Regenerative medicines have the attention of investors, industry players, and regulatory authorities. Prompted by an influx of funding in the regenerative medicines sector, drug developers have jumped forward with clinical development of product candidates. Factors that remain critical to the successful development of regenerative medicines include raw material sourcing and overcoming manufacturing bottlenecks. 

Going into the 2023 year, the biopharmaceutical industry saw 2220 active clinical trials worldwide for regenerative medicines, primarily cell and gene therapy product candidates, and of those, 202 trials are in Phase III (1). These Phase III clinical trials are expected to fuel more regenerative medicine approvals in the next few years, according to Tim Hunt, CEO, Alliance for Regenerative Medicine (ARM) in ARM’s state of the industry address (1).

Hunt further emphasized that the industry saw an 11% growth year-over-year in the number of developers working on cell and gene therapy therapies. This increase brings the total number of cell and gene therapy developers to 1457, currently.

Among the more critical factors in the development of new regenerative medicines, and specifically cell and gene therapies, is the quality of raw materials. Yet, the impact that starting materials have on the end-product quality remains ambiguous in the case of cell and gene therapies.

“To be fair,” says John Lee, senior vice-president, Head of Cell Therapy, Center for Breakthrough Medicines (CBM), “the jury is (mostly) still out on this question. Until the field is able to connect aspects of the manufacturing process to clinical readouts, it will be difficult to truly answer this question with any degree of confidence.”

Lee explains that cell selection is arguably the most important component of these types of therapies, however, since clinical efficacy is “assuredly linked to the therapeutic capacity of the source material,” which he points out the field has learned from lessons taken from the development autologous chimeric antigen receptor-T cell (CAR-T) therapies.

Regarding cell selection—in the case of cell therapies, for instance—autologous cells will have the same concerns as currently exist with FDA-approved CAR-T therapies, such as the fact that patient-to-patient variability will continue to hamper manufacturers’ abilities to standardize processes and define critical process attributes that allow streamlined production of reproducible drug product(s), Lee emphasizes. Allogeneic cells derived from healthy donors, however, offer an ability to bypass many of those concerns via donor screening and the creation of master cell banks, he explains.

“In line with this reality, the identification of ‘super donors’ who can yield cell-based products whose human leukocyte antigen (HLA) phenotype is tolerated by many recipients is an area of active investigation across the field. Likewise, induced pluripotent stem cells (iPSCs) represent an alternative allogeneic option to create a desired cellular phenotype with gene-edits that avoids immunogenic rejection or graft-versus-host response,” Lee states.

According to Randy Yerden, CEO of BioSpherix, a US-based company focused on the design and manufacture of cell culture, processing, and production equipment, the old adage of “bad protoplasm in yields bad protoplasm out” has been seen when starting cells for cell therapies are sourced from patients who have had multiple rounds of cytotoxic therapy in autologous immunotherapies. New strategies for sourcing and storing the patient’s cells before the cytotoxic therapy may improve end product quality and consistency, he notes. Other patient-specific factors, such as age or comorbidities, however, may also adversely affect autologous end-product quality.

In contrast, Yerden explains, a major allure of allogeneic therapies is sourcing starting cells from prime young healthy donors, which are then expanded and banked (i.e., stored) as a uniform, high quality end product, or as an intermediate starting cell for further processing and production. “In either case, if universal histocompatibility can’t be achieved, even a uniform high quality allogeneic end product may exhibit variable safety and efficacy due to patient immune reactivity differences,” Yerden cautions.

“Regardless of the starting cells,” Yerden continues, “innovators in the live cell industry are relentlessly finding and eliminating more and more sources of end-product variability in the production process. For example, one source of variability and end-product quality degradation has been under our noses for decades.” Yerden explains that short fluctuations in the three universal critical cell process parameters (CPPs) of temperature, carbon dioxide (CO2), and oxygen (O2) when cells are taken out of the incubator or bioreactor and exposed to room air are now known to be harmful (2). However, technology exists today that can now eliminate these fluctuations over the entire cell production process (3), he notes.

Sourcing starting material for regenative medicines overall is moderately easier than sourcing starting material for other cell-based therapies, says Lee.The reason being that most regenerative medicines are created from healthy material, he notes.

“While some platforms utilize somatic cells, the majority of products in development are derived from different types of stem cells, including embryonic, iPSC, hematopoietic, mesenchymal, and endothelial progenitor stem cells. Each of these cell types has unique differentiation and proliferation profiles that guide their respective utility for therapeutic usage,” Lee adds.

With 2220 regenerative medicine clinical trials actively in progress worldwide, the types and categories of therapies are broad and varied. Yerden points out, however, that as certain categories prove successful, more and more developers will move into these categories. “For example, with the success of CAR-T cell therapies, there might be more developers focusing on CAR-Ts than most other categories put together,” he states.

As for Lee, he notes that some of the most promising categories of clinical candidates include engineered skin grafts to treat burn patients; customized bone grafts for implants, joint replacements, and injuries; restoration of heart function in cardiomyopathy patients; restoration of muscle function in patients with urinary incontinence, rotator cuff surgery rehabilitation, etc.; restoration of eye function in disorders such as retinitis pigmentosa and/or macular degeneration; grafts to repair tears in connective tissues (i.e., tendons and cartilage); and restoration of kidney function in diabetic patients with chronic kidney disease.

As evidenced by both the number of active clinical trials currently in the industry pipeline and the growth in the number of developers, the advancement of regenerative medicines is progressing well on all fronts.

The successful development of regenerative medicine products has had its own hurdles on the road toward market approval. According to Yerden, the first obstruction happened early, at the time when the regenerative medicines industry first started to form.

Yerden recalls that virtually all discovery and intellectual property (IP) was developed in medical universities, as was the similar path of medical devices and new drugs. Unlike medical devices and drugs, however, there was no pre-existing big industry infrastructure to license the cell-based products. “Big medical device companies usually license and commercialize new devices developed in the universities. Big drug companies usually license and commercialize new drugs developed in the universities. When the first cell therapies were developed, there were no big industrial companies to license and commercialize the new cell therapies,” he explains.

