Quality System
Creatio Quality System operates under the current GMP regulations and is authorized by the Spanish Agency of Medicines and Medical Devices (PE010-1570, AEMPS, Spain). A specific organizational chart is employed at Creatio. Functions and responsibilities of each staff member of the facility are clearly defined and documented. Also, a specific document management system is implemented at Creatio and includes: risk assessment approaches, change control documentation, incidents and nonconformities records, calibration, validation and qualification annual strategy, audit programs, personnel training programs, preventive maintenance systems, reagents and starting materials record and traceability, quality control management, manufacturing management, batch certification and release system.
Facility and equipment
Creatio is an academic facility of the University of Barcelona dedicated to the validation and manufacturing of ATMPs for their use in phase I/II clinical trials. The facility has 4 B Grade cleanrooms in which all procedures can performed in a Grade A laminar flow cabinet. MCB and WCB of the HEK293T cell line (CRL-11,268; ATCC, Mansassa, VA, USA; obtained under a License Agreement between The Rockefeller University and Hospital Clínic de Barcelona; October 17, 2016) were handled in the cleanroom dedicated to cell therapy. The access to the cleanroom consists of an entrance to the pre-dressing room (Grade D), a pre-dressing room (Grade C), a dressing room (Grade B), and a distributor (Grade B) (Fig. 1). Adjacent rooms are at different pressures (Δ10–15 Pa between rooms) reaching 50 Pa in the Grade B cleanroom. Creatio facility has two storage rooms (Grade D) and a Grade D product conditioning room. Pressure, temperature, and humidity are continuously monitored and regulated by a specific control software (Controlli Delta Spain, Barcelona, Spain). The facility is validated once a year according to specific procedures of validation management. Equipment is validated and calibrated according to a pre-determined annual planning. Parameters of incubators, refrigerators, freezers, ultra-freezers, nitrogen tanks, and cell incubators CO2 levels are continuously monitored by a specific control software (Sirius, Nirco, Rubí, Spain).
HEK293T source and cell line specifications
The HEK293T cell line, which expresses the Simian Virus 40 (SV40) large antigen T, was generated at the Rockefeller University from the HEK293 cell line. According to the certificate of analysis, the product obtained from the ATCC had a passage number of 17. In addition, the certificate of analysis of the HEK293T cell line reports the results of microbiological quality control tests, Short Tandem Repeats (STRs) analysis, and PCR-based assays for human pathogenic viruses: HIV, HepB, HPV, EBV, CMV. This information was documented and used as a starting point of our process, and no further manipulations were performed between cell line purchase and its use for cell banking.
Cell line identification
The following STR profile of the HEK293T cell line was analyzed: Amelogenin: X,CSF1PO: 11, 12; D13S317: 12, 14; D16S539: 9, 13; D5S818: 8, 9; D7S820: 11; THO1: 7, 9.3; TPOX: 11; vWA: 16, 18, 19.
HEK293T cell culture
HEK293T cells were incubated for 2 min at 37 °C in a thermoblock and quickly transferred to a tube containing DMEM (Thermo Fisher Scientific, Walthman MA, USA) supplemented with 10% of Fetal Bovine Serum (FBS) (Thermo Fisher Scientific). Subsequently, cells were centrifuged (1250 rpm, 5 minutes), supernatant was removed, and cells were resuspended in fresh cell culture medium for Trypan blue-based cell counting. Cells were seeded in a sterile and pyrogen-free flask at a density of 1 × 106 cells x 25 cm2 and incubated at 37 °C/5% CO2 for 3–4 days until cells reached 80% confluence. For cell passaging, spent culture medium was removed, and cells washed with Phosphate-Buffered Saline (PBS) (Thermo Fisher Scientific). TrypLE™ (Thermo Fisher Scientific) was added and incubated for 1 min to detach cells from the flask. Once detached, cells were transferred to a tube containing fresh medium and centrifuged (1250 rpm, 5 min). Cells were counted, centrifuged again (1250 rpm, 5 min) and seeded at a density of 1·106 cells·25 cm2 in as many flasks of 25 or 75 cm2 as necessary. Cell passages were made to reach more than 200·106 cells for the MCB and 1000·106 for the WCB.
HEK293T cryopreservation
Once the required cell number was reached, cells were centrifuged (1250 rpm, 5 min) and the pellet was resuspended in cryopreservation medium to achieve a density of 10·106 cells/ml for MCB and 20·106 cells/ml for WCB. Cryopreservation medium was composed of 95% of cell culture medium (10% FBS-supplemented DMEM) and 5% of DMSO (Scharlab, Setmenat, Spain). Cryotubes were properly labelled and filled with 1 ml of the required concentration. Cryotubes were transported from the cleanroom to the cryopreservation area and temperature was monitored during transport. Cryopreservation was carried out in a controlled-rate freezer (Criomed™, Thermo Fisher Scientific) following a freezing program that lowers the temperature 1 °C/minute. The resulting cryotubes were then transferred to a gas phase nitrogen tank at -180 °C ± 20° C.
Personnel training and validations
Creatio adopts an ongoing personnel training program. Manufacturing staff is also assessed and validated once every 6 months with simulation tests of ongoing production processes. Personnel involved in the production of MCB and WCB undertook three consecutive simulation tests before starting the manufacturing process. Tryptic Soy Broth medium (TSB; Becton Dickinson, Madrid, Spain) was used to carry out simulation tests. Simulation tests were designed considering all steps of the process and emphasizing the worst-case scenarios, and were carried out with the same materials and equipment used to manufacture the MCB and WCB. During simulation tests, the presence of airborne microorganisms and particles was continuously monitored according to GMP standards. To verify the maintenance of the aseptic conditions during the simulation process, sampling points were set at the beginning and at the end of the process (negative control and simulation of the final product, respectively).
