With the help of recent investor interest, progression of advanced therapy medicinal products (ATMPs) in the clinical space continues to move forward. Buoyed by the regulatory approval of several cell and gene therapy (CGT) products for difficult-to-treat diseases, the US biopharma industry is now bracing for a new wave of CGTs in the near future as clinical pipelines reach fruition. As a result, both regulatory and industry are strengthening their relationship with the goal of preparing the US healthcare market for the entrance of these new products.

At the Alliance for Regenerative Medicine’s (ARM’s) annual industry briefing (1), Tim Hunt, CEO of ARM, characterized the CGT field as “now-maturing”. The CGT space is achieving a new normal categorized by serial approvals of breakthrough products according to Hunt in his state-of-the-industry address.

The biopharma industry has gone from seeing five gene therapy products approved within the span of five years (2017–2022) to witnessing breakthrough approvals for five gene therapies in just one year, 2023. According to Hunt, ARM was hopeful that the industry would get approval for five gene therapies for rare disorders approved within a single year, “and we called it our 5x5x5” (1).

“We got that one right: five programs approved in one year,” Hunt emphasized in his address. Importantly, the 2023 gene therapy approvals were for five programs in rare diseases that had been difficult to treat until now: one program in dystrophic epidermolysis bullosa, one in Duchenne muscular dystrophy, one in hemophilia A, and two in sickle cell disease (

“I think [this is] a glimpse of a lot of the progress we’ll see in the future,” said Hunt in his address.

ARM’s industry evaluation shows that, across North America, Europe, and Asia Pacific in 2023, the field of CGTs saw more than 2500 developers throw their hat into the ring and nearly 2000 clinical trials underway. Investment into this sector was nearly $12 billion for the year, which Hunt noted in the industry briefing as being slightly down compared to 2022, but that commitment to the development of CGTs remains vibrant. Hunt went on to explain that there is significant time, talent, and money being committed to CGTs with the goal of getting these therapies to patients with debilitating, often fatal, diseases or disorders (1).

Looking ahead to 2024, Hunt noted that ARM sees a total of 17 programs that could, in theory, get approval in the year. “We’re not going to get all 17, let’s be clear,” he stated in his address, “but we’ll get a subset of that. [However,] there’s 17 things that we can identify at this point that are at least in the discussion.”

In ARM’s estimation, the regulatory outlook for 2024 includes seven total decisions on CGT approvals pending—five in the United States and two in the European Union (EU)—and five total submissions pending—three in the US and two in the EU (1). Meanwhile, the number of regulatory submissions possible in 2024 come to five in total, three in the US and two in the EU. “We’re optimistic that we’ll see a lot of these get pulled through to the finish line,” Hunt stated. “It is also possible that we could see five more gene therapies approved in the United States for rare genetic conditions.”

Hunt also emphasized potential milestones that the industry may see in 2024. Among these are the first ever approval of an adoptive cell therapy for solid tumors in the US, and the first US approval of an allogeneic T-cell therapy. The industry can also potentially see additional therapies approved to treat hemophilia A, hemophilia B, and dystrophic epidermolysis bullosa, following the recent approval success of CGTs in these conditions in 2023 and 2022 (

“I think that’s the sign of an increasingly maturing field, right where you see more competition, which ultimately is good news for patients. If you go back in time to 2019 when FDA … said we could see 10 to upwards of 20 approvals a year starting in 2025, we think that is tracking rather nicely for that time period. It’s conceivable that we could see that [level of approvals] in the US in 2024 but we’d have to almost essentially run the tables,” said Hunt (1).

Nevertheless, there has been a significant amount of progress in this field, Hunt also noted. With success, however, comes further challenges, he continued. Pricing pressure for innovative therapeutics remains a volatile situation in the US market but, by Hunt’s assessment, the CGT market is still in its early days and working through the business model. “The good news is healthcare systems have worked through this before. They’re adapting right now, I would argue, led by the commercial payers, and we will work through these challenges yet again,” Hunt remarked in his address (1).

Meanwhile, a key goal for ARM in 2024 and 2025 is to discuss value and society and how these new therapies are perceived. ARM will do this through a new framework it is calling the Gene Therapy for Patients and Societies (GPS) framework. The GPS framework will have five components centered around the value of gene therapies, which:

target devastating, often deadly diseases

are affordable (even before taking into account cost offsets).

Because gene therapies are affordable—even before taking into account cost offsets—Hunt posited that this component would make gene therapies easily digestible for healthcare systems in the years ahead.

What’s more, Hunt pointed out that US healthcare systems are modernizing, which is beneficial for CGTs. The Center for Medicare and Medicaid Innovation’s (CMMI’s) access model, the CMMI Cell and Gene Therapy Access Model, adopted in February 2023 (11), for example, is intended to establish a voluntary partnership among the Centers for Medicare & Medicaid Services (CMS), state Medicaid agencies, and biotech companies. From these partnerships, outcomes-based agreements (OBAs) can be set up and administered, which can particularly help smaller states in the US that lack the resources to administer OBAs, according to Hunt. The model is projected to launch in 2025.

“We think this is a great development,” stated Hunt. “We believe this will be beneficial, especially to smaller Medicaid agencies that will just frankly struggle to put together the infrastructure to measure and partner with companies.” Furthermore, moving forward with this CMMI access model will be a way to modernize how the US healthcare system pays for CGTs (1).

Meanwhile, FDA has also been modernizing to prepare for an anticipated wave of CGTs to hit the market. In 2023, the agency established the Office of Therapeutic Products (OTP), a new “super office” structure that was transitioned from the former Office of Tissues and Advanced Therapies (OTAT). OTP was implemented on Feb. 26, 2023 (12). Hunt praised this transition, stating that it was “a very welcome move.”

“We are doing a lot to partner with FDA over the [p]ast year and a half to try to accelerate cell and gene therapy development and manufacturing,” Hunt emphasized in his address (1). In preparing for the future, ARM and FDA will be looking at streamlining CGT development and

manufacturing, collaborating on complex chemistry, manufacturing, and controls issues, improving societal readiness for CGTs, and scanning the horizon for future technologies.