In response to that vacuum in the United States, the US government built, staffed, and supported a handful of good manufacturing practice (GMP)-compliant cleanroom production facilities for cells with therapeutic potential, Yerden states. “They saw the tremendous potential and figured it would be well worth taxpayer money to place these around the country to provide a mechanism for the IP to advance beyond the typical limitations of universities just short of clinical trials. They would enable a pathway further down the development cycle through Phase I and Phase II clinical trials. Shortly thereafter, the big medical universities decided to build their own GMP-compliant cleanrooms for the same reason,” Yerden explains.

Yet, for years, there was no industry infrastructure to license and commercialize the cell products, despite the fact that many were advancing through early stage clinical trials, Yerden continues. A few high profile venture-funded companies were formed. Afew of the big drug companies were starting to dabble in their own skunkworks-style cell development labs.

However, it all changed when a cell therapy started to cure childhood leukemias (CAR-T). Fast forward to present time, and the industry now has a robust infrastructure, Yerden emphasizes.

The second big obstruction occurred when, instead of cell biologists making cell manufacturing decisions, it was a flood of pharmaceutical engineers making these decisions, Yerden adds. “Money was no object. They didn’t understand the significant difference between cells-as-substrate and cells-as-product. A massive amount of money and effort is still getting wasted on legacy biotech manufacturing technologies, which will never be able to produce high quality therapeutic cells. Even today, many manufactured cells are treated without regard to the science of cells,” he states.

For example, Yerden reiterates those fluctuations in the three critical cell process parameters of temperature, O2, and CO2 (universal for all cells) are routinely ignored in steps of production upstream and downstream of the incubation step.Modern science-based closed production technology eliminates these fluctuations, says Yerden, and improves cell quality.

Another example he includes is that most cell production processes in cleanrooms and traditional isolators are dependent on the heavy use of biocides to establish and maintain aseptic conditions; but he cautions that nothing is more dangerous to therapeutic cells than the biocides used for microbial contamination risk mitigation. Modern science-based closed production technologies enable aseptic production without biocides, he points outs.

A third example lays in the fact that large-scale batch production ignores the critical paracrine role in well differentiated, highly functional phenotype and results in poorly differentiated, minimally functioning phenotypes. For this challenge, modern science-based closed production technologies now enable paracrine-friendly small batch production, which can be run in parallel for large scale. Alternatively, new microencapsulation strategies may satisfy the paracrine need and result in better therapeutic cells from big vats, Yerden concludes.

“Tying the manufacturing process to clinical outcomes remains an opportunity for significant growth,” Lee adds. “The identification of critical process parameters (CPPs) and critical quality attributes (CQAs) is essential for the successful en masse establishment of regenerative medicines in the clinic.”

In regards to CPPs, Lee explains that a dedicated focus on identifying CPPs during manufacturing is merited to achieve the goal of consistency between efficacious batches of drug product. “The field is in need of automation that is capable of closing the manufacturing process while also enabling inline testing across a panel of biological readouts. By minimizing manual manipulation, variability within manufacturing can be lessened such that CPPs can be better assessed and identified,” he states.

Through the identification of CPPs, Lee further states, manufacturers can subsequently better understand how those CPPs impact the CQAs of these therapies.

Meanwhile, the absence of CQAs that are directly correlated with patient outcomes is another area that still requires substantial attention, Lee points out. “Because a portion of the manufacturing process literally occurs within the patient, researchers and manufacturers will need to prospectively derive datasets that can be retrospectively analyzed alongside patient outcomes to better define these CQAs,” he notes.

While the field collectively awaits more substantial clinical data, there may be non-human in-vivo models that can help bridge the gap in the interim, Lee posits. However, he cautions that the extraneous limitations of such models (i.e., xenograft-based biology) likely impairs their predictive utility for such applications.

Another area that Lee expects will require additional attention is how the field of regenerative medicines will address questions surrounding product-specific logistics for administration. For instance, some drug products will need to be administered within hours of completion of manufacturing, which creates hurdles around release testing and shipping conditions that must be navigated. Lee says that cryopreservation is a potential solution in some instances, but such a solution comes with other issues, including the maintenance of functionally post-thaw, and ensuring the proper handling of these valuable therapies upon receipt.

1. ARM. 2023 Cell & Gene State of the Industry Briefing. 

BioSpherix Medical. BioSpherix CEO Speaks at Perinatal Stem Cell Society. 

. April 17, 2020.
3. Manufacturing Chemist. Modernising 

Therapeutic Cell Production. Manufacturingchemist.com/Manufacturing

Feliza Mirasol is the science editor for 


Vol. 36, No. 2
February 2023
Pages: 10–13

When referring to this article, please cite it as Mirasol, F. Robust Development Fuels Drive to Finish Line. 

Articles

Cover Story

A slew of late-stage clinical trials is expected to push new regenerative medicines onto the market in the next few years.

Robust Development Fuels Drive to Finish Line

An important aspect of regenerative medicines is optimizing the supply and manufacturing of important components, such as viral vectors, which are crucial for the delivery of cell and gene therapies. 

Improving yield of viral vectors will primarily be achieved via scale-up, process improvements, and cell line development, says Emily Moran, senior vice-president of Vector Manufacturing and Supply Chain, Center for Breakthrough Medicines. Scale-up will go from the standard 200-L volume to 2000-L volume. Process improvements include alternating tangential flow filtration (ATF) on the production bioreactor, novel DNA for transfection, improved primary and polishing chromatography columns, and improved drug product formulation, Moran emphasizes.

In terms of producer or packaging cell line development, a combination of integrated adenoviral helper (i.e., Ad helper) genes, replication/capsid genes (i.e., rep and cap) and/or gene-of-interest to reduce/eliminate plasmid need will be needed to improve yields, Moran adds.

