Cytotherapy, 2017; 19: 9–18
Bioreactors for cell therapies: Current status and future advances
SHANNON EAKER1,*, EYTAN ABRAHAM2,*, JULIE ALLICKSON3,*, THOMAS A. BRIEVA4,*, DOLORES BAKSH1, THOMAS R.J. HEATHMAN5, BIREN MISTRY4 & NAN ZHANG6
1GE Cell Therapy Technologies, Marlborough, MA, USA, 2LonzaWalkersville,Inc.,Walkersville,MD, USA, 3Wake Forest University School of Medicine,Winston-Salem, NC, USA, 4Celgene Cellular Therapeutics,Warren, NJ, USA, 5PCT,a Caladrius Company,Allendale, NJ, USA, and 6National Heart, Lung and Blood Institute, NIH, Bethesda, MD, USA
Abstract The use of bioreactors in cell therapy applications is on the rise, as clinical trials and commercialization of cell therapy– based products are moving to the forefront of treatment opportunities.This review focuses on the considerations and benefits of using bioreactors for cell therapy manufacturing, with an emphasis on autologous versus allogenic, scalability, tissue en- gineering, automation and comparability and consistency in the final product. Evaluation and choice of the right bioreactor for any given process and indication is paramount when moving into scalable platforms and processes that can support the cell therapy industry’s needs.
Key Words: allogeneic, autologous, bioreactor, cell culture, cell therapy, scale-out, scale-up, stem cell, tissue engineering
Overview of bioreactors requirement.This variety is particularly useful for cell therapies, which often require complexities such as Cell culture expansion is an important unit opera- feeder cells, three-dimensional cultures, patient- tion of most cell therapy manufacturing processes. Cell specific manufacturing, controlled cell-cell contact, low culture typically contributes the largest component of oxygen concentration and undisturbed local microen- manufacturing time and profoundly affects cell product vironments. Further, most cell therapies require cells characteristics for both simple expansion processes and to be harvested from the bioreactor, whereas this re- complex processes such as cell differentiation. Ac- quirement is not always needed for many cultures used cordingly, manufacturing tools such as bioreactors, to produce cell-derived products. which maintain a culture environment for cells that The main categories of bioreactors include stirred produce (or are) a product of interest, are a valuable tanks, fixed and packed beds, rocking platforms and platform for cost-effective and consistent produc- hollow fiber systems [6]. These categories differ most tion of high-quality cell therapies. Bioreactors have been dramatically in geometry and fluid agitation methods, central in cell-based generation of products such as which include stirring by an impeller or pendulum wand, alcoholic beverages, chemicals, antibiotics, monoclo- rocking or bulk media flow. Most bioreactors are capable nal antibodies and vaccines, and have recently been of real-time monitoring and automated control of culture receiving increasing attention for cell therapies [1–3]. parameters such as control of dissolved carbon dioxide The advantages of using bioreactors in cell therapy and oxygen, temperature and pH by CO or addition include reduced manufacturing costs, improved in- 2 of acid or base. In addition, most bioreactors use closed- process control, allowing economies of scale as well system manipulations and gas filtration to prevent as product quality and consistency [4,5]. microbial contamination and cross-contamination.These closed-system manipulations also minimize the safety Bioreactor types risks of operator exposure to the product stream. A wide variety of bioreactor designs originating from The wide variety of bioreactor designs intro- the production of cell-derived compounds are avail- duces an equally wide variety of key attributes such able to accommodate nearly any scale and culture as cost and scalability, development needs and labor
*International Society for Cellular Therapy (ISCT) Process and Product Development Subcommittee. Correspondence: Shannon Eaker, Ph.D., GE Cell Therapy Technologies, 100 Results Way, Marlborough, MA 01752. E-mail: [email protected]
(Received 18 August 2016; accepted 22 September 2016) ISSN 1465-3249 Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jcyt.2016.09.011 10 S. Eaker et al. requirements. Criteria to select bioreactors include Importantly, agitation must be designed to manage culture conditions required for the cells of interest, not only damaging shear exposure of cells, but also simplicity, development investment needed to adapt the efficiency of mass transfer, suspension of cells and from static culture to a candidate bioreactor, scalabil- avoidance of inhomogeneities that cause cell incon- ity, material demand and cost. Ultimately, the simplest sistencies. Similar to agitation, mass transfer of bioreactor that can provide process needs as well as nutrients, waste and gases is well-understood for mul- cell quality is often the best choice.This is a trend that tiple bioreactor types [11]. In addition, there is strong mirrors that of the biotechnology industry where stirred experience with uniform cell seeding onto solid sub- tank bioreactors have become the industry standard strates such as microcarriers with anchorage-dependent platform for large-scale production [7]. Importantly, cells. In contrast, few studies have addressed the harvest choice of culture scale should include consider- of