1. ARM. Cell & Gene State of the Industry Briefing. 

, Jan. 8, 2024.
2. FDA. FDA Approves Omidubicel to Reduce Time to Neutrophil Recovery and Infection in Patients with Hematologic Malignancies. Press Release, April 17, 2023.
3. FDA. FDA Approves First Topical Gene Therapy for Treatment of Wounds in Patients with Dystrophic Epidermolysis Bullosa. Press Release, May 19, 2023.
4. FDA. FDA Approves First Gene Therapy for Treatment of Certain Patients with Duchenne Muscular Dystrophy. Press Release, June 22, 2023.
5. FDA. FDA Approves First Cellular Therapy to Treat Patients with Type 1 Diabetes. Press Release, June 28,2023.
6. FDA. FDA Approves First Gene Therapy for Adults with Severe Hemophilia A. Press Release, June 29, 2023.
7. FDA. FDA Approves First Gene Therapies to Treat Patients with Sickle Cell Disease. Press Release, Dec. 8, 2023.
8. EMA. Hemgenix. 

ema.europa.eu/en/medicines/human/EPAR/hemgenix 

(accessed Jan. 11, 2024).
9. uniQure. uniQure Announces License Agreement with CSL Behring to Commercialize Hemophilia B Gene Therapy. Press Release, June 24,2020.
10. FDA. FDA Approves First Gene Therapy to Treat Adults with Hemophilia B. Press Release, Nov. 22, 2022.
11. CMS. HHS Secretary Responds to the President’s Executive Order on Drug Prices. Press Release, Feb. 14, 2023.
12. FDA. Establishment of the Office of Therapeutic Products. 

Feliza Mirasol is the science editor for 


Volume 37, No. 2
February 2024
Pages: 7–9

When referring to this article, please cite it as Mirasol, F. Bracing for a Future Wave of Advanced Therapies. 

Articles

Cover Story

In the ATMP space, CGTs are hitting their stride with unprecedented approvals in the past year alone.

Bracing for a Future Wave of Advanced Therapies

Article Submitted: Nov. 8, 2022.
Article Accepted: Oct. 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 1 of the study.

Stijn H. S. Koshari is a principal investigator, Hong Zhang is an investigator, Antonio Ubiera is a senior director, and Robert G. Luo is a scientific director,all at Downstream Process Development, BioPharma R&D, GlaxoSmithKline, King of Prussia, Pennsylvania 19406.


Vol. 37, No. 2
February 2024
Pages: 26-31

When referring to this article, please cite it as Koshari, S.H.S; Zhang, H.; Ubiera, A. and Luo, R.G. Fouling Mechanisms of Filters During the Harvest Development of Monoclonal Antibody Therapeutics with Intensified Upstream Processes—Part 1. 

Peer-Review Research

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. 

Fouling Mechanisms of Filters During the Harvest Development of Monoclonal Antibody Therapeutics with Intensified Upstream Processes—Part 1

The development of chimeric antigen receptor (CAR) T cell therapies has been a cause for excitement and celebration amongst researchers and patients within the oncology space because of their ability to eradicate advanced forms of leukemias and lymphomas (1). It was Yoshikazu Kurosawa, a Japanese immunologist, and his team who initially reported on the concept of chimeric T cell receptors in 1987 (2). However, it wasn’t until 30 years later, in 2017, that the first CAR-T cell therapy was approved by a regulatory body—FDA approved tisagenlecleucel (Kymriah, Novartis) for the treatment of pediatric and young adult patients with a form of acute lymphoblastic leukemia (3).

Since the initial approval of tisagenlecleucel, there have only been a limited number of other CAR-T cell therapies that have successfully completed the regulatory review process. Challenges hindering growth of the CAR-T cell therapy sector have included the COVID-19 pandemic—which resulted in a low diagnosis rate of blood cancer, disrupted supply chains, and difficulties in monitoring patients closely—and the expense of developing the therapies and cost per treatment for the patient (4).

“The development of CAR-T therapies is a complex, costly and time-consuming endeavor, which requires specific expertise across a number of scientific disciplines as well as competencies from a regulatory, commercial and bio-manufacturing perspective,” explains Paolo Siciliano, associate partner and life sciences expert at PA Consulting—a professional services firm. “There are currently only six CAR-T therapies available on the market: all of these are autologous (i.e., personalized therapies) and target hematological malignancies, such as acute lymphoblastic leukemia, multiple myeloma, and B-cell lymphoma.”

Generally-speaking, CAR-T cell therapies either in development or those approved and commercially available came out of academic medical centers or biotech companies, Siciliano continues. Big Pharma companies have a tendency to take ownership during the development and commercialization stages, he adds.

“Very few organizations have the capabilities and expertise in-house to bring a CAR-T therapy from idea to market by themselves, and finding the right partner at the right time of the development process is key to reduce risk and increase the likelihood of the therapy to make it to the market,” Siciliano says.

When approaching development of a CAR-T cell therapy, there are some key considerations to take into account, and these can be grouped by the development stages, Siciliano notes. In the preclinical research stage, it is important to consider the selection of the therapeutic area of interest, the right target (cell and antigen), and the vector/platform for gene transfer. It is also crucial to balance potency and safety through affinity optimization, minimizing potential off-target effects, he remarks.

Moving along to the clinical study stage of development, Siciliano continues, the following need to be carefully thought about: clinical trial design; selection of locations/partnering medical centers; patient enrollment; safety and monitoring; data analysis; and compliance with regulations. Then in manufacturing, companies need to determine the manufacturing processes and technologies, as well as whether to manufacture in-house or via an outsourcing partner, while also defining the logistics and supply chain, ensuring compliance with regulations, and implementing quality control procedures and processes, he states.

Finally, for commercialization, regulations again play a significant role as companies need to make sure they are fully compliant, and they must also figure out the most appropriate market strategies and what the pricing might be or whether there will be any potential reimbursements available as these factors can have profound impact on patient access to these therapies, Siciliano specifies.

“Despite a relatively slower 2023, the CAR-T and [overall] cell and gene therapy (CGT) industry has been seeing constant growth over the past few years since the first approval of a CAR-T therapy in 2017, with increasing investment, R&D, technology development and M&A activity,” asserts Siciliano.

Providing some examples, Siciliano reveals that there has been greater collaboration between academia and pharma/biotech, more knowledge about therapy development and manufacturing, and increased access to good manufacturing practice manufacturing facilities, boosting the preclinical and clinical therapy development sectors. Further along the development lifecycle, in manufacturing and supply chain, he highlights the fact that there are more contract development and manufacturing organizations providing capacity for CAR-T and CGT drugs, more collaboration across the ecosystem, innovation in CAR-T manufacturing technologies, and greater interest in digital, automation, and advanced technologies such as artificial intelligence.

“[Furthermore], key regulations are undergoing harmonization across regulatory agencies globally, and there are increasing levels of expertise in regulatory pathways for CGT and CAR-T therapies across the industry,” Siciliano says. “And in commercialization, [there are] new reimbursement models being tested and an increasing number of countries approving and adopting CAR-T.”

