BPI’s first cell-therapy supplement was published in March 2011 (1).
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In the first cell therapy special issue of BioProcess International back in 2011, members of the International Society for Cell and Gene Therapy’s (ISCT’s) commercialization committee highlighted the need for cell-processing professionals who prepare bone-marrow and cord-blood products to collaborate with bioprocess engineers in establishing commercially-relevant manufacturing processes for a new wave of cell-based therapies (1). The emerging field of cell and gene therapy presented unique challenges for creating scalable bioprocesses under current good manufacturing practices (CGMPs) to accommodate primary cells with limited expansion potential, many of which were adhesion dependent.
The article highlighted a developing discipline, cell therapy bioprocessing. Publication of BPI’s special issue coincided with a flurry of activity within academic translational institutes and many small and large biopharmaceutical companies to establish processes that would meet the requirements of an emerging regulatory landscape. The goal was to be able to operate such processes at scales that could treat tens of thousands of patients — while maintaining the critical quality attributes (CQAs) of unique cells that showed therapeutic properties at bench scale.
Many academic culture-expansion processes were developed in multiwell plates and required scaling not just to multilayer vessels and closed-bag systems, but also to truly scalable single-use bioreactors. The challenge was to develop CGMP-compliant processes starting with donor selection and media.
The Impact and Influence of Glutamine Synthetase (GS)
The first 25 years of the bioproduction field (1985–2010) evolved from T flasks and shake flasks to roller bottles and stirred-tank bioreactor processes. Generation of standardized cell lines such as Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293 cells (2), coupled with standardized bioreactor processes and highly optimized media systems, established true platforms for CGMP therapeutic-protein manufacturing. Innovations in cell engineering led to engineered cell lines such as CHO cells that express glutamine synthetase (GS-CHO). Resulting log improvements in productivity offered protein yields exceeding 10 g/L, whereas titers in the tens of milligrams per liter were commonplace just two decades before. Further media innovations for batch, fed-batch, and perfusion cultures helped to create scalable production to achieve cost-effective manufacturing of clinical trial material. Product candidates that were successful in early-stage trials could be scaled rapidly for larger pivotal phase 3 trials and commercial production. Cell culture innovations laid the groundwork for biopharmaceuticals to be 50% of the top selling drugs in 2020 (3).
Much of early scale-up for cell therapies benefited from the experiences of the early therapeutic protein production field. Roller bottles were considered to be overcumbersome and required expensive automation, so multilayer vessels were implemented as the basic production systems for adherent cells such as fibroblasts and mesenchymal stem/stromal cells (MSCs), for which bioreactor processes for adherent cells were still in development (1, 4). Optimizing processes through complete media exchanges drove much of the improvements in cost of goods sold (CoGS) through decreased media use and labor. Only the most sophisticated of cell therapy developers implemented processes without such exchanges.
Cell therapy benefited from knowledge gained in the therapeutic protein world, but protein production was based on cell lines that could be selected for optimal growth and product characteristics. By contrast, primary cells — sometimes autologous cells coming from sick patients — created significant challenges for the early-stage cell therapy industry (4).
Standing on the Shoulders — the MSC Field Matures: MSCs have been one primary cell type making significant strides in manufacturability over the past 10 years and are arguably having a “GS-CHO moment.” Early involvement in development of MSC therapeutics by pharmaceutical companies led to demonstration of large-scale bioreactor culture expansion, but that technology was “trapped” within the walls of big pharma. MSC clinical development continued within small biotechnology companies, and eventually bioreactor processes became more common.
The MSC field has matured to the point at which “off-the-shelf” CGMP cell banks offer 10-fold higher productivities than they did just a decade before. Those productivities, along with highly optimized fed-batch cultures for bioreactor expansion and implementation of bulk MSC expansion, are laying the foundation for a host of next-generation MSC applications. In development are novel products such as cell-based gene therapies, tissue engineered constructs, and MSCs as accessory cells to other therapeutic cells such as HSCs and T cells.
Because microcarriers are required for bioreactor expansion of adherent cells, effective production at scales greater than 200 L has yet to be demonstrated. We believe that the engineering know-how that exists as a result of advances in CHO/HEK manufacturing scale-up to 20,000 L should provide the means to overcome that limitation.
The Emergence of Exosomes as a Therapeutic Modality
Exosomes are a new class of biological therapeutics that is gaining interest rapidly in pharmaceutical and academic circles alike (5). Although exosomes are technically a subset of the broader range of extracellular vesicles (EVs) that also include larger microvesicles and apoptotic bodies, the terms often are used interchangeably.