healthy, functional adherent cells from growth sub- ations of downstream processes to accommodate strates. Recently, great progress has been made in the increasing cell outputs. harvest of adherent cells from microcarrier cultures One of the primary factors influencing the choice [12]. of bioreactors for cell therapies is the anchorage Adapting cells to process conditions is an impor- dependence of the cells. Suspension cultures of tant strategy for bioreactor engineering in the field of anchorage-independent cells can be readily applied to cell-derived products. Adaptation of the cells involves stirred-tank and rocking platform bioreactors, in which engineering cells that impact bioreactor design as a sup- cells are suspended via agitation in a highly scalable plement to engineering the bioreactor itself. For manner that has been proven in the production of example, cell lines used to produce cell-derived prod- cell-based products.These systems are only effective for ucts have been adapted over many passages to achieve non-adherent cell types that do not require anchorage independence and greater shear tolerance motionless culture conditions. All bioreactor types, when to improve bioreactor scalability [13–16]. This ap- run in perfusion mode with non-anchorage-independent proach is not available to most cell therapies, which can cells require cell retention provisions. For these reason, grow for a finite number of population doublings before batch stirred-tank and rocking platform bioreactors are becoming senescent [17–19]. Because many of the pop- most frequently chosen for suspension cells. ulation doublings are leveraged to manufacture the Adherent cell bioreactors require a substrate for product, growth potential is typically insufficient to adapt cell attachment. Hollow fibers and fixed or packed beds the cells to new conditions. One potential solution to with appropriate chemistry are effective cell attach- this challenge might be to establish cell stocks under ment substrates; however, uniformly seeding and the desired conditions so that the desired properties harvesting cells from such bioreactors is challeng- become inherent to the cells of interest. ing, especially at larger scales. In stirred-tank and rocking platform bioreactors, solid substrates called Considerations for allogeneic cell products microcarriers are suspended and provide a scaffold for cells to attach. To maintain suspension and homoge- Demand for allogeneic cell products can vary greatly neity of the microcarriers (typically 100–300 m), from cell type to cell type and thus requires a large especially at high densities of microcarriers, requires range of culture scales. As a result, a variety of increased agitation to maintain suspension as com- bioreactors fulfill the needs of cell therapy cultures. pared with anchorage-independent cells. In addition, In the limiting case in which a single patient is dosed larger particles are more susceptible to shear damage from a batch of cell products derived from an unre- from turbulent fluid motion [8]. lated donor, the patient-specific manufacturing needs are identical to those for autologous therapies (see Con- siderations for Patient-Specific Manufacturing of Bioreactor engineering Autologous or Allogeneic Cell Products). At the op- The rich history of bioreactor engineering provides posite extreme are allogeneic products that treat a vast body of knowledge that can be leveraged for multiple patients and demand larger-scale bioreactors cell therapy manufacturing use. In particular, engi- as the number of patients treated increases.The largest neering methods are applied to challenges related to bioreactor demands arise from a universal donor plat- design and scale-up that arise to different degrees for form, where highly expandable cell types such as each bioreactor type. Approaches to characterization mesenchymal stromal cells (MSCs) might be used. and fundamental understanding are well-developed for These cell products approach similar scale require- common bioreactors. The design of agitation and its ments to those that might be used to make cell- scale-up impact based on fundamental understand- derived proteins/drugs. ing of fluid dynamics is particularly well-developed for For adherent cell types, microcarrier, packed bed, stirred-tank bioreactors [7,9,10]. fixed bed and hollow fiber bioreactors have been most Bioreactors for Cell Therapies 11 commonly used and can also be applied to cell prod- Batch-to-batch variability ucts requiring intermediate culture scales. Of the Routine batch-to-batch variability is inherent to all cell- available platforms, microcarrier cultures in stirred- based manufacturing processes and can be minimized tanks have emerged as the predominant choice for by the highly controlled conditions in a bioreactor. Such MSC-like cells. This mirrors the success of this plat- control is particularly advantageous for cell thera- form to generate biological products, like vaccines and pies, which exhibit more dramatic variability and more recombinant proteins, from anchorage-dependent cells. frequent donor cell stock changes than cultures used However, many such cell-derived drugs are present to produce cell-derived products. Variability is great- outside of the cell or are retrieved by destruction of est for autologous cell therapies, where the donors the cell, and harvesting of healthy cells from bioreactors change from batch to batch