“The field of CAR-T therapies is likely to see considerable advancements over the next decade,” says Siciliano. Anticipated advancements include, for example, the broadening of therapeutic targets—such as using CAR-T for solid tumors, expansion into non-oncology indications, such as autoimmune diseases, new molecular and technological approaches that reduce manufacturing times of autologous CAR-T therapies, new molecular mechanisms that enhance safety through improved specificity and efficacy, harmonized regulatory pathways, and new payment and reimbursement models, he stresses.

Additionally, the industry will benefit from new genetic transformation technologies, Siciliano remarks. “In 2023, [there has been] the first approval of a CRISPR [clustered regularly interspaced short palindromic repeats]-mediated genetically modified cell therapy,” he says. “New approaches to gene transfer will enable better transfection rates and improve therapeutic outcomes.”

As alluded to earlier, all of the currently available CAR-T cell therapies are autologous or personalized; however, allogeneic CGTs are already approved. “The first allogeneic CGT (Ebvallo by Atara Therapeutics) was approved by the EMA [European Medicines Agency] in December 2022,” states Siciliano.

Research is ongoing into allogeneic or ‘off-the-shelf’ CAR-T cell therapies, which would improve accessibility of these therapies to patients. “In the CAR-T space, there are currently no allogeneic therapies available on the market. About 130 allogeneic CAR-T therapies are currently under development,” Siciliano adds. Of these therapies in development, the majority are still only in preclinical stages with only about 15 in Phase II clinical trials, he continues.

“While we are seeing some positive results in the allogeneic space, there are still considerable challenges in the development of allogeneic (off-the-shelf) [CAR-T cell therapies], in particular with regards to their immunogenicity, the risk of GvHD [graft versus host disease] and host immune rejection,” Siciliano confirms. “It will still take a few years before allogeneic CAR-T therapies will be available on the market. It is also safe to assume that once allogeneic CAR-T therapies do reach the market, they are likely to co-exist with autologous CAR-T therapies for the foreseeable future.”

1. National Cancer Institute. CAR T Cells: Engineering Patients’ Immune Cells to Treat Their Cancers. 

, accessed Jan. 19, 2024.
2. Mitra, A.; Barua, A.; Huang, L. From Bench to Bedside: The History and Progress of CAR T Cell Therapy. 

, 14, 1188049.
3. FDA. FDA Approval Brings First Gene Therapy to the United States. Press Release, Aug. 30, 2017.
4. Fortune Business Insights. 

CAR-T Cell Therapy Market Size, Share, and COVID-19 Impact Analysis, by Drug Type, by Indication, by End-User, and Regional Forecast, 2023–2030

Felicity Thomas is the European/senior editor for 


Vol. 37, No. 2
February 2024
Pages: 10–11

When referring to this article, please cite it as Thomas, F. Developing CAR-T Cell Therapies for the Future. 

Development

Despite slow growth over recent years, the CAR-T cell therapy space is expected to see considerable advancements in the near future.

Developing CAR-T Cell Therapies for the Future

While many stem-cell therapies remain at the preclinical stage, several are progressing through the clinic toward late-stage trials. Developing successful treatments based on induced pluripotent stem cells (iPSCs) and mesenchymal stem cells (MSCs) in part depends on creating effective formulations in which cell viability is maintained during cryopreservation, storage and handling, shipment, and reconstitution prior to delivery to the patient. Excipients, therefore, play an important role in bringing novel cell therapies to the market.

Buffers, stabilizers, and cryoprotectants

As with most biologic drug products, the predominant excipient in cell therapy formulations is the buffered saline solution. In this case, the saline solution is an osmotically balanced water solution designed to keep the cells from drying out and dying, according to Ryan Guest, senior CMC translation consultant for eXmoor pharma.

In addition to physiological saline, Carole Nicco, CSO at BioSenic notes that other isotonic solutions containing chemical substances present in blood, such as Ringer’s lactate, are also used to help maintain the electrolyte balance and keep the cells stable, sterile, and viable with proliferative capacity until their application. Furthermore, these isotonic solutions offer chemical easy systemic and local application, according to Nicco.

Classically, most formulations will also have protein to balance the solution as a buffer or reservoir for smaller molecules and enable transport via active protein transporters on cell membranes, Guest notes. The standard is an albumin (0.5–2.5%), such as human serum albumin. Albumin constitutes 50–75% of the colloidal osmotic pressure of blood (1–3) and performs a very similar role in MSC and iPSC formulations, Guest says. More specifically, albumin is a useful component in cell therapy formulations for balancing water availability during product hold times, cryopreservation, and thawing prior to product administration.

In fact, Guest observes that the presence of an equivalent protein in cell therapy formulations is predicted to have a significant impact on cell survival. In MSCs, for instance, the absence of an appropriate protein can reduce viable recovery by 20–40%, he comments.

The other important excipients for cell therapy products are cryoprotectants, given that the majority of these treatments are cropreserved. The cryopreservation of MSC- and iPSC-based therapies using 2–10% dimethyl sulfoxide (DMSO) in solutions containing a high content of serum is a common procedure, Nicco observes. She adds that while DMSO has been used to reduce the formation of ice in cells stored in liquid nitrogen since 1959, it is toxic, resulting in undesired clinical and biological side effects. Serum also introduces variation and safety risks.

“There is, consequently,” Nicco says, “a move toward solutions containing bio-preservation media free of DMSO, serum, and other proteins, optimized for the preservation and distribution of these products at low temperatures, either in cold (2–8 °C) or cryopreserved conditions (-70 °C to -196 °C).” The new excipients, she explains, eliminate toxicity issues as well as the need for human product excipients (serums, proteins) that can induce a risk of contamination to the drug product. Complex formulations involving dextran-40, lactobionate, sucrose, mannitol, glucose, adenosine, and/or glutathione are examples, according to Guest.

Intended application key driver of excipient choice

The type of stem cell therapy generally does not impact the choice of excipients, although Guest does note that some iPSCs can be more acutely sensitive to the final formulation, hold times, and routes of administration. For example, anucleated products, specifically designed to deliver payloads, have a reduced capability to produce proteins or perform cellular repair. Nicco adds that iPSCs and cells differentiated from them are commonly multicellular systems, which she says also makes them more sensitive to the stresses of freezing and thawing than single cells.

Overall, however, similar types of excipients are used for the preservation of cell-based therapies, regardless of the cell type or method of manipulation, according to Nicco. “It is the route of administration, the need for transport, and the storage temperature that influence the choice of excipients,” she states.

