In the present work we demonstrated the feasibility and difficulties of generating MCB and WCB under current GMP regulations15 in academic institutions. According to the current regulation, it is crucial to first implement a risk-based assessment of the whole process in order to thoroughly evaluate every stage of the manufacturing process such as cell culture growth, cell passages, quality control and IPCs requirements as well as evaluate the need of scale up procedures, in order to reach a minimum cell number per batch. Since the main objective of the risk-based assessment is to detect those risky steps which need mitigation actions for obtaining homogeneous and robust products. In this regard, it is important to carry out this evaluation by all key personnel involved in the manufacturing process such as quality control personnel, manufacturing personnel, quality assurance personnel and qualified person. The present work shows the approach established at Creatio to validate a GMP MCB and WCB of HEK293T cell line which are being used as packaging cells in GMP-grade lentivirus manufacturing used in several CAR T-cell clinical trials.11,19
One of the main changes that a research protocol undergoes when transferred to the clinic is the quality of the reagents and starting materials used. Frequently, new ATMPs developed in academic institutions use research-grade reagents and non-tested starting materials which do not fulfil the minimum quality required to start the manufacturing step. The HEK293T cell line is widely used in research as packaging cells in different viral-vector production system such as adeno-associated viruses (AAVs), retroviruses, adenoviruses (AD) or lentiviruses.20 For this reason, before using any cell line such as HEK293T, it is mandatory to analyze its quality parameters or attributes. In this regard, although according to Chap. 8 of the current GMP for ATMP manufacturing the generation of cell banks is not mandatory,15 it is highly recommended when they are going to intervene in manufacturing processes as starting materials, since this guarantees the robustness and homogeneity of products derived from them such as viral particles.
Sterility of three consecutive batches from the MCB and the subsequent WCB was analyzed. The results showed that all batches of HEK293T MCB and WCB were sterile. Due to the nature of ATMPs, which are considered as a live-products, sterility must be kept during the whole aseptic process. Accordingly, cleanrooms must be validated in order to guarantee the adequate environment in terms of viable and non-viable particles as well as the proper pressure, temperature and humidity of cleanrooms. In addition, a personnel training program which includes the aseptic simulation of the process should be established in every pharmaceutical quality system because personnel participating in the manufacturing process is considered to be the main focus of contamination.15 Creatio’s personnel involved in MCB and WCB generation was successfully trained and validated to carry out the processes under current GMP guidelines. In addition, cleaning validation of Creatio facility was previously performed in order to guarantee and keep record of the cleaning process as a critical stage. Another critical aspect is the presence of mycoplasma in ATMPs or raw materials used in the ATMPs manufacturing. For this reason, mycoplasma test is a mandatory quality control test. According to the current GMP regulation for ATMPs manufacturing,15 the presence of mycoplasma in raw materials must be evaluated prior to starting the manufacturing process under a risk-based approach. The HEK293T initial stock was free of mycoplasma and the three consecutive batches of MCB and WCB were also free of this pathogen. In terms of safety, MCB and WCB must be also free of adventitious viruses. No cytopathic effect was detected in any batch, so we were able to ensure that the raw materials used in the manufacturing process and the handling of the cell cultures were not a focus of adventitious viruses.
Even though HEK293T cell line is never going to be used as a final product, it is necessary to characterize the identity of the cell line in order to guarantee its properties in biological terms such as growing time and packaging functions. In this sense, the presence of specific STRs after the MCB and WCB generation coincide with those given in the certificate of analysis of HEK293T cell line.Thus, no alteration of the STRs 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 was detected after the passages performed during the processes. Moreover, karyotype was also analyzed in order to detect any alteration caused by the manipulation and the passages. HEK293T has a complex karyotype. Results from HEK293T MCB and WCB show a hypertriploid karyotype with numerous aberrant chromosomic alterations such as duplication and chromosomic derivation as described elsewhere.17,18 These results are mainly due to the genetic instability of this cell line. For this reason, karyotype specification does not have acceptance criteria and is considered to be informative. Nonetheless, we consider mandatory to document this information in order to track unexpected results of viral-vectors yields and correlate with chromosomic alterations.
Other crucial aspects when cell banking is being designed are the cryopreservation and storage conditions.21,22 HEK293T MCB and WCB were subjected to a cryopreservation ramp of 1 °C/min. Thawed cryovials showed cell viability over 90% indicating that cryopreservation did not affect the quality of the product. However, different cryopreservation ramps and different cryopreservation media should be evaluated when new cell lines are planned for banking.23 On the other hand, Creatio’s cryopreservation tanks are designed to work under a gas phase liquid nitrogen that reduces cross-contamination and keeps the temperature more homogeneous than those working with liquid phase. Our results confirmed the long-term stability that cryopreserved HEK293T had over 37 months. With these data, we can assure that cell banking of HEK293T is an adequate approach to generate a homogeneous cellular product as well as to stablish an expiry date of GMP-MCB and GMP-WCB of 37 months, which can help researchers and manufactures to design ATMPs manufacturing protocols.
The present MCB and WCB approach shows the minimal requirements that academic institutions should bear in mind when a non-GMP cell line is used as starting material in a manufacturing process. Although the present work used an adherent cell line it would be equally possible to establish MCB/WCB of suspension cell lines following the same strategy.24–26 On the other hand, new technology is being developed for cell therapy products involving adherent or suspension cell culture. Bioreactors are emerging as closed systems which reduce reagent consumption and handling time.27,28 In this sense, the present procedure is applicable to generate cell banks from bioreactors systems. MCB and WCB approaches have been successfully performed in different ATMP manufacturing processes such as mesenchymal stem cells,29,30 fibroblasts,31 induced pluripotent stem cells or embryonic stem cells32,33 among others which have been used as a final product ready for administration. We would like to highlight the importance of GMP cell banking for ATMP manufacturing and its consideration for translational research.