and are unhealthy. They producing these products is, therefore, not neces- may have been treated in ways that affect the quality sary.Thus, there is relatively little experience to draw of donated cells. Donor variability can affect not only upon regarding the harvest of intact anchorage- final product characteristics, but also in-process per- dependent cells from bioreactors. Nevertheless, formance and sensitivity to bioreactor operating successful harvesting of MSCs from microcarriers has parameters. Accordingly, process screening and op- been reported using approaches that balance enzy- timization experiments should include basic matic cell loosening with control of fluid dynamics to characterization of donor sensitivity to process minimize cell damage [12]. parameters. Some allogeneic cell therapies may treat very few Other design considerations related to donor vari- patients per batch. Bioreactors used for cell culture ability include the cell expansion profile and resultant in such therapies require smaller culture scales and culture scale requirements. In particular, the avail- generally have few scale-up challenges. Instead, the crit- able bioreactor culture scale and seed train must have ical challenge is scale-out, in which many batches of sufficient flexibility to accommodate the range of cell similar cultures are performed (see Scale-Out section). growth across all batches. This accommodation can be achieved by specifying ranges for the number and Considerations for patient-specific manufacturing of scale of culture vessels (Figure 1), vessel fill volumes autologous or allogeneic cell products and seeding density for each passage. Autologous cell therapies are an emerging modality Implementation of bioreactors of treatment in which a patient’s own cells are manu- factured into a therapy for that same patient. In Although shifting from planar to bioreactor suspen- autologous therapy, each patient-specific manufac- sion culture is in many cases inevitable, to achieve turing lot generates a product used only for that patient. needed cell numbers as well as reduction of cost-of- Patient-specific manufacturing has a different cost goods (COGS), the change from planar culture structure than manufacturing for products that are used to suspension in a bioreactor is a critical decision for to treat multiple patients with a single batch. Notably, cell therapy manufacturing during the product de- few economies of scale over multiple patients are re- velopment cycle. Because such changes involve alized when a single patient is treated from a batch. physiochemical changes in the cell environment, such Thus, new approaches to minimize manufacturing costs as fluid shear, exposure to gas bubbles and change of are necessary to achieve acceptable costs for patient- substrate, there is a non-trivial risk of change to the specific manufacturing. Among these approaches are product characteristics [20]. The impact of a change the use of bioreactors that require little operator setup, to the bioreactor or culture itself on cell attributes can equipment qualification, capital investment and op- be minimized via thorough characterization of process erator manipulations. Accordingly, it is sometimes parameters, as well as a good understanding of the ther- appropriate to use bioreactors with few controlled pa- apeutic mode of action of the final desired product. rameters that require setup and calibration. In addition, Like all changes, the comparability challenge in- simple and automated liquid transfer steps reduce labor creases greatly as clinical trials advance, making it more requirements as well as improvement in quality as- desirable to implement the appropriate platform for surance and costs for patient-specific manufacturing. commercialization as early as possible. Ideally, a cell- Together, these steps serve not only to reduce costs, based therapeutic product would originate in but also to allow scale-out in commercial-scale facili- bioreactors before pre-clinical phase or when enter- ties in which many batches are simultaneously ing early-phase clinical trials, thereby avoiding manufactured. Further, the increased control af- significant process changes during important devel- forded by bioreactors serves to reduce the risk of batch opment stages. However, bioreactor development failures, efficiently using scarce donor materials and requires an investment in development time and en- reducing the time patients wait for a therapy. gineering expertise that may not be fully justified until 12 S. Eaker et al.
Figure 1. Variation in cell growth and appropriate bioreactor platforms. The red curves encompass a 20% variation around the average (blue curve) growth rate. The green curves represent a 90% variation around the average (blue curve) in the growth rate. Such variations can span several logs. The vertical red lines on the right describe the scale limits of three bioreactor platforms, illustrating how the plat- forms can accommodate different phases of cell growth for different cell lines. later stages of clinical development. Accordingly, pre- clinical and early–clinical phase cell therapies are typically developed using small-scale static culture vessels and require adaptation to bioreactors during clinical development. It is, therefore, critical to care- fully consider when to adopt manufacturing methods that are truly scalable, taking into account risks and benefits.