Special applications, for instance, such as therapies for artificial skins and wound healing may have specific properties or additional ingredients for sealing the wounds, Guest notes. Hence, the excipients or scaffolds are part of these therapies. Dosage size is another important factor, he adds. “Larger doses need to take into account any toxic side effects of the excipients, such as DMSO, and may place maximal limits upon the total volume of excipients to be administered,” he observes.

Always consider the route of administration and freezing requirements

One of the most critical issues for cell therapy manufacturers is the maintenance of the cells in an appropriate medium/excipient from the end of the culture until the time of administration to the patient. “Not only should the medium keep the cells viable with their properties intact, but the route of administration must also be taken into account,” Nicco states. She adds that a good excipient “is solvent-free and suitable for fresh or frozen suspensions of living cells formulated in a proprietary formulation adapted to the route of administration, such as intravenous, intra-articular, or intracranial.”

In addition to understanding the delivery mechanism, Guest stresses the importance of considering the container and dose when choosing excipients. The next important factor, he says, is whether the cell therapy product will be supplied frozen, as that will require selection of a cryoprotectant. Formulation development experiments should then be designed carefully to optimize potency while taking into consideration hold periods and freeze/thaw steps and eliminating or otherwise minimizing the use of diluents and wash steps. In addition, Guest recommends evaluating existing administration formulations, containers, equipment, and protocols to minimize the need for new or changes to clinical practices.

An important component of any formulation strategy is consideration of the raw materials.For excipients intended for use in cell therapy products, Guest emphasizes the need to identify available good manufacturing practice (GMP)-suitable sources with acceptable lot-to-lot controls that will enable a reproducible product formulation. It is also critical, he says, to confirm the material is suitable for the manufacturing process and will be available to meet manufacturing demand for the cell therapy throughout its product life cycle.

Equally important, Guest observes, is to ensure the availability of suitable cellular material and drug product for designing formulation experiments. Understanding the patient group and the intended administration route are essential, meanwhile, for identifying potential side effects of any excipients.

Compendial excipients are generally preferred, Guest continues, due to the risk that novel or untested solutions will require significant development to ensure the material does not impact upon the therapeutic benefit of the cellular material. In addition, all materials including excipients must be suitable for the intended route of administration and present minimal risk of inducing toxicity, immune reactions, or the transfer of adventitious agents.

Assessing formulation stability and quality

Controlling cellular material from the point of “optimal” donor selection to manufacturing under GMP conditions, commercialization, and application is critical for ensuring the quality, safety, and efficacy of the final drug product. While MSCs used as starting materials for production of advanced therapy medicinal products can only be isolated in authorized centers using globally standardized processes, the optimal conditions for culturing isolated MSCs are not standardized. That poses a major challenge in controlling their quality and therapeutic properties, Nicco contends.

It is, therefore, essential to control the cell sources (e.g., bone marrow, umbilical cord, and adipose tissue), cell density in culture, duration of culture, and cell engineering and composition of culture media and implement in-process quality controls that ensure cell efficacy and safety at all stages of manual and automated manufacturing processes, including cryopreservation, use of cell banks, and transport systems. “Ultimately, the stability and quality of the cell therapy product and the excipients it contains must always be evaluated against reference samples submitted to the same storage facility,” emphasizes Nicco.

Complicating this situation is the fact that it can be difficult to find reliable mode-of-action potency methods for cell-based therapies, according to Guest. “Often both in vitro cell culture and in vivo models are required to confirm the suitability of the formulation,” he says.

In addition, formulation strategy, hold times, cryopreservation, and thawing are intrinsically linked, and it can be difficult to determine the impact of each component of the formulation, Guest observes. “Doing so requires carefully designed experiments and suitable analytical methods that assess not only the impact of formulation changes and freezing and thawing processes, but downstream biological mechanisms such as apoptosis and necrosis, which require time in a suitable cell-culture facility or in vivo models to optimize formulations,” he explains.

“A further difficulty rests with the fact that viability determinations are often tied to the analytical methods, the technicians performing them, and whether biological mechanisms of apoptosis and true cell recoveries are accounted for in the testing strategy,” Guest comments. Therefore, it is important for cell therapy developers relying on external testing laboratories to ensure those outsourcing partners have the proper understanding, expertise, and capabilities needed for appropriate and comprehensive testing.

Advances in cell therapy formulation science underway

The cell therapy field is expanding at a rapid rate, and technology is advancing to support the increasing breadth of treatments under development. That includes formulation science and the development of enhanced excipient solutions. Cryoprotectants developed through biomimicry of natural antifreeze proteins to replace DMSO and serum protein-based media are one example highlighted by Nicco.

More work is to be done in this area, however. “There is a clear unmet need for the discovery and development of novel cryoprotectants that can either replace or reduce the required amounts of current gold standards formulated to protect and treat challenging sample types such as MSCs and, even more, iPSC multicellular systems. This multivariate problem is complex, with multiple mechanisms of damage to be addressed and subtle differences between cell types and freezing methods. Combining high-throughput testing with iterative computational algorithms is key to optimizing protocols and excipient formulations to preserve emerging cell-based therapies,” Nicco comments.

Guest, meanwhile, predicts that as the cell therapy field advances, clinical practice will naturally standardize formulation, delivery, and routes of administration through product safety and efficacy data. “That will include the use of animal-free or recombinant excipients and other fit-for-purpose additives developed through the use of artificial intelligence to minimize cytotoxicity while improving stability and hold times,” he states.

Nicco is also confident that the combination of new technologies such as intelligent library design, computational modeling, rapid screening assays, and advances in genomics will lead to a better understanding of the structure-function relationships between drug and excipient. “That greater understanding will lead to more effective and efficient excipients that afford higher-performing cell therapies and ultimately benefit both cell therapy developers and patients,” she concludes.

Valerio, C.; Theocharidou, E.; Davenport, A.; Agarwal, B. Human Albumin Solution for Patients with Cirrhosis and Acute on Chronic Liver Failure: Beyond Simple Volume Expansion. 

2016 Mar 8;8(7):345-54. doi: 10.4254/wjh.v8.i7.345. PMID: 26981172; PMCID: PMC4779163.

Hankins, J. The Role of Albumin in Fluid and Electrolyte Balance. 

. 2006 Sep-Oct;29(5):260-5. doi: 10.1097/00129804-200609000-00004. PMID: 17035887

Shah, M.M.; Mandiga, P. Physiology, Plasma Osmolality and Oncotic Pressure. [Updated 2022 Oct 3]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2023 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK544365/.

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


Vol. 37, No. 2
February 2024
Pages: 12-15

When referring to this article, please cite it as Challener, C.A. Choosing the Right Excipients for MSC and iPSC Therapies. 

