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, 2 (6), 362-376

Biomanufacturing for Clinically Advanced Cell Therapies


Biomanufacturing for Clinically Advanced Cell Therapies

Ayesha Aijaz et al. Nat Biomed Eng.


The achievements of cell-based therapeutics have galvanized efforts to bring cell therapies to the market. To address the demands of the clinical and eventual commercial-scale production of cells, and with the increasing generation of large clinical datasets from chimeric antigen receptor T-cell immunotherapy, from transplants of engineered haematopoietic stem cells and from other promising cell therapies, an emphasis on biomanufacturing requirements becomes necessary. Robust infrastructure should address current limitations in cell harvesting, expansion, manipulation, purification, preservation and formulation, ultimately leading to successful therapy administration to patients at an acceptable cost. In this Review, we highlight case examples of cutting-edge bioprocessing technologies that improve biomanufacturing efficiency for cell therapies approaching clinical use.

Conflict of interest statement

Competing interests

A.A., M.L., O.S.F., D.K., M.V.M., J.R., J.T., R.M.O. and S.L. declare no competing interests. D.S., C.L. and R.P. are employees of Hitachi Chemical Advanced Therapeutics Solutions. R.D. owns equity in BlueRock Therapeutics. D.G.A. is a founder and equity shareholder in Siglion Therapeutics. R.N.B. is an employee and equity shareholder of Sentien Biotechnologies, Inc. B.P. is a founder and equity shareholder of Sentien Biotechnologies, Inc.


