Retrospective Analysis of Autologous Chondrocyte-Based Cytotherapy Production for Clinical Use: GMP Process-Based Manufacturing Optimization in a Swiss University Hospital

Abstract

Cultured autologous human articular chondrocyte (HAC) implantation has been extensively investigated for safe and effective promotion of structural and functional restoration of knee cartilage lesions. HAC-based cytotherapeutic products for clinical use must be manufactured under an appropriate quality assurance system and follow good manufacturing practices (GMP). A prospective clinical trial is ongoing in the Lausanne University Hospital, where the HAC manufacturing processes have been implemented internally. Following laboratory development and in-house GMP transposition of HAC cell therapy manufacturing, a total of 47 patients have been treated to date. The main aim of the present study was to retrospectively analyze the available manufacturing records of the produced HAC-based cytotherapeutic products, outlining the inter-individual variability existing among the 47 patients regarding standardized transplant product preparation. These data were used to ameliorate and to ensure the continued high quality of cytotherapeutic care in view of further clinical investigations, based on the synthetic analyses of existing GMP records. Therefore, a renewed risk analysis-based process definition was performed, with specific focus set on process parameters, controls, targets, and acceptance criteria. Overall, high importance of the interdisciplinary collaboration and of the manufacturing process robustness was underlined, considering the high variability (i.e., quantitative, functional) existing between the treated patients and between the derived primary HAC cell types.

Keywords: ATMP; GMP manufacturing; autologous chondrocyte implantation; cartilage defect; cell therapy; optimization; process controls; production process; standardized transplant product; technical workflows.

Figure 1

Schematic dual (i.e., general and detailed) multi-step process overview of the development and implementation steps for the considered HAC-based injectable cytotherapeutic products for ACI, within the context of the authorized clinical trial in the CHUV. ACI, autologous chondrocyte implantation; CHUV, centre hospitalier universitaire vaudois; CPC, cell production center; GMP, good manufacturing practices; HAC, human articular chondrocytes; MRI, magnetic resonance imaging; OTR, orthopedics and traumatology service; PCR, polymerase chain reaction; UTR, regenerative therapy unit.

Figure 2

Simplified organigram specifying the roles and the responsibilities of the different CHUV professional stakeholders involved in the internal ACI clinical trial (i.e., research laboratory, GMP manufacturing platform, orthopedic clinical unit) within the CHUV. Critical importance is set on the effective collaboration, communication, and coordination between all of the stakeholders. ACI, autologous chondrocyte implantation; API, active pharmaceutical ingredient; CHUV, centre hospitalier universitaire vaudois; CPC, cell production center; GMP, good manufacturing practices; OTR, orthopedics and traumatology service; QC, quality control; UTR, regenerative therapy unit.

Figure 3

Photographic illustrative overview of the sequential mechanical and two-step enzymatic cartilage biopsy processing phases, followed by in vitro monolayer HAC expansion. (A) Procurement of the healthy cartilage tissue biopsy (i.e., size of 4 mm × 10 mm). Scale bar = 1 cm. (B) Humidification of the cartilage tissue biopsy for further processing. Scale bar = 1 cm. (C) Manual fragmentation of the cartilage tissue biopsy into < 1 mm³ fragments. Scale bar = 1 cm. (D) Two-step digestion of the cartilage tissue biopsy fragments with pronase and with collagenase. Scale bar = 1 cm. (E) Verification of complete cartilage tissue biopsy fragment digestion after overnight incubation with the lytic enzymes. Scale bar = 1 cm. (F–I) Photographic illustrative overview of sequential monitoring timepoints during the in vitro monolayer HAC culture expansion (i.e., cells at passage level 2, with photographs taken after 24 h, 2 days, 4 days, and 7 days of culture, respectively). Scale bars = 150 µm. h, hours; HAC, human articular chondrocytes.

Figure 4

Original data from GMP manufacturing records relative to primary HAC (n = 47 cell types) isolation and manufacture for human investigational cytotherapeutic use. (A) Evolution of the numbers of biopsies performed between 2017 and 2021, quantitative data distribution for the obtained HAC cell counts after enzymatic biopsy processing, and quantitative data distribution for the obtained HAC relative cellular viability after biopsy processing for cell isolation. (B) Quantitative data distributions for the manufactured HACs relative to the endpoint harvested cell yields, the endpoint cell confluency levels, and the endpoint post-harvest relative cellular viability levels. (C) Quantitative data distributions for the manufactured HACs relative to the storage time-period between cell bank manufacture and finished product preparation, as well as the breakdown of the mean cell viability values at the time of initiation of HACs cryopreserved in Biofreeze^® medium or in CryoSOfree™ medium, respectively. A p-value < 0.05 was retained as a base for statistical significance determination. API, active pharmaceutical ingredient; GMP, good manufacturing practices; HAC, human articular chondrocytes.

