The invention of human induced pluripotent stem cells (iPSCs) by Shina Yamanaka 14 years ago has heralded a new era of cell and gene therapies and clinical paradigms. The proliferative potential of iPSCs and their ability to differentiate into cells of all three germ layers has initiated many clinical trials with the goal of providing cell-replacement therapies for various diseases such as Parkinson’s disease and diabetes. However, in order to commercialize these therapies to treat a large number of individuals at an affordable cost, it is necessary to demonstrate the safety and efficacy of these therapies and industrialize the manufacturing process to generate therapeutically viable iPSC-derived cells in a scalable and controlled manner. Lonza (Basel, Switzerland) is at the forefront of this effort and in this webinar we have summarized the major steps that have been made, at Lonza, to support the clinical manufacturing of iPSCs and their therapeutic derivatives.
Good manufacturing practice (GMP)-compliant iPSC reprogramming, differentiation under GMP, efficient scale-up and suitable process control are common challenges faced when developing iPSC-based therapeutics. Directed iPSC differentiation in particular is a complex process involving multiple steps, from tissue acquisition, cell isolation and reprogramming to expansion and banking, before your iPSCs can be differentiated.
Many factors can affect the success of your therapy; understanding the quality of your starting materials, and cell microenvironments and interactions, is crucial. However, it’s also important to have developed a robust manufacturing protocol applying the latest insights for efficient and effective scale-up, whether at 2D, 3D or beyond.
Generating, expanding and differentiating therapeutic iPSCs
iPSCs are showing great potential for cell and gene therapies due to their propensity for self-renewal and their ability to be differentiated into any of the hundreds of cell types found in the human body. However, there are a number of challenges involved in producing quality iPSCs at scale.
Great care should be taken during the initial iPSC reprogramming stage; these are the cells that will be used throughout the rest of your manufacturing process. Differentiation under GMP is a complex process and working to a robust protocol is critical. Switching processes and materials can sometimes be unavoidable but, when done at a late stage, can cause delays on your path to regulation and commercialization. Finally, the basis for any successful product and procedure is suitable process controls and characterization protocols, enabling dynamic and accurate quality assurance.
Developing a suitable cGMP-compliant manufacturing process
Development of a robust system that enables high quality iPSCs to be grown and maintained will ensure the application of standardized processes to the manufacture of your iPSCs. Lonza’s non-viral 4D-Nucleofector™ Transfection Technology, together with Lonza’s L7™ hPSC Culture System, is an excellent approach for the reprogramming and culturing GMP-grade iPSCs.
The L7™ system combines quality primary cells, nucleofector technology, suitable mediums and matrices for cell growth, passaging solution and cryosolution. This complete system supports iPSC generation, stem cell maintenance and a seamless transition from the research laboratory to clinically-directed development.
Figure 1: Using iPSCs generated by the Lonza L7 system, it is possible to produce cells from all three germ layers.
Combining a GMP-compliant iPSC manufacturing process alongside a comprehensive iPSC characterization platform has been shown to successfully direct differentiation into all three main germ layers – the ectoderm, endoderm and mesoderm – in an efficient and unbiased manner (see figure 1).
Large-scale expansion of iPSCs using an end-to-end platform
Producing high quality iPSCs is difficult in large numbers. Through process development from traditional 2D cell culture to bioreactor-based culture and finally, in the later or commercial phases, computer-controlled bioreactors, you can devise an end-to-end platform that allows large scale expansion of iPSCs.
Figure 2: Expansion data from three bioreactor runs inoculated. In all runs, more than 100-fold expansion was achieved in 12-14 days.
The typical banks generated with our process yields around 1×109 cells for the master cell bank and working cell bank each. For allogeneic cell-replacement therapies, the number of cells required for a commercial process may be a thousand to a million times more.
An automated and scalable stirred tank bioreactor platform has been established, with which the process of high fold expansion of high quality iPSCs, followed by cell harvest, can be sustained in a fully closed (figure 2). iPSCs were expanded on plastic microcarriers, which were removed at cell harvest. This process reduced the manufacturing time as cell adaptation to 3D or cell passaging was not required.
Resultant iPSCs had typical hPSC morphology, were positive for markers of cell renewal and also had the ability to directly differentiate. With this set-up, harvested cells could be directly inoculated into a subsequent expansion or differentiation process, or cryopreserved at closed cell concentration as a high cell density seed bank for use in future, large-scale bioprocesses. Direct inoculation of either fresh or frozen cells into the bioreactor eliminated the need for a 2D seed train, saving time and reducing process risk.
Best bioprocessing practice for computer-controlled bioreactors
Computer control, whether process control, monitoring, data gathering and processing, is a useful tool to manage your manufacturing processes, enabling greater quantities of cells with lower batch-to-batch variance, fewer deviations and human errors, and less manpower, in a shorter period of time. Although not commonly studied in the life sciences, computational fluid dynamics (CFD) can be applied to bioreactor control in a 3 L computer-controlled bioreactor system.
Figure 3: Demonstration of shear fields present a spinner flask compared with the Eppendorf BioBlu.
CFD can effectively describe necessary scale-up parameters by fully taking into account the geometry and configuration of bioreactors, or for bioreactors with smaller volumes than 50 L, enabling efficient scale up in a serum- and carrier- free process (figure 3). CFD modelling demonstrates there are significantly different shear forces above, at and below the impeller, and between the two products. Therefore, the CFD modelling enabled scale-up of this process based on average aggregate dissipation rates, to ensure uniform and improved aggregate size.
De-risking your path to commercialization
Working with Lonza’s iPSC manufacturing services means benefiting from over 8 years of iPSC development and the world’s first iPSC GMP bank, established in 2014. Our end-to-end service offers process qualification and full characterization services to a pre-determined timeline and reassurance of full GMP compliance throughout the iPSC reprogramming, expansion and differentiation cycle. This service means support from tissue acquisition to formulation and fill & finish, and use of our proprietary cell lines will prevent commercial and regulatory challenges.
Based on the work discussed in this webinar, we have sought to address the key challenges that are faced by our clients/industry and are well poised to support the industry in the future commercial applications of iPSC-derived therapies.
Find out more by contacting us today at [email protected].