Tissue engineering and combination products Tissue-engineered products require a multi-step manufacturing process (Figure 2). Compared with con- ventional cell therapy, tissue-engineered products usually involve scaffolds with favorable microenviron- ment as structural and mechanical support for cells to adhere, proliferate, migrate, differentiate and func- tion. Due to the complexity of tissue-engineered product, bioreactors are the key to bridging the gap between two-dimensional (2D) cell culture and Figure 2. Steps required for tissue engineering processes. Tissue- three-dimensional (3D) tissue. By mimicking the mi- engineering bioreactors can be custom-designed for specific structure- croenvironment in vivo, bioreactors allow metabolically function relationships and incorporated throughout the entire tissue active cells to organize spatially and temporally into engineering process, which leads to (1) homogeneous, batch-to- functional cell-based constructs. batch consistency, and efficient cell seeding into the 3D scaffold; To date, a variety of bioreactor systems have been (2) adequate gas exchange; (3) improved mass transfer; (4) easy media change and waste removal; (5) temperature and pH control; developed in research, such as rotation-wall vessels, (6) physiological stimuli and product maturation; (7) in-process and fluidized or fixed bed bioreactors, spinner flasks, per- release testing; (8) product logistics and transport; and (9) a func- fusion bioreactors and hollow-fiber devices [21]. tional closed system. Bioreactors for Cell Therapies 13
Rotation-wall vessels provide a constant circulation flow replication process can be labor intensive and re- around the scaffold when rotating the whole device. quires use of planar cell expansion for upstream In various fluidized bed systems, scaffold rotates while processing, and highly manual and open processes for the device remains static. In other systems a solid is downstream processing, such as centrifugation, final suspend by counteracting convective fluid flow against filling and visual inspection.These manual processes gravitation (or centrifugal) force. Culture media are lead to process inefficiencies and increased human circulated by stirring in spinner flasks. In a perfu- error, which are costly, time consuming and severely sion system, cells, oxygen and nutrients are transported limiting in the lot size and total cell number. Process by flow throughout the medium and/or scaffold. automation is a familiar concept in cell therapy manu- Finally, hollow-fiber systems help cell seeding within facturing. It is considered in two different situations. a scaffold traversed by fibers, which protects sensi- First, automation can be used to efficiently perform tive cells from direct mechanical stimulation. repetitive tasks. It represents a strategy to increase Bioreactor design also varies greatly according to manufacturing capacity by using robotics to efficient- the specificity of cell type and tissue structure. For ly perform repetitive process operations.This minimizes example, cartilage, an avascular and acellular tissue, the labor requirement and increases the operational is highly dependent on convection and molecular dif- efficiency of the processing step. Second, automa- fusion with loading-enhanced transport rates when tion can be implemented as a part of a complex designing the bioreactor system. Conversely, the bioprocess unit operation developed for cell expan- bioreactor system for cardiac muscle, a densely packed sion or cell concentration and washing. Cell expansion tissue, focuses on sufficient oxygen transport; cultur- can be automated based on feedback loops to control ing bones and ligament usually benefit from mechanical temperature, pH and dissolved oxygen. Additionally stimulation, such as torsion, tension and shear stress; control loops can be programmed to temporally change culturing blood vessels require fluid flow to align en- set points according to process needs. For cell con- dothelial cells along the lumen [22]. Due to different centration and washing steps can be automated based emphasis of a specific tissue, generating a standard on volumetric set points. These types of automation model of bioreactor for all tissue-engineered prod- strategies are limited to a single unit operation. Manu- ucts is challenging. facturing systems with automation integrating multiple When transferring a research-grade system to the unit operations are generally limited to proprietary clinic, there are multiple considerations unique to systems developed for specific manufacturing processes. tissue-engineered product. First, bioreactors need to be compatible with quality control in-process and Forward-looking solutions release testing [23]. A variety of tissue-engineered prod- ucts are manufactured as a single lot. Release testing One of the major solutions to the challenges with the for such products includes metabolic activity for cell current state of cell therapy manufacturing is the de- growth, immunoassays for cell phenotype and bio- velopment and adoption of automated and scalable chemical assays for extracellular matrix formation. manufacturing processes for cell therapies and tissue These assays are usually destructive and, therefore, ne- engineering. The