The scale-up and process improvements are expected to result in significantly increased batch yields and significant reduction in cost of goods (COGS), Moran says. These improvements would enable cost reduction for developing a single product and enable the ability to treat higher and/or repeat dose targets, she explains. In the end, patient access to these therapies will likely improve as the COGS goes down and overall throughput is improved across manufacturing facilities.

Another goal in the quest for improving viral vector yields is the standardization of manufacturing, Moran points out. Standardization includes implementing a similar order of unit operations, process technologies, and control strategies across manufacturing processes. Such standardization would enable improved consistency of the quality profile of viral vectors (e.g., adeno-associated virus-based vectors) going into humans as well as the ability to connect product attributes to clinical outcomes, Moran explains.

Other benefits of standardization include the improved ability to compare clinical outcomes across products from different companies; reduced investment in process/analytical/preclinical development, allowing for investment of more products; and the streamlining of the supply chain from a materials perspective for both development and manufacturing activities, which would increase speed to market and decrease cost, Moran states.

Currently, near patient manufacturing is an increasingly urgent goal. Proximity to patients reduces vein-to-vein time, which, in return, should improve patient access and outcomes, Moran emphasizes. Furthermore, having decentralized networks can improve product access and availability. The latter may come at a higher cost and increased variability in quality, however, due to distributed manufacturing, Moran concludes.

When referring to this article, please cite it as Mirasol, F. Optimizing Vectors to Standardize Success. 

Improvement in viral vector yield has become integral to new cell and gene therapy product development.

Optimizing Vectors to Standardize Success

 Protein A membrane and its characteristics were introduced. In part 2, a next generation of the convecdiff membrane was tested in a second cycling study by AGC Biologics. This second cycling study was performed over the course of 100 cycles using clarified harvest of a recombinant monoclonal antibody produced at AGC. In both studies, the analyzed critical quality attributes, yield, and LRV of different contaminants are constant over all the cycles performed. No loss in performance was observed. The performance indicators and the viral clearance abilities of the material are comparable to state-of-the-art materials. These aspects underline the robustness and applicability of the 

Submitted: March 2, 2022
Accepted: July 21, 2022.

Chaz Goodwine is lead downstream scientist at AGC Biologics. Katrin Töppner*, Katrin.Toeppner@Sartorius.com, is product development (PD) scientist; Patrick Adametz*, Patrick.Adametz@Sartorius.com, is process engineer; Ricarda A. Busse is product manager for membrane chromatography consumables; and Volkmar Thom is head of PD purification materials; all at Sartorius Stedim Biotech.

*To whom all correspondence should be addressed.


Vol. 36, No. 2
February 2023
Pages: 22–27

When referring to this article, please cite it as Goodwine, C.; Töppner, K; Adametz, P.; Busse, R.A.; Thom, V. Purification of Antibodies Using Novel Convecdiff Membranes Part 2: Robust Performance at Comparable Product Quality. 

Peer-Reviewed Research

In part 2 of this article, results from the testing of a next generation convecdiff membrane are discussed.

Purification of Antibodies Using Novel Convecdiff Membranes Part 2: Robust Performance at Comparable Product Quality

There is no doubt that chimeric antigen receptor (CAR) T-cell therapies have had a tremendous positive impact on patients that have received them; however, autologous cell therapies produced in centralized manufacturing facilities have inherent limitations that inhibit widespread availability and access. Allogeneic, or off-the-shelf versions, are one potential solution, but developers of these therapies must overcome different challenges, such as graft-versus-host disease, the potential for introduction of chromosomal abnormalities, ensuring successful gene editing of all donor cells, and problems with T-cell exhaustion (1).

Another option is to produce autologous therapies at the point of care (POC) (2). Companies such as Miltenyi Biotech, Lonza, SQZ Biotech, Orgenesis, and Ori Biotech have developed or are commercializing closed, automated cell- and gene-therapy production platforms with small footprints that can be installed at hospitals and institutions for good manufacturing practice (GMP) production of CAR T-cell therapies onsite. There are various university hospitals around the world exploring the potential of these systems. Orgenesis and aCGT Vector are establishing networks of POC sites leveraging their own production technologies.

A third approach being explored by several companies eliminates the need for gene editing of immune cells outside the body altogether. These solutions involve in vivo generation of CAR cells. This approach also has its challenges, but offers the potential to combine some of the benefits of both autologous and allogeneic cell therapies.

Autologous cell therapies today require collection of immune cells from the patient that are then shipped to a central manufacturing facility. Most patients are extremely ill and have endured multiple previous therapies, leading to variability in the starting material and the potential for manufacturing failures. Production slots are also limited, often creating delays that such ill patients cannot generally afford. Facilities that offer cell therapy are limited as well, and as a result, patients must travel to them, adding to the burden placed on them and their families. Furthermore, the cost of gene-modified cell therapies is high.

An IQVIA survey (3), notes Gregory Frost, chairman and CEO of EXUMA Biotech, revealed that the patient’s health status, cost to patient, and geographic distance for the patient to travel were the top three reasons why community oncologists do not refer patients for CAR-T therapy.

Allogenic cell therapies offer compelling benefits relative to autologous CAR T-cell treatments for certain indications. As off-the-shelf products, they are available immediately, eliminating the logistics complexities that add time and cost to autologous therapies. They also do not suffer from the variability associated with patient-derived cells.

Avoiding the natural immune response to foreign CAR-T cells has, however, proven much more challenging than past industry hurdles such as the advancement from mouse antibodies to human antibodies, according to Frost. “Although many different strategies are being explored, the only clinically validated solutions to this challenge to date has been sustained immunosuppressive treatment regimens to eliminate the body’s T and NK cell function, thereby increasing the risk of opportunistic infections without transplant-ward-level care,” he says. Generally as a result, the time that must be spent in the specialized care setting is often extended and introduces additional burdens for patients and their families that do not live near one of these specialized facilities.

