem cells (18) and immune cells (e.g., Jurkat [19]). Various insect cell lines, such as Sf9, also grow in suspension. CHO and similar production lines have been adapted for growth in suspension (20). However, not all adherent cell lines are amenable to the process of suspension culture adaptation. Consider epithelial cells which lose polarity and the ability to form junctions in suspension culture, affecting their functionality (21).

Generally, suspension cells require some form of agitation during culture to keep them from settling to the bottom of the vessel and to facilitate gas and nutrient exchange. They can be cultured in a few different dynamic culture platforms. Shaker flasks, which resemble Erlenmeyer flasks fitted with a gas-permeable cap, swirl media as flasks are rotated on an orbital shaker. Spinner flasks and bioreactors use impellers to stir cultures to keep them aerated and in suspension. Suspension cultures can also be grown in culture bags on a rocking-motion bioreactor platform. Depending on scale and vessel type, gas exchange can occur passively by diffusion of the culture incubator atmosphere—or actively by delivery of a controlled gas mixture.

Because suspension culture is dynamic, there is greater shear stress exerted on these cultures compared to static adherent cultures. Some cell types, in particular, primary cells and stem cells, are more sensitive than others (2). Frequently, suspension culture media is supplemented with pluronic or other surfactants to protect cells from shear stress (22,23). Likewise, different suspension platforms exert varying levels of shear. Design variations can lessen shear stress—for example, flasks with or without baffles (24), different impeller designs (25), and traditional stirred versus vertical wheel bioreactors (e.g., Vertical-Wheel, PBS Biotech [26]). Gas sparging method and rate can also affect shear stress for actively gassed cultures (23,27). Suspension culture requires a careful balance between sufficient agitation to provide media aeration and prevent culture settling and minimizing the shear stress on cells.

In addition to shear stress limitations on cell expansion, media volume and cell concentration can constrain suspension cultures (15). However, suspension cultures are simply diluted with fresh medium to subculture, maintaining a specific density of viable cells per unit volume to sustain growth and keep the cells in a logarithmic growth phase (15). Provided that culture vessel minimum and maximum working volumes are met, suspension cells can be grown in as little or as much medium as desired. With significantly high yield for the spatial footprint, especially in the production stirred-tank reactors, suspension cell culture is substantially more scalable than adherent cultures. These platforms operate as closed systems, with in-process monitoring and controls. With enough process development to characterize and understand how each suspension line grows, in-process monitoring can accurately track progress of cell expansion in the absence of direct cell visualization.

Suspension platform advantages of scalability and operational efficiency may come at the cost of time and work upfront. On the one hand, suspension cell cultures are integral to biopharmaceutical production due to their scalability. CHO cells grown in suspension are widely used for production of monoclonal antibodies (mAbs), treating conditions from cancer to autoimmune diseases (20,28,29). Furthermore, suspension cultures of insect cells facilitate rapid vaccine production, as seen with recombinant protein vaccines against viruses such as human papillomavirus (30). However, other cell types require adaptation to suspension culture. Time and resources are necessary for cell line and application development, though the boundaries between adherent and suspension cell cultures are becoming blurred because of new technological advances. There has been a shift toward more scalable suspension culture for some traditionally adherent applications. For instance, adeno-associated viral vectors for cell and gene therapy applications, once reliant on adherent HEK293 cultures, are shifting toward suspension cultures for improved yields with suspension HEK293 lines and suspension-ready transfection reagents now commercially available (31,32).

Microcarrier technology is a hybrid approach that enables growth of anchorage-dependent cells in a scalable suspension environment. Microcarriers are small beads used to provide a growth substrate for adherent cells to grow in suspension (33). Microcarriers are manufactured from a variety of materials including polystyrene and dextran-based microcarriers (34–36). Other microcarriers are made from biodegradable polymers that simplify downstream processing by eliminating the need for separation steps (35). Surface modifications on microcarriers can range from simple protein coatings, such as collagen or fibronectin, to more complex synthetic peptides that mimic extracellular matrix components (37), each designed to optimize the growth and functionality of specific cell types. Importantly, microcarriers are enabling traditionally adherent-dependent cells to be cultivated in suspension for mAb production (38).