use of bioreactor-based manufac- cessitate further manipulation of the final product. turing during scale-up/scale-out is a central aspect of Developing non-destructive methods for release testing these automated and scalable processes. Bioreactor- can be technically challenging, however, it can be ac- based cell expansion is inherently scalable, better complished by investing adequate amount of time and controlled and more automated than 2D planar cell resources. To enable non-destructive methods, it is expansion. This is due to the fact that the ability to crucial for the bioreactor to have easy access to the scale-up bioreactor volumes is much easier than scale- product and by-product. Second, with scaling up, up of 2D platforms, which usually need to be scaled- closed systems are usually preferred to minimize the out as their volume and surface area are limited. risks of cross-contamination. Advanced design is desired Moreover, bioreactors coupled with closed down- to incorporate seeding, sampling and transporting. stream automated systems reduce the number of open Consideration of these challenges in the early process steps in the process, thus decreasing the risk of con- of bioreactor design is ideal. tamination and shortening overall processing time. Automation can be defined as the use of pro- Current and future needs around automation cesses that do not require direct human intervention and, therefore, reduce the need for manpower and the Current state probability of human error affecting the process and Current processes for both allogeneic and autolo- product. Optimal automation should increase process gous cell therapy manufacturing primarily involve efficiencies. An example of an inefficient automation replicating laboratory-scale methods and hardware.This is the semi-automation of 40-layer cell factory 14 S. Eaker et al. manipulation, which is expensive, cumbersome and operation, including at the bioreactor level and across in most cases does not allow the manufacture of suf- the workflow. ficient cell numbers required by medium to large An efficient way to enable scaling and process clinical indications. control of autologous therapies to the extent needed The advantages of automating bioreactors can be is through the use of a bioreactor platform that also realized in a production environment where they offer combines additional automated unit operations such better process and production efficiencies. Automa- as cell isolation, addition of cytokines and/or viruses tion can also include cell enrichment and/or gene and downstream processing. modification within the process.The development of novel biosensors that enable feedback control for Overall advantages cell therapy manufacturing, such as measuring inhib- For both allogeneic and autologous applications, the itory cytokine levels [24], and dielectrophoresis use of automated bioreactors and associated compo- cytometers, which can detect physiological proper- nents should allow automation of the following unit ties of the cells label free [25], can be useful targets operations: for technology development. The use of bioreactors includes many aspects of • Seeding protocol for cells (including mixing regime automation; the intent should be to automate as many to maximize the attachment efficiency and ho- unit operation steps in manufacturing as possible, as mogeneous distribution of cells onto substrates) well as the automation of analytical data gathering, • Control of mixing rate during the expansion phase analysis and data management. Importantly, while the • Addition and removal of media throughout the need for automation exists for autologous, allogene- process (e.g., batch, semi-continuous or contin- ic and tissue engineering, the specifics of this uous perfusion) automation vary between therapeutic modalities. • Online monitoring of critical process parameters • Addition of components (e.g., growth factor or Allogenic–automation considerations cytokines) The goal with allogeneic therapies is to have an au- • Harvest procedure tomated bioreactor platform that is scalable with as • Transfer of cells to the appropriate equipment for many automation aspects as possible. The actual washing and concentration process of initially seeding the bioreactor is usually • Vialing and visual inspection manual; however, the expansion process, which should • Data acquisition and analysis throughout workflow be conducted in a bioreactor (or a bioreactor seed train), as well as some of the downstream processes, All of these steps require a degree of human su- should be fully automated. Automation modalities pervision (e.g., process monitoring) and in some cases that can be integrated into an extended bioreactor intervention (initial seeding, certain transfer steps) but platform include expansion, media replacement, moni- can be partially or almost fully automated. toring and control over dissolved oxygen (DO) and In addition, automation should include the ability pH (see below). Implementation of such automation of the system to self-adjust (e.g., perfusion rate based is technically challenging because this requires very on critical components) and maintain the system pa- good understanding of the process and the product. rameters, such as pH, DO or glucose, at a set-point.