Figure 5

Original data from GMP manufacturing records relative to primary HAC (n = 47 cell types) isolation and manufacture for human investigational therapeutic use. (A) Quantitative data distributions for the manufactured HACs relative to the evolutive cell confluency level assessments at the various timepoints (i.e., defined as the microscopic observation of cultures during the medium exchanges) within each in vitro culture expansion phase, as well as the total culture time periods for each of the considered in vitro culture expansion phases. (B) Quantitative unitary data distributions for the manufactured HACs relative to the used materials and to the obtained harvest cell yields within MCB manufacture, as well as to the used materials and to the obtained harvest cell yields within finished product manufacture. API, active pharmaceutical ingredient; GMP, good manufacturing practices; HAC, human articular chondrocytes; MCB, master cell bank.

Figure 6

Comparative quantitative results of functional parameter (i.e., chondrogenic gene evolutive expression levels) QC assays for the cellular APIs, outlining inter-patient variability. (A) Induction fold values for the Acan gene expression upregulation (i.e., using the ΔΔCT method) between day 1 (i.e., baseline) and day 16 (i.e., endpoint) of three-dimensional cell cultures in chemically induced chondrogenic conditions. (B) Induction fold values for the COL2A1 gene expression upregulation between day 1 and day 16 of three-dimensional cell cultures in chemically induced chondrogenic conditions. The quantitative data from the functional QC assay were presented for 42 patients. Several patients were excluded from the analysis due to a change in the chondrogenic medium composition. API, active pharmaceutical ingredient; QC, quality control.

Figure 7

Standardized parametric and controlled process overview for the obtention of the preliminary HAC cell population from the cartilage tissue biopsy. (A) Mechanical cartilage tissue dissociation process, in preparation for the enzymatic tissue treatment. (B) Two-step enzymatic cartilage tissue treatment for in vitro HAC cell dissociation. (C) In vitro isolation of the preliminary HAC cell population. The established CPPs, KPPs, IPCs, and PPCs are further defined in the supplementary document–Process Parameters: Table SPP1. API, active pharmaceutical ingredient; CPP, critical process parameter; HAC, human articular chondrocytes; IPC, in-process control; KPP, key process parameter; PBS, phosphate-buffered saline; PPC, post-process control.

Figure 8

Standardized parametric and controlled process overview for the obtention of the MCB from the preliminary HAC cell population. (A) Initial in vitro culture expansion of the preliminary HAC cell population for the obtention of the cell seed. (B) Secondary in vitro culture expansion of the cell seed for the obtention of the cells used in MCB batch constitution. (C) Harvest and cryopreservation of the obtained cells for the establishment of the MCB. The established CPPs, KPPs, IPCs, and PPCs are further defined in the supplementary document–Process Parameters: Table SPP2. API, active pharmaceutical ingredient; CPP, critical process parameter; HAC, human articular chondrocytes; IPC, in-process control; KPP, key process parameter; MCB, master cell bank; PPC, post-process control; QC, quality control; RH, relative humidity.

Figure 9

Optimized parametric and controlled process overview for the obtention of the WCB from the MCB. (A) Culture initiation of MCB materials for in vitro cell expansion. © CHUV-CPC. (B) Single in vitro cell expansion. (C) Harvest and cryopreservation of the obtained cells for establishment of the WCB. The established CPPs, KPPs, IPCs, and PPCs are further defined in the supplementary document–Process Parameters: Table SPP3. API, active pharmaceutical ingredient; CHUV, centre hospitalier universitaire vaudois; CPC, cell production center; CPP, critical process parameter; HAC, human articular chondrocytes; IPC, in-process control; KPP, key process parameter; MCB, master cell bank; PPC, post-process control; QC, quality control; RH, relative humidity; WCB, working cell bank.

Figure 10

Standardized parametric and controlled process overview for the obtention of the finished product from the MCB or the WCB. (A) Culture initiation of MCB/WCB materials for the final in vitro cell expansion. © CHUV-CPC. (B) Single in vitro cell expansion. (C) Harvest and formulation of the obtained cells for the obtention of the finished product. The established CPPs, KPPs, IPCs, and PPCs are further defined in the supplementary document–Process Parameters: Table SPP4. API, active pharmaceutical ingredient; CHUV, centre hospitalier universitaire vaudois; CPC, cell production center; CPP, critical process parameter; HAC, human articular chondrocytes; IPC, in-process control; KPP, key process parameter; MCB, master cell bank; PPC, post-process control; QC, quality control; RH, relative humidity; WCB, working cell bank.