Detailed Characterization of Human Induced Pluripotent Stem Cells Manufactured for Therapeutic Applications

Stem Cell Rev and Rep DOI 10.1007/s12015-016-9662-8 Detailed Characterization of Human Induced Pluripotent Stem Cells Manufactured for Therapeutic Applications Behnam Ahmadian Baghbaderani 1 & Adhikarla Syama2 & Renuka Sivapatham3 & Ying Pei4 & Odity Mukherjee2 & Thomas Fellner1 & Xianmin Zeng3,4 & Mahendra S. Rao5,6 # The Author(s) 2016. This article is published with open access at Springerlink.com Abstract We have recently described manufacturing of hu- help determine which set of tests will be most useful in mon- man induced pluripotent stem cells (iPSC) master cell banks itoring the cells and establishing criteria for discarding a line. (MCB) generated by a clinically compliant process using cord blood as a starting material (Baghbaderani et al. in Stem Cell Keywords Induced pluripotent stem cells . Embryonic stem Reports, 5(4), 647–659, 2015). In this manuscript, we de- cells . Manufacturing . cGMP . Consent . Markers scribe the detailed characterization of the two iPSC clones generated using this process, including whole genome se- quencing (WGS), microarray, and comparative genomic hy- Introduction bridization (aCGH) single nucleotide polymorphism (SNP) analysis. We compare their profiles with a proposed calibra- Induced pluripotent stem cells (iPSCs) are akin to embryonic tion material and with a reporter subclone and lines made by a stem cells (ESC) [2] in their developmental potential, but dif- similar process from different donors. We believe that iPSCs fer from ESC in the starting cell used and the requirement of a are likely to be used to make multiple clinical products. We set of proteins to induce pluripotency [3]. Although function- further believe that the lines used as input material will be used ally identical, iPSCs may differ from ESC in subtle ways, at different sites and, given their immortal status, will be used including in their epigenetic profile, exposure to the environ- for many years or even decades. Therefore, it will be impor- ment, their mitochondrial content and perhaps X chromosome tant to develop assays to monitor the state of the cells and their inactivation [4]. These differences are intrinsic to the source of drift in culture. We suggest that a detailed characterization of starting material, and such differences may be further ampli- the initial status of the cells, a comparison with some calibra- fied by the pluripotency induction process [5]. It is important tion material and the development of reporter sublcones will to note, however, that current studies have shown that these intrinsic differences between ESC and iPSC do not necessarily Electronic supplementary material The online version of this article reflect in their functional utility; in fact, several large-scale (doi:10.1007/s12015-016-9662-8) contains supplementary material, analyses have verified that the differences seen are more re- which is available to authorized users. flective of the allelic diversity of individuals [6, 7]. The degree * Mahendra S. Rao of difference seen between iPSC lines from different individ- [email protected] uals is in the same range as differences seen between iPSC lines made from the same donor but different tissues and be- 1 Lonza Walkersville, Inc., Walkersville, MD 21793, USA tween ESC and iPSC [8, 9]. Equally important, the changes 2 Centre for Brain development and Repair, Institute of Stem Cells and introduced by the process of iPSC generation using non- Regenerative Medicine (InSTEM), Bangalore, India integration methods are in the same range as those changes 3 Buck Institute for Researching on Aging, Novato, CA, USA that are seen when cells are maintained in culture for prolonged periods [10]. 4 XCell Science, Novato, CA, USA These differences, while of importance to the academic 5 NxCell Inc, Novato, CA, USA community, would be largely irrelevant to the regulatory au- 6 Q therapeutics, Salt Lake City, UT, USA thorities and for the development of an allogeneic or Stem Cell Rev and Rep autologous product, provided these differences did not alter We have assumed that the regulatory authorities will con- the potency or efficacy of the differentiated cells that were sider a similar logic for other autologous products or HLA- derived from these lines [11]. Indeed, this concept has been matched products, including those derived from iPSC [12]. used for hematopoietic stem cell transplants where different Therefore, like cord blood, autologous or matched cells will donors (which differ in their allelic background and presum- be regulated differently than allogeneic products derived from ably in the efficacy) have been transplanted without showing iPSC. We further reasoned that if other groups wished to gen- that each sample was functionally identical by in vivo or erate new lines they could use the same process and the com- in vitro testing [12]. It was presumed that the cells were func- munity could define functional equivalence of these new lines. tioning normally in the donor