Fig. 1 |
Fig. 1 |. Cell-therapy pharmacoeconomics and manufacture.
a, Number of cell-therapy clinical trials started yearly in the United States, from 2000 to 2016. The two inflection points correlate with the publication of two phase I human trials: MSCs to treat graft-versus-host disease (GvHD)19 and CAR-T cells against chronic lymphocytic leukaemia (CLL). b, Schematic of the supply-and-demand curve for a hypothetical CTP as it evolves from preclinical testing to commercialization. Disease prevalence, or demand, is shown by the green line; CTP production, or supply, is shown by the pink line. The dashed lines represent trajectories for which the scale of CTP production does not match clinical needs. The y axis represents an arbitrary number of units. c, The bioprocesses for the manufacturing of CTPs discussed in this Review, with the boxes illustrating the case studies used. The scalability of each bioprocess, which is designed to meet a quality target-product profile (QTPP), can improve the production efficiency of a specific CTP towards meeting clinical and commercial-scale demands. LN2, liquid nitrogen.
Fig. 2 |
Fig. 2 |. Process optimization for the expansion of cells and for cell collection from microcarriers.
a, Production of clinical lots by using adherent MSCs in 2D cell-culture plates. Issues with the scaling of costs and labour efficiency make 2D culture unlikely to meet an estimated demand of >1012 viable cells per year, necessary for treating prevalent adult indications. b, Suspension culture systems for MSCs use microcarriers and stirred tank bioreactors and are a scalable and sustainable approach for cell expansion at high density. c, Unit operations identified as major bioprocessing bottlenecks: (1) bead-to-bead transfer for MSC subculturing and expansion; (2) the need for enzymatic digestion and centrifugal separation to isolate the MSCs from the microcarriers. d, Materials-science innovations in microcarrier substrates can improve product purity, identity and potency through degradable and temperature (T)-sensitive materials (such as poly(N-isopropylacrylamide), PNIPAM) that remove the need for additional enzymatic dissociation processes.
Fig. 3 |
Fig. 3 |. Towards high-throughput label-free purification.
Cell-separation techniques, in which cells are first identified and labelled, and then separated and recovered, can currently be broken down into two main categories: magnetic sorting and flow sorting. a, Magnetic sorting uses magnetic beads coated with an antibody to separate cells from a mixed population (green, purple and grey). Current magnetic methods result in a positively selected population (green) that still has magnetic beads attached, which is undesirable (bottom right, recovered population). b, This issue has been circumvented by the MagCloudz QuadGel technology, which embeds magnetic particles into a hydrogel coated by antibodies, thereby effectively eliminating direct contact between the cells and the magnetic beads by using a release buffer that separates the magnetic particles from the hydrogel, which can then be recovered by magnets. c, Flow-cytometry sorting is a widely used method, based on fluorescently tagged antibodies (top right), that allows populations of cells to be selected according to antibody binding. The method is expensive and costly to set up in parallel; hence, it is typically used at low throughput. d, In microfluidic methods, which increase throughput for unlabelled cells, a mixed population of cells is passed through microchannels, where cell separation is driven by a sequence of events, such as size filtering, acoustic separation and dielectrophoresis (DEP) sorting. A DEP trap can be set to collect the desired cells. n-DEP, negative dielectrophoresis.
Fig. 4 |
Fig. 4 |. Streamlining the genetic modification of cells for therapy.
a, An overview of the traditional method used for the genetic modification of T cells for CAR-T-cell transformation. Cells are incubated (at conditions for optimal multiplicity of infection) with viruses carrying an engineered vector that enables the expression of a desired antigen receptor, providing the CAR-T cell with recognition specificity. b-d, More efficient methods in various states of use and development to generate CAR-T cells include flow through electroporation (MaxCyte STX), where pores in individual cells are generated by an electrical pulse (b), mechanical membrane disruption by forcing cells through a narrow pore (CellSqueeze, SQZ Biotechnologies; c), and the use of permeabilization solutions where the target vector diffuses through the cell membrane and a stop solution then reverses the permeabilization (Avectas; d). Engineered vectors are displayed in orange and the CAR expressed on the membrane surface is displayed in multiple colours.
Fig. 5 |
Fig. 5 |. Overview of current tools for differentiating PSCs into retinal and neuronal lineages.
a, PSCs are initially seeded in cell-culture plates. b, Feeder cells, required to maintain proper PSC propagation, are constrained by having to maintain adequate media nutrients (which usually results in frequent media changes). c, The use of partially defined xeno-free media with the required growth factors necessary for lineage-specific cell culture removes the need for the feeder cells. d, Current state-of-the-art processes for differentiation (for example, PSCs that undergo anterior neuroectoderm differentiation and that need to undergo eye field specification before being induced as retinal pigment epithelial (RPE) cells) are expensive and time-consuming, requiring frequent media changes with specific growth factors for several months. e, The use of small molecules (bone morphogenetic protein (BMP), activin and SMAD inhibitors) greatly reduces costs at each differentiation step compared with the use of growth factors, and improves culture efficiency. f, The use of regulatory RNAs could further reduce culture costs by reducing the number of differentiation steps.
Fig. 6 |
Fig. 6 |. Islet encapsulation.
a, Islet-entrapment devices, such as crosslinked alginate capsules, are semipermeable, protecting pancreatic islet cells from immune cells while still permitting oxygen and nutrients to enter, and insulin and glucagon to escape. b, A common method of encapsulation is the formation of droplets of polymer material and islet cells via extrusion dripping into a crosslinking solution. Although effective, this method is relatively low throughput. c, Pulsating extrusion heads increase the frequency of droplet formation proportionally to pulsation frequency. d, Jet-cutting technology mechanically creates droplets by passing a blade through a continuous extrusion stream, whereby droplet formation is proportional to the rotation frequency and the separation between blades.
Fig. 7 |
Fig. 7 |. Supply chain for CTPs.
The CTP supply chain is a complicated flow process comprising a series of dynamic components starting in a clinical environment, going through bioprocessing and then returning to the clinic. Initial seeding products are derived from patients or donors. These are screened for health and for safety (identification). Once cleared, sample collection begins. Proper inventory must then be made for tracking purposes. The cells are then put into storage (either short term or long term, depending on whether they are meant for banking or for immediate use). Transportation of the cells proceeds to the manufacturing facility, where purification, modification and/or expansion can take place. Once processing is complete, the product is moved onto the end location, for administration to the end patient. Supply-chain logistics are crucial to the overall success of CTPs.
Fig. 8 |
Fig. 8 |. Segmented costs for translating cell therapeutics.
Costs associated with the development of cell therapies, broken down in steps, from manufacturing to commercialization.

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