Environmental monitoring
A laser particle counter (CLiMET, Redlands, CA, USA) was used to monitor airborne particles in the cabinet (Grade A) during the manufacturing process and during simulation tests. According to the current GMP regulations, the maximum permitted airborne particle concentrations in Grade A areas are as follows: particle size equal or greater than 0.5 µm, 3520 particles per m3; particle size equal or greater than 5 µm, 20 particles per m3 [15].
Settle plates (Becton Dickinson) were used to monitor the presence of airborne microorganism (bacteria and fungi) in the cabinet (Grade A) during the manufacturing process as well as during the simulation tests. In addition, glove prints (finger dabs) of Grade A personnel were sampled for microbiological analysis (Becton Dickinson) at the end of each working day. In accordance with current GMP regulations, acceptance criteria of less than 1 colony forming units (> 1 CFU/gloves and > 1 CFU/plates) were established [15].
Animal origin-derived products and adventitious viruses risk assessment
In accordance with GMP regulations, the use of animal products in the manufacturing process should be avoided. Furthermore, the use of animal-derived substances must be controlled to avoid the transmission of pathogens to the final product. In this sense, the only animal-derived product used in this work was the Fetal Bovine Serum (FBS) (Thermo Fisher Scientific) of Australian origin, whose certificate of analysis indicates the absence of bluetongue virus, bovine adenovirus, bovine parvovirus, bovine respiratory syncytial virus, bovine viral diarrhea virus, hemadsorbing agents, rabies virus, reovirus, and spongiform encephalopathy virus. Given that FBS is the only animal-derived reagent used in the cell bank production process, it can be guaranteed that the manufacturing process is not introducing animal viruses into the final product. However, for greater safety, an adventitious virus test is carried out on the final product, as described below.
MCB and WCB process validation
One cryovial containing HEK293T cells was thawed and split into three HEK293T populations which were independently expanded in order to obtain three cell stocks for the MCB. Each HEK293T batch was expanded to reach a concentration of 200·106 cells. Every batch was composed of at least 20 cryotubes totaling ≥ 60 cryotubes for MCB. Quality control tests were carried out for every MCB batch. Three consecutive and independent batches of WCB were prepared from the MCB. HEK293T cells from the MCB were thawed and expanded to reach a concentration of 1000·106 cells. Every batch of the WCB was composed of at least 50 cryotubes totaling ≥ 150 cryotubes for WCB. Quality control tests were run for every WCB batch.
Sterility test and growth promotion test
Cell cultures and TSB media were subjected to a growth promotion test according to Chap. 2.6.1 of the European Pharmacopoeia to ensure that growth media met the proper sterility requirements.
Sterility tests on TSB, MCB, and WCB samples were performed by using the direct inoculation method described in Chap. 2.6.1 and 2.2.27 of the European Pharmacopoeia [16].
Mycoplasma test
Venor®GeM qOneStep kit (Minerva Biolabs®, Berlin, Germany) was used for the detection and identification of Mycoplasma in TSB, MCB, and WCB samples. The method is based on the amplification of the Mycoplasma 16S rRNA coding region in agreement with the method described in Chap. 2.6.7 of the European Pharmacopoeia [16].
Karyotype test
Karyotype was determined by G-band staining. HEK293T were seeded and incubated until 80% confluence was observed. 12 µl of KaryoMAX™ Colcemid™ (Thermo Fisher Scientific) was diluted in 6 ml of DMEM. Subsequently, 6 ml of KaryoMAX™ Colcemid™ working solution was added to the cell culture and incubated at 37 °C/5% CO2 for an hour. Cell culture medium was removed, and cells were washed with 3 ml of PBS (Thermo Fisher Scientific). After removing PBS, 1 ml of TrypLE™ (Thermo Fisher Scientific) was added and cells were incubated for 1 minute at 37 °C. Then, 5 ml of culture medium was added, and cells were collected by centrifugation for 5 minutes at 1250 rpm. After removing the supernatant, the pellet was washed with PBS and centrifuged again for 5 minutes at 1250 rpm. 10 ml of pre-warmed (37 °C) 0.075M KaryoMAx™ KCl solution was gently added to the cells and, after 10 minutes of incubation at 37 °C, cells were fixed with Carnoy fixative solution.
Adventitious viruses test
Adventitious viruses testing is based on the analysis of cytopathic effects in host cells. HEK293T cell samples from MCB and WCB were disrupted by shaking with 0.2- and 3-mm stainless steel beads. Cell homogenate was centrifuged (1250 rpm, 5 min, 4 °C) and 0.9 ml of supernatant was added to MR5, VERO and RD cell cultures (Vircell, Granada, Spain) and incubated at 35–37 °C. Cell cultures were analyzed 14 days after inoculation. Positive and negative controls were included in the analysis. Real Time (RT)-PCR for Enterovirus identification was performed on samples with no cytopathic effects.
In-Process Controls (IPCs): cell morphology, cell confluence and viability
Cell morphology, cell confluence and cell viability were used as IPCs during the entire process of MCB and WCB generation. Morphological inspections of HEK293T cells and cell confluence were carried out visually under a microscope. Trypan blue exclusion test was carried out at every cell passage step to test cell viability. Samples with a viability < 80% were not used in the following production steps.
MCB/WCB stability after cryopreservation: viability and cell number
Trypan blue exclusion test was carried out on each batch of MCB and WCB to test cell viability after cryopreservation. To ensure the optimal cryopreservation conditions, the acceptance criteria of the viability test after thawing were set equal or greater than 50%.
A stability program has been carried out over a period of 37 months after WCB generation to determine the optimal storage conditions of cell banks. Trypan blue exclusion assay was used to determine cell viability and cell number of the WCB during the ongoing stability program.