 CAR therapies have the potential to offer the benefits of both autologous and allogeneic therapies. “The most apparent advantage that 

 CAR therapy could provide over autologous or allogeneic cellular therapy is to capture the durability benefits of autologous cell therapy with the reduced cost and complexity of an off-the-shelf product,” contends Frost.

If other safety parameters can be maintained clinically, Frost explains a CAR therapy that delivers a cost-of-goods-per-patient-treated on par with biologics may change the efficacy bar from 30–50% durable complete responses to a 30+% improvement in progression-free survival or overall survival, a particularly relevant goal for indications in solid tumors. “Moreover,” he adds, “lymphodepletion may not be necessary for 

 therapies, thereby potentially expanding the setting of care beyond tertiary care centers with intensive-care units and extending CAR therapy into indications not previously addressed (e.g., autoimmune disease) due to an unfavorable benefit-to-risk ratio.” Furthermore, as a true off-the-shelf option

 CAR therapy products are unburdened by the complex logistics for cryopreservation of a cellular drug product.

 CAR therapy “may address the significant concerns associated with autologous and allogenic gene-modified cell therapies and thus function to expand patient access to CAR therapy by making it safer, potentially more efficacious, less financially toxic, and available in settings more convenient to patients and their families,” Frost comments.

It is also worth noting that in addition to the reduced cost and complexity of therapy and the potential to expand to a wider array of care settings

 CAR/T-cell receptor (TCR) therapies reduce the complexity of cell processing validation, according to Frost. “While the product is the process,” he observes, “the 

 approach does not require the associated costs and time to develop an optimized and validated manufacturing cell process, which can introduce new challenges in comparability assessment with subsequent process changes.”

Frost also points out that enhancements and minor modifications to the transduction program encoded in the vector can easily be made, allowing for iterative evaluation preclinically and potentially clinically. “The versatility of the technology is anticipated to extend the technology broadly into academia and small and large biotechnology companies, which could result in the accelerated discovery and development of transformative therapies at substantially reduced costs,” he concludes.

 gene-modified cell therapies face their own set of technical challenges. A key issue, according to Frost, is the inability to perform preparative lymphodepletion of the patient prior to the infusion of the engineered cells. This procedure removes other immune cells that would otherwise compete with the CAR cells for growth factors and other nutrients, reducing their chances to survive, expand, and exhibit antitumor function. “Lymphodepletion is not a viable option for 

 CAR therapy since it would kill off the very cells targeted for in vivo engineering. As such, any 

 approach will need a means for ensuring robust proliferation and survival in an environment that is rife with competition for the same nutrients,” Frost says.

It is also essential that the vehicle delivering the genetic material for reengineering of the target immune cells is highly selective for the right cells. “Whether the vehicle is a viral vector or lipid nanoparticle (LNP), it must target delivery to the specific cell type to be re-engineered (e.g., T cell, natural killer cell) in order to prevent any unintended gene modification,” comments Frost.

Viral vectors and lipid nanoparticles represent two different approaches being explored for in vivo engineering. Nonviral approaches typically involve LNPs encapsulating nucleic acids, while viral approaches are generally based on the same retroviral vectors used in current 

Non-viral approaches, notes Frost, have the possible advantage of a more defined regulatory pathway based on the recent approvals of the COVID-19 vaccines. Non-integrating approaches may, however, not provide a sustained clinical response given that CAR transcripts become diluted (and eventually lost) as the cells divide, he observes.

Such therapies would likely require persistent retreatment in the absence of a clinical response, especially for the treatment of solid tumors, which require a prolonged T-cell response. “Integrating vectors, such as lentiviral (LV) vectors, may offer the benefit of stably
integrating into the host-cell genome to allow for a sustained response with a single dose,” says Frost. As such, he anticipates further investigations into the tropism of viral approaches to reduce the risks of adverse events associated with non-target cell integration.

The level of investment in in vivo gene-modified cell therapies suggests that this new approach truly does have real potential. Several startups have been established in the past few years with strong financial backing.

 CAR/TCR therapy platform, GCAR, comprises a lentivector with a CD3- targeting/activation element on its surface to target T cells and provide them with their initial proliferation stimulus. The genetic payload within the LV encodes for either a CAR or TCR, as well as EXUMA’s intracellular “DRIVER” signaling domain. Once integrated and expressed by T cells, DRIVER signaling and initial CD3 activation causes robust in vivo proliferation with persistence and antitumor activity in a lymphoreplete environment occurring from continued antigen engagement, according to Frost.

“Our proprietary LV design elements address the challenges faced by 

 CAR therapy, as the DRIVER allows for proliferation and persistence in the midst of competing cells, and the CD3-targeting moiety directs the lentivector to T cells,” Frost states. In preclinical animal models, EXUMA has confirmed enhanced tropism to T cells, circulating CAR-positive cells, and a potent, dose-dependent elimination of target cells upon 

Umoja Biopharma has developed its VivoVec surface-engineered LV vector-based off-the-shelf viral-vector particles for the generation of CAR-T cells 

 (4). The particles include rapamycin-activated cytokine receptors (RACRs) designed to selectively provide costimulatory molecules to CAR-T cells in the presence of rapamycin. Preclinical study results indicate the particle design demonstrates enhanced T-cell binding and activation both in vitro and 

, as well as generation of large numbers of CAR-T cells, thus enabling enhanced antitumor activity at lower doses.

Vector BioPharma’s Shielded, Retargeted Adenovirus (SHREAD) platform uses engineered adenoviral vectors combined with exogenous, high-avidity adapter proteins (in the form of virus-like particles) to achieve delivery to defined biomarkers on specific cells or tissues of DNA encoding various genes and regulators, allowing local production of potent drugs with reduced risk of systemic toxicities (5,6). The company was established in August 2022 to commercialize technology developed in the laboratory of Andreas Plückthun, PhD at the University of Zürich (7).

