Piloting a scale-up platform for high-quality human T-cells production

Abstract

Cell and gene therapies are an innovative solution to various severe diseases and unfulfilled needs. Adoptive cell therapy (ACT), a form of cellular immunotherapies, has been favored in recent years due to the approval of chimeric antigen receptor CAR-T products. Market research indicates that the industry's value is predicted to reach USD 24.4 billion by 2030, with a compound annual growth rate (CAGR) of 21.5%. More importantly, ACT is recognized as the hope and future of effective, personalized cancer treatment for healthcare practitioners and patients worldwide. The significant global momentum of this therapeutic approach underscores the urgent need to establish it as a practical and standardized method. It is essential to understand how cell culture conditions affect the expansion and differentiation of T-cells. However, there are ongoing challenges in ensuring the robustness and reproducibility of the manufacturing process. The current study evaluated various adoptive T-cell culture platforms to achieve large-scale production of several billion cells and high-quality cellular output with minimal cell death. It examined factors such as bioreactor parameters, media, supplements and stimulation. This research addresses the fundamental challenges of scalability and reproducibility in manufacturing, which are essential for making adoptive T-cell therapy an accessible and powerful new class of cancer therapeutics.

Keywords: T-cell; adoptive cell therapy; biaxial rotary bioreactor; bioprocessing; scale-up; stirred-tank bioreactor.

FIGURE 1

Human T-cell expansion timelime. T-cells were stimulated on day 0 using ImmunoCult™ Human CD3/CD28/CD2 T-cell Activator in ImmunoCult™-XF T-cell Expansion Medium supplemented with IL-2 in static culture. Total culture volumes were increased by 8-fold using fresh medium on day 3. Total culture volumes were further increased by 4-fold on day 5 and transferred to the respective bioreactor systems. Basal media was added to the cultures by 4-folds on day 7 and day 10 until harvest on day 12 of the process.

FIGURE 2

T-cell growth kinetics in bioreactors. The growth of human T-cells from independent donors was compared against the bioreactors over 9 days is displayed. Cells were seeded equally for three healthy donors with high viability (∼98%). All donors had variable expansion profiles across the bioreactors. Appliflex and WAVE bioreactors showed elevated fold expansion (fold change: 31–36) compared to the static control on harvest. Tisxell bioreactor had only a marginal increase due to early growth saturation for Donor 1. All bioreactors for Donor two were uniformly expanded (fold change ∼20) relative to the control T-flasks. There was no significant growth improvements for Donor three in comparison to the static cultures. Data are shown as mean ± SD. Statistical significance is indicated in the plots if applicable, p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***) or p ≤ 0.0001 (****).

FIGURE 3

pH and dissolved oxygen (DO) trends under various bioreactors. The Applikon and Wave reactors had automatic process control of pH and DO (gas sparging). The pH profiles aligned with the viable cell concentration and with the exhaustion and yield of metabolites. DO remains stable >95% throughout the culture duration while matching the oxygen demand for cell growth. The subtle spikes observed in both pH and DO are due to media addition on respective days. Data are shown as mean ± SD.

FIGURE 4

Metabolite concentrations for the bioreactors. Levels of glucose, glutamine, lactate and ammonia were monitored daily. Glucose and glutamine consumption correlates well with the cell growth kinetics data. Lactate and ammonia production reflects the expansion cell growth trend. Lactate generation peaked from Day 7 followed by rapid decline of glucose and glutamine. Data are shown as mean ± SD.

FIGURE 5

Phenotype characterization of T-cells. (A) T-cell differentiation Surface markers, (B) Exhaustion markers and (C) Intracellular cytokines. T-cells subpopulations are analyzed pre-transfer and post-harvest on all culture systems. Cell populations are gated based on isotype controls. Cell quality was assessed based on CD4⁺ and CD8⁺ T-cell subpopulations. (A) CD4 to CD8 T-cell ratio was taken as a quality indicator of the final yields. Ratio (CD4:CD8) was higher (5:1) before transfer and reduced (2:1) as the expansion progressed. Expression profiles of the memory cell types had increased progressively for all donors. (B) Potential T-cell exhaustion was assessed in the harvested cell products. The upregulated inhibitory markers (PD-1 and LAG-3) on pre-transfer indicate the poor proliferation abilities of the cultivated T-cells. It was noted that collective suppressive markers decreased significantly along with the cell expansion. (C) T-cell function was evaluated by measuring the levels of intracellular cytokines. T-cells secreted high amounts of pro-inflammatory (IL-2 and IFN-γ) and anti-inflammatory (IL-4) cytokines upon stimulation (pre-transfer). Harvested T-cells had greatly reduced background levels across all donors. Data are shown as mean ± SD.