Validation of GMP Lentiviral Vector Production for CAR T-Cell Therapy in the Spanish Public Health System: Development of New QC Required By Aemps (Spanish FDA)

The production of Lentiviral Vectors (LV) for academic CAR T-cell therapies under Good Manufacturing Practices (GMP) is the main bottleneck within the production process. We have developed a GMP facility (ViPro-IBIS) according to the AEMPS (“Spanish Drug and Health Products Agency”), including:

- Facilities and equipment: grade qualifications (main production room and changing rooms), equipment qualifications, validation of the cleanliness of the qualified facilities as well as validation of the entry of materials into them.

- Production processes: validation of Master Cell Bank (MCB) and Working Cell Banks (WCB) production of packaging cells (HEK293T “Lenti-X”), validation of LV production and validation of media fill (MF) processes (3 consecutive successful productions in all cases).

All the mentioned validations have been successfully completed, complying with all the requirements and quality controls (QC) requested by the AEMPS. Of these, some have been requested for the first time (highlighted in italics), which has required an extra effort of fine-tuning:

• MCB and WCB production: microbiological and particle process monitoring; cell viability ≥ 70% and ≥ 170x10⁶ total live cells achieved (cell viability ≥ 94% in all cases, total live cells achieved: MCB = 352,5x10⁶, WCB1 = 170x10⁶, WCB2 = 267,1x10⁶, WCB3 = 310,5x10⁶); sterility; absence of mycoplasma; absence of endotoxins; cell identity by DNA Fingerprint (Short Tamdem Repeat (STR), only for MCB); analysis of genetic stability; cell duplication calculation (only for WCB); analysis of the cytopathic effect with VERO, MRC-5 and RD cell lines for at least 14 days; analysis of human adventitious viruses by PCR and RT-PCR; analysis of porcine viruses by PCR and RT-PCR (only for MCB); control of endogenous retroviruses by detection of reverse transcriptase activity (only for MCB).

• LV production: microbiological and particle process monitoring; cell viability ≥ 70% (≥ 90% in all cases); sterility; absence of mycoplasma; absence of endotoxins; functional LV titer measured by FACS ≥1x10⁷ TU/mL (LV1 = 3.07x10⁸ TU/mL, LV2 = 9.95x10⁷ TU/mL, LV3 = 1.66x10⁸ TU/mL); analysis of DNA (by digital PCR) and protein (by WB) residues from packaging cells and residual DNA from plasmids (by digital PCR) used in the production process. All LV batches were packed in bags with 250x10⁶+10% lentiviral particles diluted in TexMACS™ GMP Medium (Miltenyi), in accordance with the specifications. Moreover, the LV stability was set at at least one year.

Currently, we have submitted the application for AEMPS inspection, which we expect to occur during the second semester of 2024. Thus, GMP LV production at ViPro-IBIS represents a robust platform within the Spanish public health system for CAR T-cell therapy clinical trials, allowing to provide LV in a more rapid and affordable way for the trials approved by the AEMPS in the region/country (including ours: SPCD19M-CAR for lymphoma and CARTemis-1 (BCMA CAR) for myeloma). Our platform may serve as a model for improvement for CAR T-cell treatments for the patients.

No relevant conflicts of interest to declare.