Buffers, stabilizers, and cryoprotectants play major roles in cell therapy formulations.

Choosing the Right Excipients for MSC and iPSC Therapies

With the rise of nucleic acid-based therapeutics, such as the mRNA vaccines, biopharma developers are working toward the next big breakthrough in personalized medicines. Over the past couple of years, the industry has seen more intense partnering activity between developers and biotech firms specializing in gene editing technologies. A look at the advances in gene editing technologies illuminates the strides gained in R&D enabling the next generation of precision medicine targets.

The approvals of Casgevy by the UK Medicines and Healthcare products Regulatory Agency (MHRA) in November 2023 (1) and FDA in December 2023 (2) mark a significant turning point in the biopharma industry. Casgevy is the first therapeutic based on clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene-editing technology to be approved by regulatory authorities anywhere in the world.

“The approval of Casgevy for sickle cell disease is a significant milestone for the industry and paves the way for a wave of gene-edited therapies, including those that leverage next-generation CRISPR-based technologies with potentially more efficient and precise genome editors,” says Tedd Elich, chief scientific officer, Life Edit Therapeutics, an ElevateBio company.

“The approval also establishes a regulatory path as more investigational therapies enter the clinic and aim to address a wider range of diseases,” Elich adds. He explains that this established roadmap is likely to yield increased attention and investment from the broader biopharmaceutical industry. An increase in investment will, in turn, further accelerate the development of novel gene-editing technologies and therapeutics.

“Most importantly,” Elich continues, “the approval of Casgevy is a monumental achievement for people living with sickle cell disease and other genetic diseases. The approval lays the groundwork for future gene-editing therapies to more specifically target—and potentially cure–disease.”

“I see the approvals for Casgevy as the opening of the flood gates for gene-editing technologies,” Kate E. Broderick, PhD, chief commercial officer, Maravai Lifesciences, affirms.

“Over 10 thousand monogenic diseases have been identified which could be positively impacted by this incredibly powerful technology, offering hope to patients with diseases [for] which, previously, symptom management was the only option.”

“There’s never been a more exciting time in medical science,” Michelle Fraser, PhD, head of Cell and Gene Therapy, Revvity, enthuses. “Casgevy is the first CRISPR-based gene therapy approved by [FDA] and UK MHRA, introducing a completely new class of therapeutic modality where the genetic cause of a disease can be permanently corrected, instead of merely treating the disease symptoms.”

Fraser points out that Casgevy, an autologous cell therapy, is the first of a healthy pipeline of CRISPR-based cell and gene therapies. “The promise of this class of therapies is a permanent reversal of the genetic cause of a range of inherited diseases and cancers,” she says.

“It’s hard to believe that CRISPR-based therapies were first used clinically only 11 years ago; the field is just getting started. As we understand more about the genetic cause of disease, gene editing technologies are rapidly evolving, offering new hope to patients by providing personalized, highly effective, and safe therapies,” Fraser notes.

A paradigm shift advancing R&D for diseases

Gene-editing technologies offer precision, and it is that precision that has allowed the creation of complex, biologically relevant disease models, emphasizes Fraser. These disease models have facilitated unprecedented, detailed investigations of the genetic causes of disease, she adds.

“The gene editing toolbox available today enables precise editing of coding and non-coding regions, and to edit multiple genes in the same cell to create realistic disease model cell lines.These cell lines can then be used to screen the safety and efficacy of drug candidates and understand their potential side effects,” Fraser explains.

In turn, an improved understanding of the genetic causes of disease and drug responsiveness has led to a new generation of precision diagnostics, under which a patient’s disease can be accurately diagnosed and an ideal drug treatment selected for that patient, Fraser reports.

“With the first regulatory approval of CRISPR-Cas9 gene therapies, the next phase of the precision medicine evolution has been realized—where an individual can be genotyped, their disease accurately diagnosed and characterized, and then specifically treated using gene editing.Genes can be turned on or off, reduced, elevated, or corrected using gene editing technologies,” Fraser adds.

Because gene editing technologies allow genetic material to be removed, added, or altered at a specific location in the genome, they allow companies to develop therapeutics directed at the underlying cause of disease for the most challenging genetic disorders—and otherwise intractable diseases, notes Elich, who also points out that many of these therapies also have the potential to be cures for these genetic disorders.

“Emerging gene-editing technologies across a range of modalities offer greater versatility for targeting genetic disorders and the potential to reach a wide range of patient populations,” Elich says. “This includes the full spectrum of editing modalities like we’re pursuing, including base editing and reverse transcriptase or prime editing, where the best editing approach can be matched to the disease.”

“The biotechnology and bio/pharmaceutical industries have seen significant progress in the advancement of technologies, and the first gene editing therapies are now being developed and approved—providing the first proof points for this new era of medicine,” Elich states.

Because of this newfound understanding of diseases with a genetic origin, the biopharma industry is realizing a re-emergence of personalized medicine, Elich continues. As such, the industry is making greater investments to further accelerate gene-editing technologies as well as establishing R&D collaborations with biotech companies that are at the cutting-edge of gene editing. Elich explains that, by specifically targeting an individual genetic mutation and addressing the underlying genetic defect, gene-editing therapies could potentially transform or cure disease for otherwise chronic and debilitating diseases.

“In addition to the approvals for Casgevy, this year has also shown some remarkable progress in the field of personalized cancer vaccines highlighting the impact that an individualized, tailored approach can have on the prognosis and survival outcomes for certain cancers,” says Broderick. “Like gene-editing where the treatment precisely and directly targets an individuals’ genes, personalized medicine takes the same approach—developing a bespoke treatment plan based on the individual’s genetic makeup.”

Meanwhile, Fraser explains that, traditionally, the bio/pharma industry created therapies that reduce the symptoms of a disease, and the modalities of these therapies have included small molecules and larger molecules, including antibodies and antibody drug conjugates. Often, however, patients need to take these drug therapies for prolonged periods of time or the rest of their life, and unwanted side effects are not uncommon.

“In this new era, gene editing is playing a pivotal role in the bio/pharma industry’s transition toward personalized medicines. The precision and specificity offered by gene-editing technologies, such as CRISPR, enable the tailoring of treatments to individual patients based on their unique genetic makeup. This approach allows for more targeted interventions, minimizing potential side effects and enhancing therapeutic outcomes,” Fraser says. “Whilst gene therapies are currently very expensive, the treatment is administered only once, changing the medical intervention and economic model for many otherwise chronic and life-threatening diseases.”