Another hybrid approach that lies between adherent and suspension culture is the cultivation of spheroids and organoids, otherwise known as 3D cell culture. There are methods that utilize extracellular matrix scaffolding to support 3D growth and scaffold-free methods that encourage cells to self-organize into structures that mimic the form and function of tissues in vivo (39). For example, human iPSCs (hiPSCs) cultured in suspension form spheroids that can be differentiated into hiPSC-derived cardiomyocytes (40). Regardless of method, cell–cell connections are formed in three dimensions that provide the contextual clues for more natural cell behavior, with improved gradients of oxygen, nutrients, and signaling molecules that are critical for cellular differentiation and function (39). The resulting tissue-like structures present in 3D cultures offer advanced models for studying disease mechanisms, tissue regeneration, and drug responses with higher physiological relevance compared to 2D systems.

The evolution of cell culture technology continues to revolutionize biological research and industrial applications. While adherent platforms that facilitate natural cell–cell and cell–matrix interactions characteristic of many cell types exemplify this endeavor, this advantage is tempered by limited scalability and higher labor requirements. In contrast, suspension cultures excel in scalability, particularly for high-yield production, provided that the cell type thrives in suspension. Advanced platforms that merge the advantages of both, such as microcarrier technology and 3D cultures, are leading the charge towards more advanced and efficient culture systems. Choosing between these platforms requires a deep understanding of their respective advantages and limitations, ensuring a strategic fit for the intended application and research objectives. Looking forward, continued innovation is expected to further enhance these cell culture technologies, broadening their application in the fields of general bioproduction, regenerative medicine, and cell and gene therapy. This progress emphasizes the critical role of strategic platform choice to leverage the full potential of cell culture advancements.

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Ann Rossi Bilodeau, PhD, is senior bioprocess applications scientist at Corning Life Sciences.


Volume 37, No. 1
January 2024
Pages: 18–22

When referring to this article, please cite it as Bilodeau, A. R. The Pros and Cons of Adherent Versus Suspension Cell Culture. 

Upstream Processing

Both adherent and suspension cell culture approaches have their pros and cons, which must be considered for process development.

The Pros and Cons of Adherent Versus Suspension Cell Culture

Cell-based therapeutics are defined as “articles containing or consisting of human cells or tissues that are intended for implantation, transplantation, infusion, or transfer into a human recipient” as per 

 Title 21, Part 1271.3(d) (1). With their unique mechanism of action, cell therapy products hold the potential to treat many diseases that are refractory to current remedies in therapeutic areas such as regenerative medicine, immunotherapy, and cancer therapy. Technological advancements and the approval and successful commercialization of several chimeric antigen receptor T cell (CAR-T) products since 2017 have promoted exponential expansion of the cell therapy landscape over the past several years.The global market size is estimated to increase from $9.5 billion in 2021 to $23 billion in 2028 (2).

As with all biopharmaceuticals, thorough characterization and a robust lot-release testing program are required to ensure product safety and efficacy of novel cell-based therapeutics. The reliable assessment of product potency, a critical quality attribute (CQA), is of particular importance. To this endeavor, bioassays that can measure relevant biological responses and/or characteristics in accordance with a product’s expected mechanism of action serve a significant role.

, animal-based bioassays, when appropriately developed, can be more relevant to clinical response than 

 bioassays but often suffer from higher inherent variability and lengthy assay time. In comparison, 

 cell-based assays tend to offer more robust assay performance with less intrinsic variability and faster turnaround of results. In addition to the general effort of adopting the principal of three Rs (replacement, reduction, and refinement), it is also recognized that for most cell-based therapeutics, a relevant 

 assay for potency measurement is understandably often not possible due to the fact that test samples are live human cells. Therefore, in this article the author focuses on discussing the unique challenges of developing and implementing 

 cell-based assays for the potency measurement of cell therapy products.

