Automation of cellular therapy product manufacturing: results of a split validation comparing CD34 selection of peripheral blood stem cell apheresis product with a semi-manual vs. an automatic procedure

The promise of automation of cell therapy processing is several-fold. A closed system might allow the process to be performed in a less strictly controlled environment than currently (class A in B or 100 in 1000), or for several processes to proceed concurrently within the same room of a cell factory. Automation is expected to be labor saving, and specifically for very complex processes it might add stability and convenience, for instance with respect to the timing of sequential process steps (especially at inconvenient hours). With respect to product/process quality, improved stability/predictability of outcomes was posited as a likely outcome; experience thus far does not unequivocally support this expectation [1–3]. Before automats will be programmed to perform very complex processes consisting of a sequence of orchestrated process steps, such as washes, stimulations, incubations, selections, etc., the individual building blocks must be programmed and put to the test. Moreover, for cost effectiveness of the automats, customers may expect to perform all standard processes on one device. Therefore, an immunomagnetic selection module was generated for the cell processing automat CliniMACS Prodigy (referred to as “Prodigy” throughout the remainder of the manuscript) to copy the CD34 “normal scale” selection module for the established semi-automatic CliniMACS Plus System, although performance of CliniMACS Plus is excellent, highly robust and not particularly challenging nor time-consuming for cellular therapy laboratories. The specific technical challenge of the overall relatively simple CD34 selection process is the near-homogeneous enrichment of stem cells with minimal contamination by non-target cells that can be achieved with magnet, column and reagent, components which are shared by both systems. The number of contaminating cells largely represents the device’s efficiency at flushing unwanted cells from the complex tubing systems and less so, non-specific immunomagnetic retention. Previous analyses comparing outcomes of CD34 selections with the “large-scale” process on Prodigy versus on CliniMACS Plus had suggested greater non-target cell reduction in CliniMACS Plus products [4, 5]. In light of these data, the manufacturer re-designed the process on Prodigy with the aim to further reduce non-target cells. At the same time, the process became scalable, as the “normal-scale” module was introduced which uses only one vial of CliniMACS CD34 reagent. This allows for magnetic labelling at reduced material costs for smaller cell products of not more than 600 × 10⁶ CD34+ cells and 60 × 10⁹ WBCs. The “normal scale” Prodigy process was tested here for the first time. By performing split validations, where cells from the same G-CSF mobilized apheresis product were concurrently manipulated with both devices, product variables affecting outcomes were controlled and direct comparison of process outcomes was for the first time facilitated. The quality of the processes is described by feasibility, process time and labor consumption, as well as CD34 recovery and the efficiency of immune cell depletion. The manuscript presents several novelties about the automatic Prodigy CD34 selection process, albeit each one modest, namely, split validation, first reported use of the re-designed “LP-34 Enrichment” process and first reported use of the “normal scale” module.

The fact that only “super-donors” were eligible for cell donations for this study implies comparatively high CD34+ cell frequencies and thus relatively favourable T-cell to CD34+ cell ratios in the apheresis products. Considering how –log T-cell depletion is calculated, the observed T-cell reduction with both systems is therefore quite satisfactory.

Work by Stroncek et al. [5] as well as our own previous work [4] with the “large-scale” CD34+ cell selection process had suggested slightly less efficient T-cell reduction on Prodigy than might have been expected based on clinical products processed with CliniMACS Plus, but as mentioned above, systematic differences between clinical and validation apheresis products did not allow for definitive conclusions. Our split validation now confirms statistically significantly less efficient immune cell depletion with the automatic device, although, as mentioned above, products meeting specification are generated by both devices. Center specific acceptance criteria for clinical stem cell products may be different from those used for this study and because of higher desired stem cell doses or lower tolerance for T-cells may require T-cell frequencies of less than 0.1 % among CD34+ cells (10 × 10⁶ CD34+/kg, 10 × 10³ T-cells/kg). 3 of 3 and 2 of 3 runs fulfil these more stringent requirements for CliniMACS Plus and Prodigy respectively, requiring a T cell depletion of more than 4.2 log with the CD34 and T cell frequencies of the leukapheresis products used in this study. Non-selected clinical stem cell products may differ considerably in composition, [9] possibly affecting separation performance additionally to impacts by design and tubing set of the system used for processing. In the largest published work to this topic, above-mentioned 4.2 logs T-cell depletion was achieved in 75 % of 139 CliniMACS Plus CD34 selected grafts [9]. With respect to generating CD34-selected stem cell products, larger studies will be required to evaluate whether the automat Prodigy results in a more consistent T cell depletion of more than 4.2 log than the currently used semi-automat CliniMACS Plus, although available data suggests that with the current software and consumable it might not. The lineage distribution of non-CD34+ cells in the final product (TARGET) differed somewhat from the starting population (ORI), in that T-cells were relatively more efficiently depleted (frequency among non-CD34+ MNCs threefold lower than in TARGET vs. in ORI) than B-cells (twofold higher) and monocytes (one-third higher), but with no striking differences between the selection methods. Thus Prodigy products contained approximately fivefold, fourfold and sevenfold greater numbers of residual T-cells, B-cells and monocytes than CliniMACS Plus products, i.e. non-target cell contamination was not specific or preferential for one lineage. While Prodigy does not reduce the total process time over CliniMACS Plus (on the contrary), the hands-on operator time is reduced by almost 2 h. Moreover, the Prodigy process could run unsupervised or supervised remotely over-night so that cells could be generated during unfavourable working hours. Thus selections could be routinely started on the apheresis day as opposed to the day thereafter, yielding a several hours fresher product. The earlier release of these products would then allow for transportation to transplant centers for same-day infusion. Avoidance of unfavourable working hours will become a more relevant issue with complex cell manipulation processes, as is already apparent with the automated process for enrichment of virus specific T cells [1] which no longer requires manual addition of peptide reagent between midnight and 2 am and will be even more accentuated as longer multi-step processes are developed on the automat. Automation of CD34+ cell selection also optimizes personnel and clean-room utilization, with the automat taking over the “second (unsupervised) shift” and by concurrently operating several automats within the same clean room. While few centers will generate sufficient CD34+ cell products to render this issue relevant for the process tested here, the same advantages will similarly apply to other, frequently requested cell products as may be established with the advent of T-cell products re-targeted against cancer antigens, as well as the availability of automatic systems may encourage centralization of cell selection processes.

Downsides, clearly, are the apparent superiority of the semi-manual process with respect to non-target cell contamination and the difficulties rescuing a partly processed product in case of a system failure.

This is the first report on performing the “normal scale” CD34 selection with the “LP-34 Enrichment” process on the Prodigy. The Prodigy is unconditionally suitable to perform the CD34 selection process, in that all validation products met the pre-defined specification of the cell product “G-CSF mobilized allogeneic HPCs, CD34 selected”, a marketing authorization for which has been issued to several cell therapy laboratories in Germany, including the one the authors are affiliated with. Recovery of target cells was equal to that reported for the semi-automatic method. Final product content of potentially allo-reactive T-cells, as well as B-cells and monocytes was statistically significantly higher. Prodigy reduces operator time by approximately half and at least theoretically allows unsupervised, e.g. nocturnal, operation (although may be limited by liability issues). Although an automat like Prodigy is simple enough for any transplant unit to use (and a clean room facility may be dispensable for its operation), the quality control (residual T-cell quantification) is quite demanding and should be performed in centers of excellence. World-wide shipping from a very small number of centralized manufacturing sites is conceivable but may not be cost-effective.