Exosomes are produced by cells for intercellular communications such as signaling, immune modulation, or molecular recycling. Exosomes are being studied as diagnostics and therapeutics in oncology, immune disorders, and tissue repair. MSC exosomes have garnered significant interest because they maintain similar therapeutic functions as their parent cells, but they are inherently safer due to their inability to replicate (5). Exosomes also are easier to handle as therapeutics than are primary human cells because they are amenable to more conventional biologics formulations, drug-product presentations, and long-term storage conditions. They’ve been used as vehicles for the in vivo delivery of small molecules, proteins, RNA, and even viral vectors given their lower immunogenicity and favorable tropism compared with lipid nanoparticles (LNPs) and viral vectors.
HEK293 exosomes can be grown in suspension cultures to high densities similar to CHO cultures in large-scale bioreactors, enabling production of large quantities of exosomes. However, HEK-derived exosomes lack the therapeutic properties of their MSC-derived counterparts, thereby limiting their applications. Because they are similar in size and composition to certain viral vectors and LNPs, exosomes can leverage much of the same downstream process technologies (depth and membrane filtration, tangential flow, and so on) as those used for therapeutic proteins — notably, large-pore chromatography resins or monoliths for enhanced purification capacity.
We believe that innovations in cell culture over the past 30 years have enabled the rapidly growing uptake of exosomes as a new therapeutic modality because many aspects of the CGMP supply chain and manufacturing platforms for those novel biologics already exist. Innovations could be accelerated further by development of clonal immortalized MSCs engineered to generate exosomes that carry specific nucleic-acid or protein cargoes or novel proteins on their surface.
Several transfection/transduction reagents, media, and techniques have been developed recently, including those that facilitate MSC engineering using plasmids, viral vectors, LNPs, and electroporation. Modern analytical technologies such as nanoflow cytometry are enhancing our ability to characterize exosomes and elucidate the mechanisms of action of exosome therapeutics, which will facilitate the identification of critical quality attributes (CQAs).
Today’s Therapies, Tomorrow’s Industries: Innovations Matter
We posit that large-scale cell culture is one of the great innovations that has emerged from the bioprocess field in the wake of monoclonal antibodies. These emerging therapies are facilitated by advances in development of cell lines, media, equipment, and their integration into comprehensive bioprocesses for greater productivity, scale, and purity. In the coming years, we expect to experience a similar paradigm shift in the manufacturing of cellular therapies. Three-dimensional bioreactor processes (even for adherent cells), off-the-shelf GMP cell banks, and fed-batch media systems for high productivity and low CoGS set the stage for multiple commercial products. We believe that MSCs will provide the cell and gene therapy industry with a robust bioproduction platform, with an impact similar to what CHO created for antibodies, with induced pluripotent stem cells (iPSCs) and allogeneic T cells, regulatory T cells(Treg), and natural killer (NK) cells close behind.
For exosomes, we envision establishment of development and manufacturing platforms such as those that emerged for monoclonal antibodies, with standard conditions for cell-line engineering and expansion, robust capture and polishing steps for purification, and consensus formulation and analytical characterization strategies. Cell-culture technologies are greatly increasing the supply of cells to meet the massive demand for tissue engineering and bioprinting, and further innovations will be needed to meet the enormous demands of future industries such as those producing cultivated meat.
References
1 Brandenberger R, et al. Cell Therapy Bioprocessing: Integrating Process and Product Development for the Next Generation of Biotherapeutics. BioProcess Int. 9(Supplement 2) 2011: 30–37; https://bioprocessintl.com/manufacturing/cell-therapies/cell-therapy-bioprocessing-314870.
2 Lai T, Yang Y, Ng SK. Advances in Mammalian Cell Line Development Technologies for Recombinant Protein Production. Pharmaceuticals (Basel) 6(5) 2013: 579–603; https://doi.org/10.3390/ph6050579.
3 Sagonowsky E. The Top 20 Drugs By Worldwide Sales in 2020. Fierce Pharma, May 2021; https://www.fiercepharma.com/special-report/top-20-drugs-by-2020-sales.
4 Rowley JA, et al. Meeting Lot Size Challenges of Manufacturing Adherent Therapeutic Cells. BioProcess Int. 10(Supplement 3) 2012: 16–22; https://bioprocessintl.com/manufacturing/cell-therapies/meeting-lot-size-challenges-of-manufacturing-adherent-cells-for-therapy-328093.
5 Phinney D, Pittenger MF. Concise Review: MSC-Derived Exosomes for Cell-Free Therapy. Stem Cells 35(4) 2017: 851–858; https://doi.org/10.1002/stem.2575.
Corresponding author Jon A. Rowley is founder and chief product officer of RoosterBio Inc., Walkersville MD; [email protected]. Michael Boychyn is senior vice president of analytical, product and process development and services; and Tim Kelly is the chief executive officer at RoosterBio.
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