Autologous–automation considerations Why automation? Automation becomes even more important for au- There are multiple benefits of automation ranging from tologous therapies, such as chimeric antigen receptor economic factors to product quality. Labor is often T (CAR-T) manufacturing, than for allogenic thera- a major cost component in manufacturing; there- pies. The complexity of processes for autologous fore, automation greatly reduces the cost of goods. therapies, as well as the need to repeat them many Automation also improves staff productivity, as well times (i.e., one lot = one dose = one patient) in a par- as recruitment and retention rates. Staff can avoid re- allel processing modality, necessitates automation. petitive strain injury with more automated processes. Moreover, with autologous therapy, there is inherent Importantly, automation reduces operator- patient-to-patient variability in the incoming start- dependent variability and improves process consistency ing material, and, unlike with allogenic therapies, there and overall product quality. Many potential sources is no option for optimal donor selection (screening and of process contamination or errors are eliminated selecting donors based on appropriate product and through automation. Automated processes allow for process characteristics.).These characteristics under- easier audit trails, which simplifies product tracking. score the importance of in-process control at each unit Process validation is also simplified through automation Bioreactors for Cell Therapies 15 by reducing the number of parameters to be tested without a recovery phase, should be validated to ensure and ease execution of validation studies. the product maintains consistent quality attributes [27]. Automation improves process stability and control, In addition to these biological factors for the man- which enables process improvement. Process changes ufacture of cell therapies, there are a number of can be made in a defined manner, and the increased considerations for the use of bioreactor technology that consistency allows for more accurate statistical anal- arise during development of scale-up processes. In par- ysis of the impact of these changes.This is important ticular, the supply of oxygen and removal of waste to overall manufacturing as process improvements in- products become increasingly important as the crease product yield and improve product quality, bioreactor process is scaled-up [28]. A summary of which ultimately lower costs and shorten timelines. these considerations can be seen in Table I. Overall, automation leads to more consistent prod- As discussed previously, there is a wealth of en- ucts, which allow for improved treatment regimes. gineering knowledge and technology that can be Automation is also important for manufacturing leveraged to support the manufacture of off-the-self throughput, where more batches can be run over a 24-h cell therapies at increasing scale, given a rigorous un- period. Additionally with a closed automated process, derstanding of the product quality profile. Lessons from multiple donors can be processed simultaneously, which current bioprocessing in large-scale stirred tank re- increases productivity within the manufacturing suite. actors include the following: Also it reduces the footprint required for the cleanroom, which is a significant cost saving. Additionally, it also • Single-use bioreactor technology reduces the risk enables rapid expansion of manufacturing capability of process contamination and avoids having to val- (i.e., opening up other production sites). idate in-process sterilization procedures for reusable technology. • Particles (cells and/or microcarriers) must be fully Current and future needs around scale-up, suspended to use the benefits of operating in sus- scale-down and scale-out pension, such as monitoring, control and improved mass transfer. Current and future needs around scale-up • Impeller agitation rate should consider a balance For the manufacture of allogeneic cell therapy prod- of sufficient oxygen supply to the cells while mini- ucts, scale-up bioreactor technology can be used to mizing the fluid dynamic stress experienced by manufacture a single batch for multiple patients, which the cells. can then be cryopreserved and stored long-term, ready • Baffles can be integrated into the bioreactor to for delivery.These off-the-shelf therapies represent a promote an axial flow pattern, allowing for ade- business model akin to current biopharmaceuticals, quate particle suspension at reduced energy allowing for economies of scale to drive down the cost dissipation rates. per dose of the final product. Preservation of cellu- • Monitoring and control of process parameters, lar quality throughout the entire manufacturing process such as temperature, DO and pH, are critical to is critical to the success of off-the-shelf cell therapies maintaining consistent cell growth and quality and will require the development of scalable harvest- characteristics. ing and downstream technologies capable of processing • Increasing cell concentrations in the bioreactor cellular material in a timely manner.To maintain cell is important to increase product yield and reduce quality in allogeneic cell therapy processes, compa- the cost per dose. nies must also consider the heterogeneity of donor • The effect of direct aeration into the culture material used to produce multiple doses, and screen- medium (sparging) on cellular quality and yield ing metrics for master cell bank material must be should be considered for cell therapy expansion established to ensure product attributes are suitable processes as this is a requirement for current large- to treat the target indication. Given the high cell ex- scale cell bioprocesses. pansion ratio required to manufacture off-the-shelf cell therapies to achieve a large batch from a single Overcoming the challenges of developing scal- donor and the fact that in some cases cell quality able manufacturing processes for cell therapies requires is known to decrease with time in culture [26], the a detailed understanding of bioreactor design and op- impact of these multiple population doublings on cell eration to balance the competing demands of the quality attributes must also be carefully considered. biological and physical systems involved. Quantify- Cryopreservation of the cells is central to achieving ing and maintaining the physical parameters of the an off-the-shelf product by de-coupling the manu- bioreactor system allow for a sound engineering basis facture from the delivery; however, the infusion of a to maintain product comparability in quality and yield cell therapy product directly from cryopreservation, as the bioreactor scale increases. These include the 16 S. Eaker et al.