Compared to protein therapeutics, cell therapy products come in a much wider range of diversity and complexity. With the large panel of analytical tools available for protein products being non-applicable, potency assessment becomes the focal point and the most challenging aspect of product characterization of cell therapeutics. While there are simpler cell therapy products for which measuring cell viability and specific cell surface markers may be sufficient as potency assessment, the majority of cell therapeutics require significant potency assay development efforts.

Potency assays first and foremost need to reflect a product’s mechanism of action. In the case of cell-based therapeutics, there are often multiple modes of action; and in many cases, not all of these modes are completely understood/delineated. Accordingly, it is crucial that potency assay development focuses on carefully selecting the appropriate assay(s) that is/are reflective of key mechanism(s) of action, which naturally leads to a “matrix approach”. For example, cytokine release and antigen-specific cell killing are considered the most relevant modes of action for CAR-T products. Correspondingly, functional tests measuring these responses, in conjunction with assays for cell viability, transgene expression copy number, and the phenotypical characterization of cell surface receptors and CAR expression, make up a typical CAR-T potency testing matrix.

It is worth noting that additional assays are often developed and used to more thoroughly characterize the product and to assist process development during early stages of clinical development. Failure to develop an adequate assay matrix to address complex mechanisms of action(s) may delay product licensure, as in the case of lifileucel, an autologous tumor infiltrating lymphocyte (TIL) therapy, for which the sponsor’s testing scheme, with a single potency assay, was considered inadequate and rejected by FDA (3). Additional assay development effort was subsequently undertaken by the sponsor and a test matrix was proposed and implemented, including a functional cell co-culture assay, to measure potency with multiple aspects of the product. In some cases, direct functional assays may suffer from difficulties, such as lengthy assay time and poor performance, that prevent them from serving as quality control (QC) methods. In such cases, upon discussions with regulatory agencies and with sufficient justification, surrogate assays may be utilized for release testing; however, a functional assay should be developed as a characterization tool, and correlation between the two methods should be demonstrated.

The complex nature of cell therapy products presents additional practical challenges for potency assay development and implementation. These may include inherent product variability due to starting materials (donor cells), limited material available for testing, the lack of reference standard and an urgency for quick product release. For protein therapeutics, sample potency is measured against a well-characterized reference standard and expressed as relative potency calculated from comparing the median effective concentration (EC50) or half-maximal inhibitory concentration (IC50) of the dose response curves. In the case of cell therapeutics, however, a true reference standard often cannot be generated, especially in individualized therapies.

In the absence of a reference standard, appropriate assay control(s) should be identified and implemented. Ideally, a significant amount of material generated within the same manufacturing process that behaves similarly to the test sample should be implemented as an assay control, along with other controls that help assure acceptable assay performance.These assay controls should be thoroughly characterized and are of great value in long-term assay maintenance and dissecting assay variability from product variability, which, in combination with other considerations, can provide critical information to help and justify setting product specifications. The reported potency results, even if not possible to be directly compared to a reference standard and expressed as relative potency, should still be quantitative wherever possible.

Another practical challenge for cell therapy products is often the need to release the product quickly. The logistics to ensure a quick turnaround time to generate test results requires efficient coordination between multiple parties involved. It becomes a particularly important consideration when the sponsor works with contract manufacturing and testing organizations.A streamlined, expedited process from sample shipment/receipt, laboratory testing, to final release of certificate of analysis should be well established and tested for potential roadblocks. In rare cases, the short shelf life of a product may warrant an abbreviated release test panel, which may be granted by regulatory agencies on a case-by-case basis. For example, products may be released after assessing key phenotypical biomarkers while awaiting results from lengthy functional bioassays. In this case, the justification for a simplified conditional release test procedure should be clearly described and explained. More importantly, the positive correlation between phenotypical assessment and functional assays should be established well in advance during product development and process validation. Notably, accessibility to this pathway highlights the importance of adopting the “matrix approach” for potency assessment, as discussed in the previous paragraph.