Next-gen editing technologies and the future

Looking toward the future, next-generation gene-editing technologies that are emerging include the “incredibly powerful” combination of RNA therapeutics (RNA guide and RNA- encoded nuclease) and CRISPR technology, says Broderick, who notes that this combination has the synergistic potential to impact genetic diseases that have previously had limited treatment options and challenging prognosis. “Utilizing next-generation delivery systems, such as cell targeting peptides, will allow these revolutionary therapeutics to reach their potential for disease outside just the blood,” she states.

Fraser discusses the original concept of applying CRISPR-Cas9 technology, which has been used to create a double-stranded DNA break and thereby disrupt gene function. This application has since led to newer generation base-editing and prime-editing systems that precisely correct the causative genetic mutation, she explains.

“Base editing relies on a partially active Cas enzyme to nick one of the DNA strands and a deaminase to introduce a controlled, specific single-base change on the opposing DNA strand at the target site,” Fraser continues, “Homology-directed DNA repair leads to a base change on the nicked DNA strand, altering both strands of DNA, for example, to correct a gene function.”

Meanwhile, prime editing uses a reverse transcriptase to insert a larger DNA template to, for example, introduce a functional copy of a defective gene, Fraser also explains. “Selection of the optimal editing system depends on the intended purpose.”

Fraser further explains that consideration is given to whether the need is for a single gene to be addressed, or multiple genes, or whether treatment requires changing a single base or introducing a larger genetic change—such as reducing the size of a repeat expansion. Other considerations include whether the target site is in close proximity to a protospacer-adjacent motif (PAM) sequence and therefore easily accessed, or if the target site is difficult-to-reach with no PAM sequence nearby, what degree of editing efficiency is needed to see a biological change, and the tolerability of the specific cell type to gene editing systems.

“There is no one-size-fits-all editing system,” Fraser asserts, “each has its own unique advantages and disadvantages. In the future, it is possible that multigene disorders may even be best addressed using a combination of editing systems delivered simultaneously.”

Diseases that could potentially see positive or further impact from the advancement of gene-editing technologies in the near future include cystic fibrosis, Huntington’s disease, phenylketonuria, and hemophilia, to name a few, says Broderick.

“However,” Broderick adds, “while the blood-based targets are moving forward rapidly, delivery challenges are hindering the progress to other tissues. Once more efficient and more directly targetable delivery modalities are co-developed, the full potential of gene-editing technologies will be realized.”

According to Fraser, using gene therapy to treat sickle cell disease marks the start of the next generation of personalized medicines. In her estimation, there are several diseases that are emerging as key focus areas, including:

 Fraser notes that there are well-described genetic risks associated with the development of mature onset neurological conditions such as Alzheimer’s disease and Parkinson’s disease. Here, gene editing can play a role in targeted therapies to reduce these risks.

 Personalized medicine is being used in oncology to drive understanding of the underlying genetic mutations that cause a patient’s cancer and allow the tailoring of the treatment accordingly. In this area, gene editing is being used to create immunotherapies, such as chimeric antigen T (CAR-T) cells that turn the patient’s own immune response against the cancer cells.

 Gene editing holds immense potential in addressing rare genetic diseases by correcting or modifying the faulty genes responsible for these conditions. As such, this approach goes beyond traditional treatments and offers a more targeted and effective solution that could be given prior to symptoms presenting, and perhaps even prior to birth.

 The use of personalized medicine in cardiovascular health involves diagnosing and then using gene-editing technologies to address genetic risk factors that influence heart disease.

 Diseases such as rheumatoid arthritis and lupus are being explored through a personalized medicine lens, according to Fraser, who explains that understanding the individual’s genetic predisposition to developing an autoimmune disease allows for early intervention with tailored therapies. She notes that initial research has suggested that CAR-T cells created using gene editing may be an effective treatment.

Fraser goes on to say that gene-editing technologies, particularly CRISPR, play a crucial role in the evolution of personalized medicines by enabling precise modifications to the DNA code. These technologies “allow researchers to edit, add, or remove specific genes, addressing the root causes of diseases at the genetic level. This level of precision is essential for developing highly targeted and effective personalized medicines,” she states.

“As these technologies advance, they are likely to become integral in the development of a new wave of therapies that go beyond traditional cell and gene therapies, offering even more tailored and individualized treatment options,” Fraser concludes.

Elich adds that many companies are currently focusing on similar diseases, such as sickle cell disease, in which numerous corporate partnerships may already exist. “As gene editing technologies become more precise, and new CRISPR systems are discovered and engineered, we expect the pool of targetable diseases to increase significantly into more monogenic diseases and ultimately expand into polygenic diseases,” Elich states.

“With this evolution, we believe that gene editing is going to be central to new therapeutic development in the future, including gene-editing therapies to specifically target disease, as well as the mechanism driving engineered cell therapies. Each step and advancement in the gene editing field will bring us closer to true personalized medicine for patients,” Elich envisages.

1. Vertex Pharmaceuticals. Vertex and CRISPR Therapeutics Announce Authorization of the First CRISPR/Cas9 Gene-Edited Therapy, CASGEVY (exagamglogene autotemcel), by the United Kingdom MHRA for the Treatment of Sickle Cell Disease and Transfusion-Dependent Beta Thalassemia. Press Release, Nov. 16, 2023.
2. FDA. FDA Approves First Gene Therapies to Treat Patients with Sickle Cell Disease. Press Release, Dec. 8, 2023.


Volume 37, No. 2
February 2024
Pages: 16–19,25

When referring to this article, please cite it as Mirasol, F. Milestone Approval to Steady Stream, Gene Editing Revs Up. 

Upstream Processing

The milestone approval of a gene-edited therapeutic paves the way for gene-editing technologies.

Milestone Approval to Steady Stream, Gene Editing Revs Up

The human digestive system provides a layer of protection from undesirable microbes and foreign bodies through acidic digestive processes. This protection allows a certain tolerance vis-à-vis delivery of oral delivery methods.

However, parenteral drugs are not subject to that same level of tolerance as they are injected directly into tissue or bloodstream. For these dosage forms, there is a particular need to guard against other risks, such as contamination and blood vessel occlusion.

Single-use systems (SUS), which are by nature more sterile, have therefore found a home naturally and with more celerity in parenteral drug manufacture. In fact, the global single-use bioprocessing market is projected to experience double digit growth between 2022 and 2030, reaching $15.1 billion by the end of that specified period (1).

As adoption of SUS in parenterals is becoming firmly established, attention turns to both process optimization of therapeutic segmentation, alongside regulation evolution. “Visible particulate contamination in SUS is typically performed using procedures adapted from USP [

] <790> (2), Visible Particulates in Injections,” says Mark Bumiller, technology manager, Entegris. “The USP <729> protocol calls for sampling General Inspection Level II, single sample plan, AQL [acceptable quality limit] of 0.65% (3). SUS products typically approach 100% of guidance goals for visual particulate contamination before final packaging. 