Table I. Considerations for the scale-up of cell therapy bioprocesses.
Challenge associated with increasing bioreactor scale Considerations during development
Achieving sufficient • As the bioreactor scale increases, the air/liquid interface surface area to volume ratio decreases, resulting in oxygen supply to the reduced oxygen transfer from the headspace. cells within the • Initially, the agitation rate can be increased to improve the oxygen transfer into the liquid phase. bioreactor • Increasing the product yield (cells per volume) will place additional pressure on achieving the required oxygen transfer rate. • Primary cell therapy products tend to have a reduced oxygen consumption rate which reduces the demands on maintaining increased oxygen transfer. • Many cell therapy products are cultured at low oxygen concentrations, which increases the oxygen concentration difference, driving the mass transfer from the headspace into the liquid. Maintaining adequate • Developing scalable in-situ cell harvesting strategies based on defined engineering parameters will be harvesting efficiency critical in maintaining desired harvest efficiencies as the process scale increases. of adherent cells from • Developing scalable technology for the downstream separation of the cells and microcarriers will be microcarriers imperative to maximize yield and quality while minimizing cell processing times. Removal of cellular • As process scale and volumetric cell yield increase, direct aeration into the liquid (sparging) may be waste products required to control the concentration of carbon dioxide, which can be detrimental to the cells. including cell • Traditional cell bioprocesses have typically utilized surfactants such as Pluronic® F68 (Gibco, Grand metabolites and Island, NewYork) to reduce the impact of cell damage due to bursting bubbles during sparging.This may carbon dioxide not be possible for cell therapy manufacture, particularly adherent cells, where the presence of a surfactant will inhibit the attachment efficiency of the cells to the microcarriers. • As the bioreactor scale and product yield increases, the build-up of waste products such as lactate must be controlled to avoid detriment to the product quality and yield. • This may require the addition of an acid or base to control the pH of the system, where a reduced mixing time will be critical to enable pH control. • If an acid or base is employed to control the system pH, careful monitoring of the liquid osmolality is required to ensure it remains within specification for the particular cell-based product. following: impeller tip speed, power input per unit therefore, be achieved by advances in engineering and volume, oxygen transfer coefficient, mixing time in the manufacturing technology to reduce the number of bioreactor, bioreactor geometry (height-to-vessel di- labor-intensive and open process steps that are routine ameter), superficial gas velocity for aerated systems and in cell therapy production. However, there are some Reynolds number of the impeller. strategies that can be implemented within the scale- Understanding these physical parameters of the out manufacturing model to control the cost per dose bioreactor system is essential for establishing scale-down and ensure scalability (Table II). bioreactor models to drive process development and Incorporating the steps in Table II will help to optimization [29]. In turn, high-throughput experi- control the top-down facility costs and the bottom- mentation and statistical techniques such as design of up process costs associated with cell therapy product experiment (DOE) can be used for process optimization manufacture. Reducing these costs will minimize the of the culture conditions. Establishing acceptable op- overall production cost for the patient-specific cell erating ranges for the physical parameters of the therapy product and allow for strategies such as the bioreactor system in the scale-down model ensures that exploitation of shared resources among multiple prod- the optimal local cell environment is maintained and ucts to realize economies of scale in the scale-out agitation conditions do not negatively impact the growth manufacturing model. Given the high fixed cost as- and quality of the cell therapy product as the scale sociated with the manufacture of cell therapies, increases. minimizing the cost of idle capacity will be critical to reducing the overall product costs when ramping up Current and future needs around scale-out to commercial production.The ramp-up of addition- al manufacturing capacity must, therefore, be carefully Patient-specific cell therapies offer a new and excit- managed and aligned with projected patient accrual ing challenge for process scalability, where the rates or product sales projections. manufacturing process must be scaled-out to produce one batch per patient.The unique challenge of scaling- Maintaining scalability and sustainability in the out patient-specific cell therapy manufacturing supply chain processes is reducing the cost per dose given that there are currently few economies of scale to exploit. Re- In addition to individual manufacturing unit opera- ducing the cost of these patient-specific therapies must, tions scalability, supply chain sustainability is Bioreactors for Cell Therapies 17
Table II. Steps to achieve economies of scale within a scale-out cell therapy manufacturing process.