Ensoma, launched in February 2021, is also leveraging adenoviral vectors (8). Ensoma’s Engenious vectors are devoid of any viral genome, thus minimizing the chance of an immune response and freeing up to 35 kilobases of DNA packaging capacity to deliver a diverse range of genome modification technologies. The vectors can be used in vivo to engineer hematopoietic stem cells as well as erythroid, lymphoid (e.g., T cells, B cells), and myeloid (e.g., macrophages, microglia) cell types with precision. The company has entered into a strategic collaboration with Takeda Pharmaceutical Company (9).

Capstan Therapeutics, which was co-founded by Carl June, one of the original innovators in the autologous CAR T-cell-therapy space, and launched in September 2022 (10), is using mRNA technology to generate CAR-T cells 

. In one example, they injected CD-5 targeted mRNA-LNPs to reduce fibrosis and restore cardiac function in a mouse model of heart disease (11).

Interius BioTherapeutics is commercializing technology developed by Saar Gill at the University of Pennsylvania (12). The company is initially focused on treating hematologic malignancies using an in vivo CAR treatment, but hopes to expand to applications beyond immuno-oncology that address diseases not amenable to current gene-therapy modalities. It has provided minimal information about the specific technology involved.

gene-modified cell therapies at the preclinical development stage. SG295 is an 

 CAR T with CD8-targeted fusogen delivery of a CD19-targeted CAR for treatment of patients with B cell malignancies and for which the company expects to file an investigational new drug application (IND) in 2023 (13). SG418 is a hematopoietic stem cell-targeted fusosome with the ability to deliver gene-editing material 

 to repair genetic abnormalities such as those that cause sickle cell disease and beta-thalassemia. Sana hopes to demonstrate preclinical proof of concept in 2023.

Gene therapy provides a potential regulatory roadmap

gene-modified cell therapies constitute a novel drug class, they are related to direct gene therapies, several of which have received regulatory approval in the United States, European Union, and other countries around the world. These gene-therapy approvals, according to Frost, provide a helpful roadmap for regulatory evaluation of 

 CAR therapies. “While each product and platform has its own unique considerations that must be addressed for a specific indication, the FDA has also published numerous guidance documents regarding gene therapy that are helpful to sponsors developing 

As is the case with recent regulatory reviews of retroviral gene vectors in cells other than T lymphocytes, Frost expects that regulators will expect developers of 

 therapies based on retroviral vectors to demonstrate that those vectors are indeed transducing the target cell type (e.g., T cells) and not other cell types, such as hematopoietic stem cells or gametes. In this context, he notes that significant consideration must be given to the tropism of the delivery vehicle, whether viral or non-viral.

Clinical candidates anticipated in the next few years

Given the growing number of companies developing 

 CAR therapies using many different approaches, Frost anticipates candidates entering clinical trials within the next several years. The data gathered during those trials will serve to better refine the science. “With the advances that will be achieved and the growing body of evidence that will be generated as a result of these studies, it can be expected that the overall industry will become more comfortable with 

 gene therapy, ultimately facilitating the progression of other products to the clinic and eventually the market. The widespread use and breadth of enhancements to monoclonal antibodies, which were first discovered in 1975 and first approved in 1986, is suggestive of the potential for in vivo CAR therapy. With today’s technology and know-how, the innovation cycle for this technology is expected to be dramatically accelerated,” he concludes.

Preparing for the Allogeneic Cell Therapy Tipping Point

Alvaro, D. and Challener, C.A. Bedside Cell Therapies: Point-of-Care Manufacturing Is Slowly Becoming a Reality. Pharma’s Almanac, Nov. 15, 2022.

Saez-Ibañez, A. R.; Upadhaya, S.; Partridge, T.; Shah, M.; Correa, D.; and Campbell, J.Landscape of Cancer Cell Therapies: Trends and Real-world Data. Nature Reviews Drug Discovery, 2022. 21, 631-632, Supplementary Fig. 9,. doi: https://doi.org/10.1038/d41573-022-00095-1.

Umoja Biopharma. Umoja Biopharma Presents New Preclinical Data on its Integrated In Vivo CAR T and Engineered Induced Pluripotent Stem Cell Platform Technologies at the Society for Immunotherapy of Cancer (SITC) 37th Annual Meeting. Press Release, Nov. 10, 2022.

Freitag, P. C. et al. Modular Adapters Utilizing Binders of Different Molecular Types Expand Cell-Targeting Options for Adenovirus Gene Delivery. Bioconjug Chem. 2022 Sep 21;33(9):1595-1601. doi: 10.1021/acs.bioconjchem.2c00346.

Smith, P. C.et al. The SHREAD Gene Therapy Platform for Paracrine Delivery Improves Tumor Localization and Intratumoral Effects of a Clinical Antibody. Proc Natl Acad Sci U S A. 2021 May 25;118(21):e2017925118. doi: 10.1073/pnas.2017925118.

Vector BioPharma AG. Versant Ventures Launches Vector BioPharma AG. Press Release. August 10, 2022.

Ensoma. Ensoma Launches to Pioneer Next-Generation 

 Approach to Deliver First “Off-the-shelf” Genomic Medicines. Press Release, Feb. 11, 2021.

Ensoma. Ensoma Announces Strategic Collaboration with Takeda to Accelerate Next-Generation 

 Gene Therapies. Press Release, February 2021.

Capstan Therapeutics. Capstan Therapeutics Launches with $165 Million to Deliver on the Clinical Promise of Precise 

 Cell Engineering. Press Release, Sept. 14, 2022.

Rurik, J.G. et al., CAR T Cells Produced 

 to Treat Cardiac Injury. Science. 2022. 375 (6576), pp. 91-96, Jan. 6, 2022. DOI: 10.1126/science.abm0594

Interius BioTherapeutics. Interius BioTherapeutics Raises $76M Series A Financing to Advance Breakthrough Cell and Gene Therapy Platform. Press Release, May 18, 2021.

