by Bart Vaes, Group Leader, ReGenesys
Therapeutic products require thorough quality testing to demonstrate safety and efficacy. For stem cell based products this is of particular importance, as they are of extreme complexity in comparison with other biologics such as proteins or peptides. Although cell characterization is essential for lot release, its role in the product and process development path is at least of similar importance.
Clinical studies with diverse types of autologous as well as allogeneic products are increasing in number and are advancing towards markets of unmet clinical needs. In particular when large numbers of patients may be treated with an allogeneic off-the-shelf product, the industry faces multiple challenges, of which increasing production scale and the corresponding material supply chain are among the most important for process development.
The large cell numbers needed to treat large cohorts of patients, e.g. 1-10 million per kg body weight (Maziarz et al 2015, Hess et al 2013), require the exploration of novel production platforms. Various types of expansion platforms have been developed, such as multilayer vessels and diverse types of bioreactors. Although the bioreactors may be based on very different techniques (e.g., multiplate, hollow fiber nanotubes or microacarriers), and each method requiring its own scale-out or scale-up strategy, they have one thing in common: the dynamic 3D micro-environment in which the cells need to grow may alter significantly from static 2D culture conditions, and it is therefore subject of investigation to what extent cells are influenced by such changes.
Also, the supply chain may be a significant point of concern when large amounts of (GMP grade) materials are needed for stem cell manufacturing. This is in particular relevant for cell expansion procedures that require bovine serum. It is generally known that batch to batch variation is a drawback for the use of serum. Thus in addition to ethical issues using foetal bovine serum, the availability of batches of consistent quality may be a limiting factor in upscaling to large bioreactors. As an alternative, multiple serum-free medium formulations have been developed and are commercially available. As the constituents in serum and their precise effects on cells are often not known, care must be taken to ensure that serum-free expanded cells maintain their features as known for serum expanded cells.
Demonstrating that applying changes to expansion methods does not alter cellular properties is a key step in process development in order to maintain the therapeutic properties of the final product. Therefore, cell comparability testing is of most importance to demonstrate that the proper product is obtained after switching expansion platforms. For comprehensive comparability testing, a panel of assays is needed that establishes cell identity and functionality. As such, the comparability panel provides analytical tools to: 1) Support consistency in product manufacturing and process change towards commercialization 2) Ensure safety and potency when large scale manufacturing moves towards extended population doubling.
A diversity of characterization methods is available, with the purpose to demonstrate cell identity (e.g. by means of flow cytometry) or therapeutic activity in so-called potency assays. Although different types of markers and cell characteristics have been identified to characterize different types of therapeutic cells, e.g. ISCT criteria for MSC surface marker expression and trilineage differentiation (Dominici et al 2006), it can be argued whether these are immediately relevant for the therapeutic function. Some of the mechanisms by which stem cells exert their regenerative function (e.g. cell trafficking, immunomodulation, induction of blood vessel formation, tissue regeneration) are becoming more and more understood nowadays. Research on the regenerative mechanism in disease models is key to unravel the mechanisms by which therapeutic cells exert their action in vivo, for instance via secretion of cytokines. The measurement of such molecules may then serve as surrogate potency assay. For example, a number of cytokines have been identified that correlate well with pro-angiogenic function and their expression levels may be used as pass/fail criteria (Lehman et al 2012).
The more precise we understand the biology of therapeutic cells, and the more we know about their identity at the molecular level, the better we may correlate molecular markers with therapeutic efficacy. Promising techniques such as deep sequencing and proteomics enable the identification of molecules that may be used as highly specific identity and/or potency markers that can subsequently find their way in comparability testing during process development or eventually for clinical lot release.
Dominici, M., Le, B.K., Mueller, I., Slaper-Cortenbach, I., Marini, F., Krause, D., Deans, R., Keating, A., Prockop, D., & Horwitz, E, 2006, Cytotherapy., 8, (4) 315-317
Hess DC, Sila CA, Furlan AJ, Wechsler LR, Switzer JA, Mays RW., 2014, Int J Stroke. 9(3):381-386
Lehman, N., Cutrone, R., Raber, A., Perry, R., van’t Hof, W., Deans, R., Ting, A.E., & Woda, J. 2012. Development of a surrogate angiogenic potency assay for clinical-grade stem cell production. Cytotherapy. 14(8):994-1004
Maziarz RT, Devos T, Bachier CR, Goldstein SC, Leis JF, Devine SM, Meyers G, Gajewski JL, Maertens J, Deans RJ, Van’t Hof W, Lazarus HM. 2015, Biol Blood Marrow Transplant. 21(4):720-728.