Steps to achieve economies of scale Implications
Understand the product • Enables management of comparability risk as the production rate is scaled and unit operations are quality profile modified. • Ensures the cost of failed batches is minimized by in process monitoring, allowing unsuccessful batches to be ended as early as possible. Minimize the number of • Reduces labor and equipment requirements by reducing process complexity, which reduces variability process unit operations and costs, particularly as the number of batches increases. Avoid peak capacity points • Peak capacity points increase the overall labor cost as additional resources are required at peak times. by evenly distributing • Reduces the cost efficiency of the process as the personnel cost per batch is increased. labor requirements across the process Drive development to • Multiple input products derived from primary patient material are inherently variable. minimize variation and • Reduced variation allows for defined process times and effective scheduling maximize product yield • Maximizing product yield reduces process times which reduces the cost per batch. Closed process steps • Allows for the concurrent manufacture of multiple patient-specific products. • Potentially reduces the grade of clean room, which significantly reduces operating cost as well as the cost of idle manufacturing capacity. • Reduces the potential for contamination and therefore the risk of batch failures. Automated process steps • Reduces the labor requirements which becomes important as the production rate increases. • Reduces in-process variation and creates more reliable process transfer. Shared infrastructure across • Manufacturing infrastructure such as quality testing, logistics and management can be shared across multiple product multiple processes. manufacturing processes, • Reduces the overhead cost per batch for each of these services. in-house or externally Development of scale-down • Process optimization can occur at reduced scale, allowing for increased throughput. process models • Scale-down models must be based on sound engineering principals, to ensure they are comparable with commercial scale processes.
fundamental to the success of both scale-up and scale- Another example of ensuring scalability in the out cell therapy processes.The supply of materials that supply chain for cell therapy manufacturing pro- service the process must be available at the scale de- cesses is the production of viral vectors for gene- manded by each stage of development, thereby modified products such as CAR T-cell therapies.These ensuring clinical and commercial sustainability, and viral vectors are critical to the transduction process at the required cost-of-goods. As an example, good for modified cell therapies, therefore, their cost- manufacturing practice (GMP)-grade fetal bovine effective supply must be maintained at the necessary serum (FBS) is currently in short supply and cannot volume and quality to service an increasing number cost effectively service the entire cell therapy indus- of manufactured product doses. try [30]. Therefore, switching to serum-free and sustainable alternatives at an early stage of develop- Conclusion ment will likely reduce the product cost in late-stage development when the medium manufacturing process The use of bioreactors in patient-specific and off-the- itself can be effectively scaled and the cost of the shelf cell therapy manufacturing presents unique medium per volume can be reduced rather than in- challenges and potential opportunities for the indus- creased. In addition, moving toward chemically defined try.These bioreactor systems must allow for the robust, media will further increase sustainability by reduc- scalable, sustainable and high-quality production of ing the risks associated with a single supplier for critical cell therapy products at a reasonable COGs to meet manufacturing reagents because the media manufac- the patient need over the commercial lifecycle of the turing process can be transferred if necessary. To be product. As the future state continues to emerge a viable solution in the long term, expensive serum- it will be important for cell therapy and technology free media components such as growth factors, must developers to understand the particular quality, com- also be produced in a scalable manner to drive down parability and cost drivers for the unique product needs costs as the volumes increase. (e.g., patient-specific or universal off-the-shelf) 18 S. Eaker et al. described in this article, so that the development of [13] Wurm FM. Production of recombinant protein therapeutics bioreactor systems continues to build inherent value in cultivated mammalian cells. Nat Biotechnol 2004;22(11): for the industry. Although much can be learned from 1393–8. [14] Gallo–Ramírez LE, Nikolay A, Genzel Y, Reichl U. Bioreactor bioreactor development in established bioprocessing concepts for cell culture-based viral vaccine production. 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