In general, development and implementation of potency bioassays is an ever-evolving process, and it is particularly true for cell therapy products. Development of relevant, quantitative, and robust QC potency methods has proven to be challenging and time consuming. Typically, sponsors are expected to “initiate potency assay development by way of product characterization during preclinical and early clinical investigations to obtain as much product information as possible” as recommended in FDA’s guidance for industry on 

Potency Tests for Cellular and Gene Therapy Products

 (4). Over the course of product development, potency testing strategy is refined, leading to a partition between methods intended for lot release versus characterization based on both scientific and practical considerations. Lot-release potency assay(s) should be fully validated, and assay and sample acceptance criteria tightened in preparation of the biologics license application. Once validated and deployed for routine testing, continuous assay monitoring and maintenance is necessary, especially given the assay complexity and the implementation of multiple controls.

The bridging between different lots of controls and other critical reagents needs to be planned well in advance and guided with a clearly defined testing scheme and criteria. In the case where lot release testing is performed in a laboratory different from where it was originally developed, method transfer may post significant challenges. Expertise in some of the highly specialized assays may be difficult to source.

Some assays, such as flow cytometry methods, can present unusual difficulties due to highly customizable instruments and manual input for data analysis. Because the intensity of the fluorescent signal is influenced by the assay-specific instrument settings, data analysis and even acceptance criteria may need to be adjusted, which in turn can lead to the need for a more substantial comparability study. Therefore, a gap analysis should be performed to guide the selection of a compatible partner lab and the implementation of a sufficient training program.

Ideally, the same instruments and filter configurations are utilized and cross standardized between the originating and receiving laboratories. Advanced planning that allows co-validation may allow better resource utilization and an expedited timeline. In the case where substantial modifications are introduced to the methods due to instrument differences, a full validation by the receiving laboratory coupled with a comparability study may be necessary.

The excitement around the potential of cell therapeutics has fueled the race for developing the next breakthrough therapy. These complex biological products can present unique technical and regulatory obstacles with respect to potency bioassays as discussed. Initiating potency assay development early on allows sponsors to gain useful information on multiple aspects of their products’ function so that a sufficient and practical lot release and stability testing program can be developed that are both scientifically sound and regulatory compliant.

Continuous monitoring and sufficient control of potency bioassay performance is of paramount importance to ensure accurate potency measurement and meaningful interpretation of clinical results. Challenges such as the lack of relevant reference standards, need for rapid product release, method transferability, and a requirement for specialized assay expertise can be mitigated through careful planning and close communications between all stakeholders.

Given the still-emerging regulatory guidance in this new field, discussions with regulatory authorities should be initiated early on to seek necessary advice whenever difficulties in potency testing are observed to prevent delay in the advancement of clinical development.

 (Government Printing Office, Washington, DC), 

www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/cfrsearch.cfm?fr=1271.3

Cell Therapy Market Size Analysis Report, 2021–2028

. Market Research Report (2021).
3. Iovance Biotherapeutics, Iovance Biotherapeutics Provides Regulatory Update for Lifileucel Potency Assays. Press Release, May 18, 2021.
4. FDA. 

Guidance for Industry, Potency Tests for Cellular and Gene Therapy Products

Weihong Wang is technology development director at Eurofins BioPharma Product Testing.


Volume 37, No. 1
January 2024
Pages: 31–33

When referring to this article, please cite it as Wang, W. Navigating Challenges in Cell Therapy Potency Assays. 

Analytics

Developing cell-based potency assays for cell therapies requires meticulous coordination.