Sana Biotechnology. Sana Biotechnology Confirms Key Program Timelines and Announces Portfolio Prioritization. Press Release, Nov. 29, 2022.

Cynthia A. Challener, PhD has been a freelance technical writer for over 20 years, leveraging her education from Stanford University (BS) and University of Chicago (PhD) and 10+ years of industry experience. She currently focuses on pharma/biopharma topics, writing technical articles, white papers, blogs, and other content for a variety of clients in addition to contributing regularly to 


Vol. 36, No. 2
February 2023
Pages: 14–17

When referring to this article, please cite it as Challener, C.A. Technology for 

Development

In vivo CAR-T gene therapies could overcome the challenges faced by autologous and allogeneic treatments.

Technology for In Vivo CAR T-cell Therapy Advances

As downstream bioprocessing operations gradually implement single-use, biomanufacturing facilities are also being brought into alignment. What hurdles do biomanufacturers face in converting facilities to accommodate single-use systems (SUS) in downstream processing?

Certain key innovations in technology and equipment have helped to advance the performance of SUS in downstream bioprocessing. Jay Harp, director of Single-Use Product Management at Avantor, notes that the higher product yields and greater cell densities resulting from the increased acceptance of perfusion technology in upstream bioprocessing has put a strain on downstream processes. However, advancements in chromatography and filtration techniques have been implemented to meet these challenges.

“Advancements in sensing technology and the ability for single-use monitoring tools give manufacturers a greater degree of control over their processes,” Harp states. “Tangential flow filtration systems use this advanced process technology to better control flow rates and achieve better process performance.”

Ati Alizadehbirjandi, manager of Manufacturing Science and Technology (MSAT) at Avid Bioservices, notes that continuous processing technology, such as the multi-column chromatography platform (BioSMB) has been a key innovation in biomanufacturing. The development of continuous inline viral inactivation skids (by Pall Sciences), single-pass tangential flow filtration, and other multiple components that have been built all address bottlenecking issues in the downstream, Alizadehbirjandi emphasizes.

Bioprocess skids have the capability to process larger volumes in a shorter period of time, where previously only hard-piped systems could handle such volume, adds Gene Yoshioka, senior director of Manufacturing at Avid Bioservices.

“The rise of multi-modal chromatography allowed for higher precision in purification efficiency. Membrane chromatography was also developed to act as a more size-efficient replacement for high-capacity packed bed resin chromatography,” Alizadehbirjandi says. Furthermore, in-line process analytical technology development will advance performance and minimize bottlenecks to a higher extent by providing instant essential results, she adds.

The implementation of SUS has had its share of challenges, including the stress of high-volume products, high facility cost, and supply chain vulnerability, according to Alizadehbirjandi.

Alizadehbirjandi explains that if a high-volume product used a stainless-steel system for its purification, then it would be costly to switch to SUS, and the manufacturing process for the product would need revision. Moreover, stainless steel is generally more cost-efficient for a high-volume product. In terms of facility cost, companies that have established stainless-steel facilities would also face a high cost for the switch to SUS.

In addition, single-use facilities require a much larger warehouse footprint to store consumables, says Yoshioka, who mentions that managing the supply of consumables becomes critical as the reliance on vendors becomes more prominent.

Another aspect to be wary of is safety concerns regarding extractables and leachables (E&L) from plastic-derived or polymer material, which comprise SUS, adds Alizadehbirjandi. As expected, regulatory concerns for E&L are elevated for SUS systems, vs. transitional stainless-steel production, she says.

Tim Korwan, director of New Product Introduction at Avantor, explains that biomanufacturing facilities differ greatly from those that are fully single use. One of the key differentiators that he notes is the operating expenses and reliability of supply.

“In standard biomanufacturing facilities, the ability to change something in the manufacturing process relies upon the manufacturer to manage through extensive investments (both capital and time). With fully single-use facilities, manufacturing technicians and process engineers need to have a deeper knowledge about new technologies because the supplier is responsible for the managing the supply,” Korwan emphasizes.

Because the supplier is responsible for components in a fully single-use facility, if one component of the manufacturing process isn’t met by the supplier, then the process cannot move forward, cautions Korwan. Coupled with the flexibility inside fully single-use facilities, a biomanufacturer could end up with a stalled manufacturing process that could cause delays to the supply chain.

“To address this challenge, facilities can take a hybrid approach where the standard stainless-steel footprint is used (where appropriate) and is supplemented with single-use products.” Korwan states.

Korwan also explains that there is greater demand placed on the warehouse in a single-use facility, with lead times being a main issue. Because single-use products do not have an infinite lead time, one cannot overstock these products or risk having them expire. “As a result, single-use facilities require robust inventory management and coordination with vendors,” he says.

In terms of advantages, single use offers the greatest degree of flexibility, says Harp, who highlights the fact that biopharma manufacturing is constantly evolving with new processes and modalities. “Scientific advancements would not be capable without the equal advancements of single-use technology and the response of single-use integrators,” he states.

Korwan says that the need for supply chain professionals has never been greater because managing the supply of the entire fluid pathway in biomanufacturing is an extremely rigorous exercise. “As a result,” he explains, “the warehouse infrastructure needed to support this has never been greater.”

Facilities have been moving away from a stainless-steel footprint in recent years. Facilities are transforming into more of a shell-type structure, which is a substantial change for how these buildings have operated in the past, says Korwan.

The former stainless-steel footprint requires mass amounts of infrastructure and immovable piping, Korwan adds. Whereas now, SUS have allowed for the unit of operations to be placed in unclassified rooms, which minimizes the requirement for electrical sterilizing equipment, and thus allowing the ability for flexibility within the suite.

