Compliance with Good Manufacturing Practice in the Assessment of Immunomodulation Potential of Clinical Grade Multipotent Mesenchymal Stromal Cells Derived from Wharton's Jelly

Abstract

The selection of assays suitable for testing the potency of clinical grade multipotent mesenchymal stromal cell (MSC)-based products and its interpretation is a challenge for both developers and regulators. Here, we present a bioprocess design for the production of Wharton's jelly (WJ)-derived MSCs and a validated immunopotency assay approved by the competent regulatory authority for batch release together with the study of failure modes in the bioprocess with potential impact on critical quality attributes (CQA) of the final product. Methods: The lymphocyte proliferation assay was used for determining the immunopotency of WJ-MSCs and validated under good manufacturing practices (GMP). Moreover, failure mode effects analysis (FMEA) was used to identify and quantify the potential impact of different unexpected situations on the CQA. Results: A production process based on a two-tiered cell banking strategy resulted in batches with sufficient numbers of cells for clinical use in compliance with approved specifications including MSC identity (expressing CD73, CD90, CD105, but not CD31, CD45, or HLA-DR). Remarkably, all batches showed high capacity to inhibit the proliferation of activated lymphocytes. Moreover, implementation of risk management tools led to an in-depth understanding of the manufacturing process as well as the identification of weak points to be reinforced. Conclusions: The bioprocess design showed here together with detailed risk management and the use of a robust method for immunomodulation potency testing allowed for the robust production of clinical-grade WJ-MSCs under pharmaceutical standards.

Keywords: cell culture; cellular therapy; good manufacturing practice; immunomodulation; multipotent mesenchymal stromal cell; proliferation assay; quality by design.

Figure 1

Schematic of bioprocess and established in-process controls (IPCs) for the follow up of the critical quality attributes for starting material and drug product. Although initial setup included an intermediate working cell bank, the competent regulatory authority allowed the possibility to expand drug product (DP) directly from the master cell bank (MCB). UC: Umbilical cord; WJ-MSC: Wharton jelly–mesenchymal stromal cells; IPC: In-process controls; Ph: Pharmacopea.

Figure 2

Immunophenotypic analyses of eight batches of clinical-grade WJ-MSC. The resulting values were always higher than 95%, in accordance with the established product specifications: 99.9 ± 0.2% CD45⁻/CD105⁺(■), 99.8 ± 0.2% CD31⁻/CD73⁺(▲), 99.7 ± 0.3% CD90⁺ (▼), 99.1 ± 0.6% HLA-DR⁻(♦).

Figure 3

Results from lymphocyte proliferation assays (potency) in eight batches of clinical-grade WJ-MSC. Bright field microscopy images revealed clumping of peripheral blood MNC after 5 days in the of presence of 25 ng/mL PMA and 0.5 µM ionomycin (A), as opposed to same cells in co-culture with WJ-MSCs showing a dramatic decrease in the presence of such cell clumps (B). Values (in %) of the inhibition of the proliferation of activated lymphocytes are shown in (C). Scale bars = 100 µm.

Figure 4

Risk analysis. (A) A Pareto chart was generated and plotted according to the classification and the failure group. (B) Graphical representation of the impact of risks from each failure group on critical quality attributes. The graphical view of the risk analyses for each of the failure groups provided invaluable assistance to focus efforts in the mitigation of critical risks affecting the specifications of MSCs in the final product. This methodology permits to streamline the identification of the weakest points of the process that deserve the implementation of further in-process controls or to preserve existing ones. MCB: Master cell bank; WCB: Working cell bank; DP: Drug product; FP: Final product; RPN: Risk priority number.