Navigating Challenges in Cell Therapy Potency Assays

Targeting enhancement with two active antibody arms makes bispecific antibodies (bsAbs) highly attractive for the treatment of cancer and other diseases. Their purification can be more challenging than traditional monoclonal antibodies (mAbs), however, due to the increased level of product-related impurities with high similarity to the desired bsAbs. Advances in chromatography resins are helping to simplify downstream operations and improve separation performance.

In addition to process-related impurities like those seen with mAbs, bsAb manufacturing often results in the production of product-related impurities that can be difficult to separate from the desired product. These impurities, according to Russell Overbeck, director of global technical and CMC at Lonza, largely comprise mispaired variants, including homodimers, halfmers, and others. “Depending on the format of the bispecific, often homodimers, fragments, or half antibodies need to be removed during downstream processing,” agrees Leslie Wolfe, vice-president of process development with KBI Biopharma.

“The biggest challenge for purification of bsAbs, therefore, is separating the target molecule from mispaired variants,” Overbeck states. The difficulty can be enhanced for some bsAbs due to their tendency to aggregate and/or sensitivity to lower pH conditions, which can lead to degradation, misfolding, or other structural changes, comments Eva Heldin, a principal scientist at Cytiva.

On a positive note, Wolfe observes that how a bispecific is designed and/or how the cell line expresses the bsAb can potentially reduce purification challenges. For example, she points out that separation of homodimers with isoelectric points (pIs) different from the heterodimer will be simpler than separation of those with similar charges. Meanwhile, the removal difficulty for half antibodies or other product-related impurities can depend on their levels in the clarified harvest.

Purification approaches employed for standard mAbs can often be applied to bsAbs to a certain extent. Processes typically include Protein-A, polishing I, and polishing II chromatography, according to Overbeck. For a given bsAb, he notes that depending on the molecule complexity, pI, and other factors, these steps can look different, but mainly still consist of ion exchange (IEX) and/or hydrophobic interaction chromatography (HIC), and mixed-mode chromatography.

Wolfe says that host-cell proteins and host-cell DNA are typically removed by anion and cation exchange chromatography, while mispaired variant impurities may require an alternative affinity capture resin to remove half antibodies or mixed-mode and/or HIC chromatography to remove homodimers depending on the design of the bsAb.

For Heldin, it is the Protein A capture step that removes most of the host-cell proteins and host-cell DNA. She does observe, however, that often differences in avidity can be imparted to affinity resins that can also provide selectivity for product-related impurities. “We have seen, for instance, that with MabSelect PrismA resin, which possesses both Fc- and VH3-interactions, the avidity effect may be leveraged for an asymmetric bsAb if designed for asymmetry in respect to one of these interactions,” she explains.

A chromatographic step with anion exchanging properties of the resin is almost always included to facilitate good viral clearance and to remove any residual DNA after the capture step, Heldin continues.

High resolution and selectivity are essential

For purification of bsAbs, selectivity over the chromatographic steps is essential, according to Heldin. High resolution is best, adds Overbeck, followed by a combination of high dynamic binding capacity and good pressure/flow characteristics.

“To achieve these goals,” Heldin explains, “the purification process is typically designed with individual steps using orthogonal interaction principles.” She notes that it is important to start with an affinity step that has selectivity for product-related impurities in order to simplify concurrent polishing steps. Furthermore, she says that to achieve a large-scale process with good economy, resins should, in addition to having good pressure/flow properties and good binding capacities, be cleanable between runs to enable several cycles of purification.

One key to ensuring good downstream purification performance for bispecifics does not relate to the actual chromatography resins. It has to do with designing stable bsAbs. “Carefully investigating bsAb stability is a good starting point,” states Heldin. Doing so helps determine pH limitations and acceptable salt conditions. That information can in turn be used to select process conditions that ensure good stability of the targeted bsAb, she adds.