and that the harvesting process Since the lines themselves are merely input material to make would not alter the cells (i.e. the cells were minimally manip- fully differentiated cells, we felt that no animal tests were ulated), and one could reasonably infer that no change had required at the iPSC stage; rather, criteria for use for further occurred. This concept was extended to sibling or related downstream processing could be established by in vitro dif- transplants and then to matched unrelated donors (MUD) ferentiation assays and agreed-on quality control (QC) criteria transplants. Here the inference was that one could reasonably for pluripotency. Functional characterization and equivalency extend the idea of functional equivalency even though the of the end product with any necessary in vivo or human stud- transplanted cells were functioning in a different setting. ies would occur on the final manufactured product. As with This concept of functional equivalence was extended to other products that may be used for autologous or allogeneic cord blood, which is used for the same purpose as bone mar- manufacture, we assume that the tests required will be differ- row but differs in that cord blood is processed differently from ent and the regulations likely different, but in both cases it will bone marrow and is considered more than minimally manip- be critical to have comparability data. ulated. The regulatory authorities reasoned that if functional Given most groups were initially focusing on allogeneic equivalence in in vivo studies showed that cells could be therapy, we initiated a program to generate clinically compli- manufactured reliably and reproducibly, then different groups ant cells and have reported on the generation of two such lines using different processes and manufacturing at different sites [1], which we presume will be used to generate a variety of could be approved under a Biologics License Application products from a MCB. Although the process development (BLA). Indeed, five public cord blood banks have been ap- was expensive and time consuming [1, 14], we reasoned that proved to provide MUD-type transplants for individuals using the cost would be amortized over a large number of patients a commonly accepted release criteria for functional equiva- [15]. Our data suggest that these lines could be used to com- lence. Given the extent of in vivo human data available, no mercialize iPSC-based cell therapy following a standard animal studies were required for the approval process. It is Investigational New Drug (IND) path. important to note that the regulatory authorities in the United However, it became evident that this was not a viable mod- States recognized that such licensure requirements should not el for autologous cells and haplobank-derived cells, as had be extended to autologous or related cord blood use, such as become clear with other cell therapies (see above). One would that proposed by private cord blood banks; indeed, those have to reduce cost and the regulators would need to develop banks are not subject to the same BLA licensure requirements. models akin to those they have developed for cord blood This logic has been extended to other autologous therapy banks. Therefore, we evaluated what would need to be done where cells are more than minimally manipulated, such as to reduce cost should cells be used for autologous therapy[13] autologous T-cells, B cells, dendritic cells, NK cells and mac- or if a Haplobank was established[16]. Moreover, since these rophages [12]. Each of the cell populations is manufactured in lines may be used by a number of individuals and utilized to a lot that is sufficient for one individual, and each lot is intrin- generate a number of different products – each of which will sically different from another lot and is transplanted in a host be likely manufactured in a different site by different compa- at different stages of illness, where the cells likely encounter nies – we reasoned that additional characterization may be different environments. The authorities have not required that necessary and that a database to monitor changes in cells in each lot undergo testing, as would be required for an alloge- culture needs to be established. In this manuscript, we de- neic product that would be used for hundreds or thousands of scribe the detailed characterization of two cGMP-compatible patients. Rather, they have asked people to demonstrate that iPSC lines using WGS, array-based analysis and aCGH SNP the end product obtained after processing is functionally analysis. One of these lines - LiPSC-GR1.1- generated during equivalent [12]. In some cases the authorities have required GMP manufacturing runs and the other line - LiPSC-ER2.2 - that eight or ten samples manufactured be shown to be effec- generated during engineering runs using the same GMP com- tive in an animal model, and in some cases have required patible process described before [1]. Our goal is to provide human safety studies of a limited nature [12].

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