GMP Production and Scale-Up of Adherent Neural Stem Cells with a Quantum Cell Expansion System

Abstract

Cell-based therapies hold great promise for a myriad of clinical applications. However, as these therapies move from phase I to phase II and III trials, there is a need to improve scale-up of adherent cells for the production of larger good manufacturing practice (GMP) cell banks. As we advanced our neural stem cell (NSC)-mediated gene therapy trials for glioma to include dose escalation and multiple treatment cycles, GMP production using cell factories (CellStacks) generated insufficient neural stem cell (NSC) yields. To increase yield, we developed an expansion method using the hollow fiber quantum cell expansion (QCE) system. Seeding of 5.2 × 10⁷ NSCs in a single unit yielded up to 3 × 10⁹ cells within 10 days. These QCE NSCs showed genetic and functional stability equivalent to those expanded by conventional flask-based methods. We then expanded the NSCs in 7 units simultaneously to generate a pooled GMP-grade NSC clinical lot of more than 1.5 × 10¹⁰ cells in only 9 days versus 8 × 10⁹ over 6 weeks in CellStacks. We also adenovirally transduced our NSCs within the QCE. We found the QCE system enabled rapid cell expansion and increased yield while maintaining cell properties and reducing process time, labor, and costs with improved efficiency and reproducibility.

Keywords: GMP; HB1.F3.CD; adherent cells; bioreactor; clinical grade; manufacturing; neural stem cells; quantum cell expansion system.

Figure 1

Lactic Acid Monitoring of Run A and Characterization of CD-NSCs that Were Propagated in the QCE System (A) Lactic acid concentrations in culture media collected from CD-NSCs grown using the QCE system (run A). Lactic acid levels were maintained at 8–12 mmol/L by increasing the feed rate in the QCE system to limit metabolic stress during the propagation of CD-NSCs. (B) Comparison of QCE-grown or flask-grown CD-NSCs expressing the cell identity marker human nestin (red bars) and CD transgene (blue bars), as assessed by flow cytometry. Samples were run in triplicate (percent positive cells, mean ± SD: 99.94% ± 0.08% [nestin]; 93.75% ± 1.3% [CD]). These identity tests are part of the cell bank release criteria for CD-NSCs.

Figure 2

QCE-Based Production of a Clinical Bank of CD-NSCs under GMP Conditions (A) Schematic of production flow for CD-NSCs manufactured under cGMP (ISO-7) conditions. (B) Lactic acid levels for CD-NSCs for 36–171 hr after cell loading in 7 QCE units that were used to generate a pooled clinical cell bank. Multiple t test of repeated treatments showed no statistical differences among lactic acid levels for the 7 reactors.

Figure 3

Adenoviral Transduction of NSCs Using the QCE System CD-NSCs were transduced with Adv.hCE1m6 at MOIs of 13.2 and 20 (runs E and F, respectively) on day 7 and harvested the next day. Percent cell recovery (91.12% ± 10.2%), viability (83.1% ± 0.74%), and cell identity test, CD expression (94.3% ± 1.6%) are shown (values mean ± SD). Cell products from runs E and F passed the cell bank release criteria of recovery (>80%), viability (>70%), and CD expression (>70%).

Figure 4

Functional CE Activity of CE-NSCs CD-NSCs were transduced with adenovirus-hCE1m6 at MOIs of 13.2 and 20 (runs E and F, respectively) on day 7 and harvested the next day. Functional CE activity of CE-NSCs expressing the transgene protein hCE1m6 from days 2 to 4 after thaw in culture is shown (CE activity, mean ± SD: 749 ± 28 [run E] and 882.1 ± 56.7 [run F] on day 4). The CE activity levels of both cell products corresponded to MOIs used during transduction. Both runs yielded cell products that passed the cell bank release criteria for CE expression of 300 nmol/min/mL on day 4.