As an example, Heldin notes that while the capture step is operated at a neutral pH and is typically straight forward, instability can be observed at elution. In this case, need for instantaneous adjustment to a higher pH may be necessary upon elution. She also notes that “if an HIC-step is found to be more selective than ion-exchange, it may be advantageous to run that step at non-binding or partial-binding conditions to avoid high salt concentrations.”

Product and process knowledge drive resin selection

Understanding the characteristics of a bsAb product as well as the potential impurities that its production process may generate and their properties is necessary for effective chromatographic resin selection.

“When developing a purification process for a bsAb,” Wolfe emphasizes, “it is important to understand the biochemical properties of the product-related impurities that may be present. Specifically, the isoelectric point of the bsAb compared to the potential homodimers is important in selecting not only the resins to screen but also the pH range to evaluate.”

Wolfe goes on to note that depending on the design of the bsAb and product-related impurities present, a specific affinity capture resin may be beneficial to separate half antibodies from the target bsAb. “In these instances, selecting an affinity resin that binds to a region of the bsAb that is not present in the product-related impurities is important,” she remarks.

Similarly, Wolfe notes that understanding the biochemical properties of the product-related impurities that may be present during the polishing step is important for selecting these resins.

The type of bispecific and its intrinsic molecular properties will typically dictate which resin is needed, agrees Overbeck. “Ninety percent of the time, high-resolution cation exchange is effective,” he states. In cases where it is not, he observes that hydrophobic interaction and mixed-mode resins should be screened with high-throughput robotics and scaled-up as appropriate.

Heldin agrees that resins for polishing steps should be screened and stresses that the necessary orthogonality be kept in mind. However, she recommends including a resin with anionic interactions (only anionic or multimodal anionic), and notes the order of the polishing steps can be decided later, always with the goal of minimizing the required buffer adjustments and sample preparation.

Screening of chromatography resins for bsAb purification should be achieved, according to various experts, using a combination of physical experimentation and computational modeling.It may, in fact, be necessary to screen for more polishing resins that potentially can provide the selectivity needed for closely related impurities than when working with mAbs, particularly for process developers that have not previously used multimodal resins, according to Heldin.

For instance, according to Wolfe, often product-related impurities have a different surface charge and/or hydrophobicity from the target bsAb. “Through either in silico or experimental characterization of these impurities, one can determine how the target bsAb may differ from the impurities and select resins and operating conditions to screen that will yield the best separation,” she says.

In addition, Wolfe recommends selecting a resin with some level of hydrophobicity, even if the contribution comes from the base bead, to help with development of a robust separation step. She also contends that given the sometimes-challenging separations presented by bsAbs, leveraging multimodal properties on the surface of the bsAb usually results in a more robust separation. Overbeck adds that if a typical high-resolution cation exchange resin does not work, then high-throughput screening using robotics is the best strategy for identifying the best resin.

Of course, screening methods are not effective without good accompanying analytics. “Access to an analytical assay during process development that can distinguish the bsAb product from similar impurities is critical to ensuring a robust separation step,” observes Wolfe.

New resin chemistries for bsAb purification have focused, not surprisingly, on improving their separation from similar product-related impurities. Some have also focused on providing effective solutions for more sensitive molecules.

Cytiva has introduced several products addressing the first need. “The development of resins with affinity to other domains than the Fc-domain of bsAbs has enabled selectivity towards product-related impurities in the capture step and not just the polishing steps,” Heldin comments.

Product and process understanding are essential to effective resin selection.

BioPharm International, January 2024

Leap Year

New Therapies Cry Out for New Skills

Right the First Time: Bioreactor Scale and Design Translation

From Vaccines to Oncology and Beyond: Tracking mRNA’s Progress

The Pros and Cons of Adherent Versus Suspension Cell Culture

Navigating Challenges in Cell Therapy Potency Assays

Selecting Chromatography Resins for Bispecific Antibody Purification

Are You Ready for Inspection?

Reimbursement Precipitating Shortages