“Coupled with the lack of immovable piping, the facilities are able to flex between different modalities quickly and efficiently without having to change the infrastructure of the building,” Korwan states.

Meanwhile, Alizadehbirjandi points to a quicker turnaround time as being a key advantage of SUS. SUS allows for better time management by avoiding steam-in-place cleaning. This time advantage has resulted in achieving better speed-to-market.

Another benefit is water usage conservation, in which water intake for biomanufacturing facilities has been cut down by >50%, resulting in decreased plumbing, Alizadehbirjandi emphasizes. Other benefits include agile process development, process intensification, as well as better ability to deliver personalized medicine.

Yoshioka adds that for a contract development and manufacturing organization working with multiple products, eliminating the risk of cross-contamination is vital to the safety of the products that are produced. SUS allows for quick changeover while removing the risk of product residuals on product contact equipment surfaces.It has also eliminated the need for equipment cleaning and cleaning validation/verification which in itself is a large resource commitment.


Vol. 36, No. 2
February 2023
Pages: 20–21

When referring to this article, please cite it as Mirasol, F. Benefits of Technologies Facilitate Adoption of Single-use Systems. 

Downstream Processing

Single-use systems hold key benefits for biomanufacturers, despite certain limitations.

Benefits of Technologies Facilitate Adoption of Single-use Systems

In a tightly regulated industry, certifying that a drug is authentic, safe, and effective requires passing through several different thresholds of proof. In the 2011 

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

 (CGT) (1), FDA states, “No lot of any licensed product shall be released by the manufacturer prior to the completion of tests for conformity with standards applicable to such product, (21 

 610.1), which include tests for potency, sterility, purity, and identity (21 

 Part 610, Subpart B).” These requirements apply to all biological products, including autologous and single patient allogeneic products, where a lot may be defined as a single dose.

The need to meet this guidance, and especially for rising levels of instances of single dose production for CGT, can create a burden in terms of time, economics, and staff training. The biopharmaceutics classification system (BCS)-based biowaiver approach is intended, along with other measures, to reduce the need for 

 bioequivalence studies (i.e., it can provide a surrogate for 

 bioequivalence). The International Council for Harmonisation’s (ICH) M9 guideline states, “

 bioequivalence studies may be exempted if an assumption of equivalence in 

 performance can be justified by satisfactory 

data. The BCS is a scientific approach based on the aqueous solubility and intestinal permeability characteristics of the drug substance or substances” (2). In practical terms, CGT fields are moving so rapidly many identification and potency tests are being invented alongside the drug therapies themselves. It is an increasingly hopeful time for critically ill patients and also for the pharmaceutical industry, but this growing trend places a strain on scientists, suppliers, manufacturers, hospital staff, regulators, and insurers. In a industry insider survey (3), nearly half of all respondents replied that CGTs represented the largest opportunity for growth in terms of potential new therapies. While the surge is clearly a highly positive step, it also means taking care to solve inherent present challenges becomes more urgent.

Only a certain amount of past knowledge is useful

A typical strategy is to apply lessons learned from more mature therapies, particularly the use of monoclonal antibodies (mAbs), where much has been optimized due to popularity, length of use, and to some degree (arguably), less complexity residing in these systems. But even here more work on potency assays is evidently required. An article offering a novel mathematical approach for fitting concentration-response curves on mAbs for potency data concludes, “the future strategy for bioassay analysis should include investments both in the development of the best Mechanism of Action (MoA) reflecting assay and the identification of cost-effective techniques for implementation; this investment should begin in the early phases of clinical development. There have been some noteworthy achievements in the utilization of mathematical models in the bioassay evaluation strategies in various segments, but there is a long way left to go. Because the mechanism behind monoclonal antibody development is complex or poorly understood, mathematical models, which are relatively simple, could be used to reduce time and expenses” (4).

Vibha Jawa, executive director, head of Biotherapeutics Bioanalysis in Nonclinical Disposition and Bioanalysis (NDB) Organization, Bristol Myers Squibb, describes which types of products require the closest watch for potency fluctuations. “I would say the novel complex modalities where we don’t know what expression levels are needed for these new targets in order to validate that the drug is adequately binding to the intended target. So, definitely novel modalities like multidomain biologics and CGT therapies require more consideration for potency.” So, ab initio, increased biological complexity itself, plays a central role in making these therapies more difficult. If we cannot match on a one-to-one basis the expression levels required for MoA, how then does one model an effective potency regime at the point of origin?

Jawa goes on to build on this observation, saying, “a common thread for these new modalities is that the drug product itself is a cell. And it’s a patient’s cell that we are talking about. So, it’s not a Chinese hamster ovary cell or antibody that comes to us already being well defined. In this case, the Chimeric antigen receptor T cell (CAR-T) for example, where we are taking cells from the patient, we are going to engineer them outside of the body, and we have to understand if the CAR we have transduced into these cells, are they going to be able to bind the tumor target, so for potency you are using this CAR-T cell, or a cell line which is very similar, which can go kill a tumor, and we have to do all potency testing upfront to know this receptor is dedicated or efficient enough.”

And as if to put an exclamation point on the issue of complexitiy, Jawa concludes, “so herein lies the complexity—each person has their own T cell, with its own level of T-cell antigen receptor variability. We just don’t know how the T cell receptors are going to interact with the tumor cell, and same for major histocompatibility complex class one and class two molecules which are the proteins present, which present the peptides. So, potency is, shall we say, a completely different question to answer for CAR-T therapies.”

There is some good news, however, when it comes to viral gene therapies. There is an intrinsically more consistent manufacturing base to pull from, and the adeno-associated viruses or lentivirus being used to transduce the modification into a cell of interest can therefore be manufactured more consistently, and with less variability, than for CAR-T therapies. Another direction would be to question if there is a significant rise in complexity, yet again, when dealing with emerging therapies such as allogenic CAR-Ts, Natural Killer cell-based CARs, or armored CARs. These new modalities require ingenuity in setting up biorelevant test systems for target cells, because the screening of multiple new test cells lines will be required. The further we go on this path, the more variability we need to account for in order to engineer consistency into these new products.