 <790> suggests testing against black and white background with illumination between 2000 and 3750 lux (2). Many SUS products are tested using normal fluorescent cleanroom lighting on a light table and against a black background.”

Bumiller goes on to state that the extraction method to collect loose particles from the SUS surfaces for testing components and assemblies comes from the ASTM E3230-20 validating extraction process (4). “The approach was inspired by an automotive industry standard used for parts which require critical cleanliness,” he explains. “The guidance on the method for counting the particles is not provided.”

A wide-range of high-performance polymer materials are used for SUS as a result of their ability to withstand the varying and challenging bioprocessing conditions. For example, polyethylene and polypropylene can withstand a range of conditions; polyethersulfone is used when heat resistance is required; polytetrafluorethylene (also known as Teflon) are useful as inner layers of SUS when using more aggressive materials; cellulose acetate is a hard material that is popular for filtration cartridges; and polyamide (nylon) is a suitable material for outer layers in composite components (5).

As a result of the unique properties of each polymer and the challenging characteristics of the compounds being processing in bioreactors, it is common for SUS to consist of more than just one plastic material. It is common for bioreactors to comprise several layers that all work to contain and protect the expensive and sensitive materials being processed (5).

Initially, SUS were employed for larger batch volumes in the production of vaccines, biosimilars, and monoclonal antibodies. Nowadays, in line with the industry’s increased focus on developing novel therapies to treat conditions such as cancer and autoimmune diseases, more highly potent drugs are being processed (6).

While useful therapeutically, highly potent drugs are challenging from a manufacturing perspective as they are dangerous not only to the manufacturing operators but also to the environment, which means they must be contained effectively. Furthermore, such potent compounds are difficult to remove from stainless steel surfaces, leading manufacturers to favor SUS for their bioprocessing needs (6).

In addition to the greater use of highly potent compounds, industry is also witnessing a vast movement into cell and gene therapy applications—or simply for cell expansion activities. With these shifts, SUS is creating a need for accurate yet inexpensive disposable sensing equipment, so that a closed environment is maintained across the whole production process. PreSens, for example, is tackling this issue through the development of non-invasive online optical oxygen sensors for use in perfusion systems (7).

Across the world, all industries are facing decisions around how to be more environmentally friendly, which usually involves using less disposable plastic and more reusable materials. Ironically, within the biopharma industry, using plastics a single time, then throwing it all away, can be a more sustainable and environmentally friendly option, particularly when compared with traditional stainless-steel technologies—once all the water costs and energy budgets for cleaning and decontamination are taken into account. It has been commonly reported throughout the industry that SUS reduces or eliminates requirements to clean and then validate that cleaning, between batches.

Additionally, it is widely believed that SUS sidestep the age-old problem of cross batch contamination, as everything is thrown away. The disposable nature of SUS also provides the flexibility and nimbleness to act quickly when either opportunity knocks, or when there is an unexpected need to quickly scale back down. Furthermore, manufacturing costs can be reduced with SUS, which will ultimately transfer to more cost-effective options for patients.

Single-Use Bioprocessing Market Size and Forecast

USP. USP General Chapter <790>, Visible Particulates in Inspections. 

 <729>, Globule Size Distribution in Lipid Injectable Emulsions. USP-NF (Rockville, Md., December 2007).

ASTM. E3230-20, Standard Practice for Extraction of Particulate Matter from the Surfaces of Single-Use Components and Assemblies Designed for Use in Biopharmaceutical Manufacturing. 

Seifert, D. Single-Use Bioprocessing: Why it Pays Off to Switch to Single-Use Systems Now. 

ESCO Pharma. Disposables—Single Use System for Aseptic Processing and Filling Operations. 

PreSens. Online Oxygen Monitoring in Perfusion Systems: Single-Use O2 Flow-Through Cell FTC-SU-PSt7-S. 

Chris Spivey is the editorial director of 

When referring to this article, please cite it as Spivey, C. Stepping Up Process Control with Single-Use Systems. 

Downstream Processing

SUS aids biopharma manufacturers to overcome the rigidity of more traditional stainless-steel technologies.

Stepping Up Process Control with Single-Use Systems

Messenger RNA (mRNA) molecules are inherently unstable and readily degrade when exposed to ubiquitous enzymes such as RNase and undergo pH-dependent hydrolysis of phosphorus-oxygen bonds when exposed to water. That instability creates challenges for manufacture, formulation, storage, and transport of mRNA-based vaccines and therapeutics. While encapsulation of mRNA drug substances in lipid nanoparticles (LNPs) enhances their stability (and facilitates their delivery into cells), mRNA-LNP products still typically require low-temperature storage and present distribution challenges in areas where cold-chain management is limited.

One solution for overcoming mRNA instability is lyophilization, or freeze-drying, which is used in the manufacture of many biologic and small-molecule drugs that exhibit instability in aqueous solutions. Lyophilization involves the removal of frozen water (ice crystals) via sublimation under vacuum at low temperature to ideally produce a solid cake that typically can be stored at room temperature for extended periods. The challenge with mRNA-LNP products is to find the right lyophilization conditions that do not harm the product particles (change their size and polydispersity, among other attributes) and produce a solid with desirable properties.

While there was insufficient time during development of the mRNA-LNP COVID-19 vaccines to identify an effective lyophilization process, several companies have since then developed effective lyophilization solutions. Pfizer/BioNTech and Moderna are two well-known firms with candidates in the clinic. But it was the small biopharma company Arcturus Therapeutics, in collaboration with CSL, that received the first marketing approval of a lyophilized mRNA-LNP product. Its self-amplifying mRNA COVID-19 vaccine ARCT-154 was approved by Japan’s Ministry of Health, Labor, and Welfare (MHLW) in November 2023 (1).

The main challenge: maintaining integrity

While lyophilization is an important means of addressing stability issues associated with mRNA therapeutics, allowing the transport and storage of these products without the need for ultracold chain infrastructure, there have been challenges to effectively harnessing the benefits of lyophilization for these products, according to Vincenza Pironti, strategic marketing director at Recipharm.

The sensitivity of large, highly charged mRNA molecules, even when encapsulated in LNPs, to oxygen, light, heat, and temperature must be taken into consideration, contends Joseph E. Payne, president and CEO of Arcturus Therapeutics. The temperature transfer during lyophilization and the use of a vacuum places a strain on the LNPs. “It is necessary to develop a subtle and sophisticated method that does not perturb or discombobulate the nanoparticles during both freeze-drying and reconstitution,” he says.

