GMP-Compliant Production of Autologous Adipose-Derived Stromal Cells in the NANT 001 Closed Automated Bioreactor

Abstract

In recent years mesenchymal stromal cells (MSCs) have received a great deal of interest for the treatment of major diseases, but clinical translation and market authorization have been slow. This has been due in part to a lack of standardization in cell manufacturing protocols, as well as a lack of biologically meaningful cell characterization tools and release assays. Cell production strategies to date have involved complex manual processing in an open environment which is costly, inefficient and poses risks of contamination. The NANT 001 bioreactor has been developed for the automated production of small to medium cell batches for autologous use. This is a closed, benchtop system which automatically performs several processes including cell seeding, media change, real-time monitoring of temperature, pH, cell confluence and cell detachment. Here we describe a validation of the bioreactor in an environment compliant with current good manufacturing practice (cGMP) to confirm its utility in replacing standardized manual processing. Stromal vascular fraction (SVF) was isolated from lipoaspirate material obtained from healthy donors. SVF cells were seeded in the bioreactor. Cell processing was performed automatically and cell harvesting was triggered by computerized analysis of images captured by a travelling microscope positioned beneath the cell culture flask. For comparison, the same protocol was performed in parallel using manual methods. Critical quality attributes (CQA) assessed for cells from each process included cell yield, viability, surface immunophenotype, differentiation propensity, microbial sterility and endotoxin contamination. Cell yields from the bioreactor cultures were comparable in the manual and automated cultures and viability was >90% for both. Expression of surface markers were consistent with standards for adipose-derived stromal cell (ASC) phenotype. ASCs expanded in both automated and manual processes were capable of adipogenic and osteogenic differentiation. Supernatants from all cultures tested negative for microbial and endotoxin contamination. Analysis of labor commitment indicated considerable economic advantage in the automated system in terms of operator, quality control, product release and management personnel. These data demonstrate that the NANT 001 bioreactor represents an effective option for small to medium scale, automated, closed expansion of ASCs from SVF and produces cell products with CQA equivalent to manual processes.

Keywords: GMP—good manufacturing practice; autologous; automation; bioreactor; mesenchymal stromal cells.

FIGURE 1

Bioreactor design. (A) The closed, automated NANT 001 bioreactor with thermostatically controlled compartment, touch screen interface, barcode reader and with single use fluid reservoirs attached. (B) Configuration of the single use components of the bioreactor, comprising of a cell culture flask, reservoirs for storage of media, wash buffer, cells prepared for seeding, detaching agent (typically an enzyme preparation), bag for waste fluids and receptable for collection of the expanded cell population. (C) Fluidics design of the NANT 001 bioreactor system incorporating a sterile, single-use cell-culture chamber with a tilt and shake mechanism connected in a closed system to a series of bags containing complete culture medium, wash buffer (WB), detaching agent, cell suspension for seeding, a waste bag and a harvesting bottle. Continuous monitoring of temperature and pH is achieved by the inclusion of sensors. Cell morphology and confluence are monitored using an integrated, self-operating and auto-focusing microscope.

FIGURE 2

Flowchart describing the process steps and testing involved from donor qualification to cryopreservation of ASCs. ASC, adipose-derived mesenchymal stromal cells; AT, adipose tissue; GMP, good manufacturing practices; SVF, stromal vascular fraction.

FIGURE 3

Expansion characteristics of ASCs. Representative phase contrast images of cell morphology at (A) 50% and (B) 90% confluency respectively for automated and manual processes. (C) Expansion characteristics of cells from automated and manual processes for each validation run. Scale bar = 100 µm.

FIGURE 4

QC analysis. (A) Flow cytometry immunophenotyping of ASCs expanded in automated or manual processes over three validation runs (n = 3). Expression is shown as percentage positive cells. Results are expressed as mean ± SEM. (B) ASCs expanded in both automated and manual cultures tested negative for microbial contamination. Endotoxin concentration was assessed in automated cultures only and was <1 EU/ml for all. (C) ASCs expanded using the automated and manual processes were capable of undergoing adipogenic differentiation for all three validation runs, representative images of oil red O staining in automated and manual process test samples and a control (non-differentiated) sample are shown. Semi-quantification of oil red O staining by measurement of absorbance at 520 nm was performed, results are expressed as mean ± SEM. Magnification = ×10, scale bar = 200 µm. (D) ASCs expanded using the automated and manual processes were capable of undergoing osteogenic differentiation for all three validation runs, a representative image of alizarin red staining in automated and manual process test samples and a control sample are shown. Calcium quantification was performed for each validation run, results are expressed as mean ± SEM. Magnification = ×10, scale bar = 200 µm. Calcium deposition was not detected in control wells (data not shown).

FIGURE 5

A cost-effectiveness analysis comparing the cost of a manual expansion process with an automated process using the NANT 001 system to produce 130 batches of an autologous ASC therapy per year. The following assumptions were applied: personnel consists of a production team of two manufacturing technicians, one QC technician and one Qualified Person (all FTE), one Class A/B cleanroom facility available and a production process of 7 ± 1 days. Upstream, downstream and all manual expansion processes would be performed in a Grade A/B cleanroom with the bioreactors operating in a Grade D area. All relevant manufacturing costs including direct and indirect fixed costs and direct variable costs for the expansion phase and all upstream and downstream processes were calculated and compared.

FIGURE 6

Labor commitment analysis comparing an open, manual expansion process with an automated expansion process using the NANT 001 system for an autologous therapy with a production process of 7 ± 1 days. Results are presented as hours of labor required per batch by a production team consisting of two manufacturing technicians, one QC technician and one Qualified Person for all upstream, downstream and expansion processes. The percentage reductions in hours of labor for the automated expansion process vs. the manual process are indicated.