M9 Biopharmaceutics Classification SystemBased Biowaivers Guidance for Industry

3. Playter, G. Crossing the Same River Multiple Times. 

Mathematical Modeling of Cell-based Potency Data for mAb Biotherapeutics, 


Vol. 36, No. 2
February 2023
Page: 18-19

When referring to this article, please cite it as C. Spivey. Potency Assays for Novel Complex Modalities. 

Analytics

Variability instills consistency in advanced therapy medicinal products. 

Potency Assays for Novel Complex Modalities

Biologics, especially protein drugs, are often produced in cultured cells. Those cultured cells, called host cells, synthesize the target proteins along with the proteins coded by the cell genome for the cells’ own survival, function, and reproduction. Purifying the target protein from the other host cell proteins (HCPs) is the main task for downstream processing of biologics manufacturing.

HCP removal is crucial, as HCPs are often immunogenic and can affect the potency and stability of biologics. Therefore, detection and quantification of residual HCPs are categorized as obligatory critical quality attributes in the biologics manufacturing process, and HCPs are strictly monitored throughout production to ensure safety and potency of the products.

There are thousands of types of HCPs within one cell. For example, Chinese hamster ovary (CHO) cells, which are often used in biologics production, have more than 24,000 coded genes (1), making HCP detection a challenging technique, as both assay breadth (number of HCPs detected) and sensitivity are required. In this paper, the authors lay out some commonly accepted HCP analytical methods, the challenges of HCP assay, especially during late clinical stages (process characterization and validation stages), and provide recommendations on what can easily be accomplished in-house and when it may be better to outsource, due to time, cost, and expertise factors.

ELISA and HCP antibody coverage evaluation

The most common assay method for HCP detection is enzyme-linked immunosorbent assay (ELISA) which utilizes pre-raised HCP antibodies to detect HCPs in samples. As it utilizes enzymes to amplify the signal, ELISA is highly sensitive and can detect HCPs at a level of 1-3ng/mL. ELISA is quality control friendly and is used as a product release assay for nearly all biological products.

The difficult part of HCP ELISA is the coverage assessment of HCP antibodies. HCP antibody coverage data are often requested by regulatory agencies to support the use of an HCP ELISA as early as investigational new drug application (IND) stage. Two-dimensional (2-D) gels followed by Western blot analysis or immunoaffinity purification followed by 2-D gel analysis are often the choices for HCP antibody coverage evaluation.

Two-dimensional gels separate proteins by size and charge, and those proteins are then transferred to a membrane that is incubated with HCP antibodies for Western blot detection (

 courtesy of Xiaoqing Zhu, WuXi Biologics). The technical challenges here are the large size of the gel, which holds thousands of proteins, and the need of transferring those thousands of proteins to a membrane under one voltage and time condition, plus the alignment of each protein on the gel with the corresponding protein on the membrane, making it a task more suitable for experienced and well-equipped labs. There are many experienced contract laboratories available to assist in this more complex analysis.

Coverage evaluation using immunoaffinity purification followed by 2-D gel analysis involves generation of HCP antibody conjugated resin and running 2-D gels for total HCPs and affinity-purified HCPs separately (

, courtesy of Muchen Li, WuXi Biologics). Both the antibody conjugation step and the HCP purification step need multiple experiments for optimization, and often consume mg level of HCP antibodies. This analysis could take a long time and become quite costly. Outsourcing to an experienced lab might be a good option.

The total HCPs needed for coverage evaluation are usually produced in bioreactors using null cell lines (cells transfected with gene of interest [GOI] deleted plasmids) under conditions similar to product manufacturing. Because both plasmid transfection for null cell generation and cell growth in a bioreactor are typically available for most biological drug development labs, those activities could be conducted in-house instead of outsourcing.

Platform- and process- specific HCP ELISA

Several commercially available ELISA-based kits are on the market for HCP detection. These “off-the-shelf” kits are ideal for preclinical process development support, as most protein analytical labs have the requisite equipment and trained personnel to utilize these kits. The cost of using these kits can potentially be more affordable than outsourcing.

However, as biologics development moves towards late stage (i.e. Phase III and beyond), it is a regulatory expectation that more thorough analytical characterization and assay qualification activities be applied to ensure process robustness and product quality. For HCP detection, the commercial HCP ELISA is expected to be replaced by either a platform HCP ELISA (developed from the use of the same CHO host cell line across multiple products) or process specific HCP ELISA (2). A concern from regulators, as well as empiracal observation from industry, is that the commercially available kits do not have breadth of antibody coverage of the HCPs that are produced by the specific production host cell line in use and therefore may miss the ability to detect certain low level HCPs that may co-purify with the product.

To develop a platform- or process-specific HCP assay, three key reagents are needed: HCP standards, anti-HCP antibodies, and enzyme conjugated HCP antibodies. The process (

) of making the key reagents can take a year or longer and requires animal facilities, specialized equipment, and expertise. To ensure this process goes smoothly, it is recommended that the activities of developing those specific HCP ELISA assays to be outsourced to the more experienced labs.

In this paper, the authors lay out some commonly accepted HCP analytical methods, the challenges of HCP assay, and provide recommendations on what can easily be accomplished in-house and when it may be better to outsource.

BioPharm International, February 2023

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Purification of Antibodies Using Novel Convecdiff Membranes Part 2: Robust Performance at Comparable Product Quality

Technology for In Vivo CAR T-cell Therapy Advances

Benefits of Technologies Facilitate Adoption of Single-use Systems

Potency Assays for Novel Complex Modalities

Current Trends in Host Cell Protein Detection for Biologics Manufacturing

FDA Interactions with Industry Under Scrutiny

The Commercial Message mRNA Products Send

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