Risk-based framework for GMP-compatible low-density platelet lysate-supplemented expansion of human cartilage cells for tissue repair

Advanced therapy medicinal products (ATMPs) require robust, GMP-compliant manufacturing processes and well-defined critical quality attributes (CQAs) to ensure safety, reproducibility, and clinical applicability. Chondrocyte-based therapies represent a promising approach for cartilage repair, yet their translation into clinical practice is limited by challenges in scalable cell expansion and preservation of chondrogenic potential. This study aimed to develop a translational, GMP-compatible, risk-based framework for the expansion of human chondrocytes enriched in progenitor cells using human platelet lysate (hPL). Chondrocytes were expanded under low-density conditions in media supplemented with two different hPL formulations. Proteomic characterization of hPL batches was performed using high-resolution mass spectrometry followed by bioinformatic analyses to identify differentially abundant proteins and enriched pathways. Cellular phenotype was assessed through proliferation assays, flow cytometry, and marker expression analysis. Chondrogenic potential was evaluated using three-dimensional spheroid cultures, followed by histological and immunohistochemical analyses. A risk-based manufacturing strategy was implemented using Failure Modes and Effects Analysis (FMEA) to identify critical process parameters (CPPs) and associated risks. Proteomic analysis revealed significant differences in protein composition between hPL formulations, although both supported efficient cell expansion. Chondrocytes maintained a low immunogenic profile and expressed progenitor-associated markers, including CD146 and CD166, particularly under low-density culture conditions. Spheroid formation demonstrated preserved chondrogenic capacity, with optimal extracellular matrix deposition observed at a seeding density of 200,000 cells per well. Both hPL conditions supported type II collagen production, while differences were observed in glycosaminoglycan and type I collagen content. Risk analysis identified critical steps. Identification of targeted mitigation strategies supported the definition of CQAs linked to CPPs. This study establishes a GMP-compatible, risk-based framework for the expansion of chondrocyte-based ATMPs. The integration of biologically optimized culture conditions with a structured risk assessment approach enabled the generation of functionally competent cartilage-forming cells. These findings support further preclinical validation and contribute to the development of scalable and clinically applicable therapies for cartilage repair in patients with early osteoarthritis.