Doing so, Payne adds, requires a considerable amount of resources and years of innovation, and typically involves the use of the right combination of buffers and excipients to stabilize the mRNA-LNPs during dehydration and rehydration.

These challenges, Pironti says, include ensuring the lyophilization process is effective at optimizing product stability and protecting the integrity of the LNPs. “In practice this means there needs to be a good final cake within the vial that can be readily reconstituted when it is time to administer without detrimental effects to the formulation,” she explains.

That can be particularly difficult to achieve for low-dosage formulations such as mRNA-based therapies due to the ratio of ingredients to dose volume, observes Pironti. “What is needed in these cases is the use of appropriate cryoprotectants or bulking agents within the formulation, which must be compatible with the formulation and the vial and ensure the stability of final drug product,” she observes. She also notes that comprehensive analytical testing of both the formulation and the LNPs well ahead of commercialization can help identify the right lyophilization methods to support product stability and shelf-life once the therapy is in commercial use.

Equally important, according to Christian Dohmen, executive director, technology development & CMC with Ethris, is to ensure that the LNPs are fully intact after reconstitution. There are many factors that contribute to product deterioration of lyophilized mRNA/LNPs, including particle size, mRNA integrity, and encapsulation efficiency. “If left unaddressed, the combined effect of these factors leads to deterioration of the product and decreased biological activity as well as undesired immune recognition (e.g., due to formation of aggregates), ultimately leading to reduced therapeutic potency,” he explains. In addition, he comments that aggregation of LNPs during the reconstitution process after lyophilization is another challenge for lyophilized mRNA products.

Pironti recommends using long freeze-drying cycles to prevent any damage to the LNPs within the formulation that could undermine performance and stability. Ethris has, Dohmen says, optimized its product design and lyophilization process to account for these factors and improve the potency of the resulting product as well as a reconstitution process that prevents aggregation from occurring.

One approach for overcoming lyophilization challenges that is not appropriate, warns Pironti, is modification of the mRNA structure, as that would change the nature of the treatment and potentially undermine the efficacy of the finished therapy, she states.

The most important strategy is to ensure the chosen lyophilization process is as mild as possible, which requires accurate study of each freeze-drying phase to gain in-depth process knowledge and understanding. “While an accelerated method might save time, the risk of damage to the formulated mRNA product is much greater,” Pironti concludes.

In addition to mild conditions, formulation development is essential to establishing an effective lyophilization process for mRNA products. For instance, Pironti points out that the ionic strength of the chosen buffer can have a significant impact on the resulting cake post-lyophilization, potentially leading to better stability and easier reconstitution. She also notes that the concentration of the mRNA formulation pre-lyophilization can also affect the overall freeze-drying process, while use of an appropriate surfactant may address challenges with respect to LNP aggregation.

Lyophilization does more than increase the stability of mRNA-LNP products for simpler storage and distribution. It also, observes Dohmen, is the next step toward user-friendly drug products that physicians and patients can use without any concerns about stability and potential aggregates. “As we design future mRNA medicines, especially those for chronic conditions that will be administered on a regular basis, optimizing for stability in the hands of physicians and patients and reducing the potential for an immune reaction is of utmost importance,” he states.

Lyophilization technology in general has advanced in recent years, with improvements in automation, such as automated loading and unloading, which according to Pironti has significantly reduced the time required for manufacture and filling of mRNA products. Automated unloading is important for these materials, because even post-lyophilization, mRNA formulations remain sensitive to temperature. “By automating and accelerating unloading, it is possible to minimize the risk of formulations being exposed to temperatures that are impactful on final stability,” Pironti says.

The advances in lyophilization technology are being leveraged by contract development and manufacturing organizations (CDMOs), particularly those invested in acquiring relevant experience in the production and handling of mRNA therapies and vaccines, Pironti adds. “CDMOs with this expertise have the knowledge and infrastructure to support pharmaceutical companies in accessing advances in lyophilization so they can harness it in a well-structured way to support the efficient commercialization of their mRNA products when they reach this stage,” Pironti contends.

The most successful CDMOs, according to Pironti, have deep knowledge of their mRNA manufacturing platforms, allowing the development of effective lyo cycles and identification of optimal formulations based on approaches that are well-known and already established.

Pironti also stresses the importance of working with an expert CDMO with experience in mRNA process and formulation development and manufacturing and the infrastructure for both lyophilization and subsequent sterile fill/finish activities. “Partnering with such CDMOs allows mRNA vaccine and therapy developers to benefit from existing knowledge about effective approaches, streamlining the development journey and eliminating the need to reinvent the wheel,” she observes.

Best tip for success: address lyophilization early in development

The development of optimal lyophilization processes takes time for even the simplest molecules. For complex mRNA-LNP products, it can be longer still. The lyophilization process itself also takes time, and as mentioned above, longer, and milder approaches are best for sensitive mRNA formulations.

Consequently, Pironti suggests that if a pharmaceutical company has a very short window in which to pass the mRNA candidate from early development into the clinic, it might be necessary to avoid lyophilization at the clinical manufacturing stage to accelerate the route to the clinic. However, Pironti believes that the earlier lyophilization is considered in the mRNA-LNP formulation development journey, the easier it will be to create an effective process that affords the maximum stability and simplified storage capabilities offer by lyophilization.

Lyophilization is a great step toward further advancing the mRNA field, according to Dohmen, and he is excited to see lyophilized products reaching and advancing through clinical development to commercialization. “In the end, we need more user-friendly drugs, especially if mRNA technology is tofind use beyond vaccines,” he says.

For Pironti, the stability afforded by lyophilization has clear benefits for mRNA vaccines designed to tackle future pandemics, particularly when it comes to logistics. “A lyophilized product can simplify logistics in a scenario where vast numbers of doses of vaccine need to be manufactured and distributed across the globe, as there is no need for special cold-chain transport or storage,” she comments.

Developing freeze-drying processes requires patience and deep product and process understanding.

BioPharm International, February 2024

Bracing for a Future Wave of Advanced Therapies

Fouling Mechanisms of Filters During the Harvest Development of Monoclonal Antibody Therapeutics with Intensified Upstream Processes—Part 1

Developing CAR-T Cell Therapies for the Future

Choosing the Right Excipients for MSC and iPSC Therapies

Milestone Approval to Steady Stream, Gene Editing Revs Up

Stepping Up Process Control with Single-Use Systems

Increasing mRNA Product Stability with Lyophilization

Addressing PK/PD Study Design Early in Development

J.P. Morgan Losing Weight

Computer Validation Confirms Evidence