The ATU described in this study is a platform shared by the La Fe University and Polytechnical Hospital (HUP La Fe) and the La Fe Healthcare Research Institute (IIS La Fe). It was designed under the regulatory framework governing the conduct of clinical and preclinical studies as a unit responsible for supporting different research groups in the development of academic ATMPs.

The ATU, established in 2017, covers an area of 177.25 m2, distributed into 3 independent class B compounding rooms, 5 class C rooms, an area dedicated to vapor-phase LN2 cryopreservation, and a quality control laboratory (Fig. 1). The unit meets all the requirements established in the EU's GMPs, assuring the quality, safety, and efficacy of the medicines produced.

Fig. 1.

Layout and equipment of the advanced therapies unit.

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The ATU boasts a highly-qualified multidisciplinary team steeped in the particulars of GMPs, which includes hematologists, biologists, and pharmacists, both from IIS La Fe and from HUP La Fe. At present, the unit is led by a hospital pharmacist, who is responsible for authorizing the production of every batch of ATMPs once its compliance with the approved specifications has been ascertained. AMTP-producing units must meet the following requirements to ensure compliance with GMPs:5

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  Personnel: Highly qualified staff with clear attribution of responsibilities.

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  Facilities and equipment: The facilities and equipment must be fit for purpose and regularly maintained. Facilities producing more than one ATMP (multi-product facilities) should either assign a separate area for each ATMP produced, or distribute the production of different ATMPs across different time slots, ensuring the implementation of strict cleaning systems to avoid cross-contamination.

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  Documentation system: Facilities must be in possession of comprehensive specifications for all bulk and finished materials and intermediate products, as well as of a detailed description of the production process and all the associated records.

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  Compounding process: The consistency of the production process, the quality of the products compounded, and the compliance with all specifications must be duly guaranteed.

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  Quality assurance: Quality assurance systems must be independent from the production system.

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  Prospective evaluation of change: Any changes must be planned for and be approved prior to their implementation.

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  Quality defects and deviations from the required specifications must be identified as early as possible and action must be taken to investigate the causes and implement the corrective and/or preventive measures.

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  Traceability: It is indispensable to ensure full traceability of final products and of starting and critical materials.

Along the same lines, new Annex 1 of the EU GMPs seeks to tighten the requirements related to the prevention, control, and tracking of pharmaceutical products, and to improve the quality assurance and information management systems used. With regard to cleanrooms, it emphasizes the importance of cleaning systems, of maintaining an aseptic and controlled environment, of properly designing facilities and materials to avert the risk of contamination, and of ensuring that the staff are properly trained and qualified.7

Qualification of air treatment systems (according to ISO 14644-1 and ISO 14644-2 standards) and cleanrooms is carried out yearly by an external company. In addition, an environmental monitoring program exists that allows evaluation of the effectiveness of the contamination control measures adopted. The environmental monitoring program includes viable and non-viable particle monitoring:

Surface sampling: Carried out by means of contact plates to control for aerobic bacteria, fungi, and yeasts.

Environmental sampling: Performed by specialized teams, it is used to control for aerobic bacteria, fungi, and yeasts. It may be carried out with an air sampler or with sedimentation plates.

Non-viable particle counts: This procedure is aimed at checking for 0.5–5-μm particles. The system used must allow continuous control of the cleanroom.

Differential pressure: The theoretical pressures defined for each cleanroom must be verified with a system that allows for continuous monitoring.

Number of air changes per hour: A system is used to ensure that the minimum number of air changes established for each room is complied with.

The ATU staff monitors for the presence of viable particles every 3 weeks. The results of the analysis are examined by the HUP La Fe Microbiology Department, with which an internal collaboration agreement has been concluded.

Aseptic process simulation tests, also known as media fill tests, consist in the performance of an aseptic manufacturing routine based on a sterile microbiological growth medium and/or a placebo to determine if the procedures employed during drug compounding are capable of avoiding microbial contamination.

Media fill tests must follow the usual compounding procedure and be carried out in the same place as the actual production is performed, with a particular focus on processes involving non-sterile stages, and considering all possible interventions.

Securing the approval of the Spanish Medicines and Health Products Agency (AEMPS) to produce an ATMP requires performing 3 consecutive media fill tests per operator and production process, with satisfactory results. In addition, a media fill test must be conducted every year in active ATUs, with an additional test being required if the ATU has not produced ATMPs for 6 months and 3 additional tests being mandatory if a whole year has gone by without the ATU producing an ATMP.

The application procedure established by HUP La Fe for ATMPs for which the ATU holds an AEMPS-issued compounding authorization comprises the following stages (Fig. 2):

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  The attending physician submits a standardized application report to the hematopoietic stem cell transplantation (HSCT) and cell therapy committee. The committee, made up of a multidisciplinary team, meets weekly to analyze applications for HSCT and ATMPs, including both commercial T-cell therapies with chimeric antigen receptors (CAR-Ts) and in-development or academic ATMPs. At those meetings, the different therapeutic alternatives available are discussed and the potential indication for an ATMP is evaluated. In case of acceptance, a search for the most appropriate donor commences.

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  The HSCT and cell therapy committee selects the most appropriate donor, which may be the previous HSCT donor, a family member with appropriate HLA compatibility, or a third-party donor from the REDOCEL cell donor network, a collaborative project with the Transfusion Center of the Valencia Region (CTCV) and the Blood and Tissue Bank of Cataluña (BST). The donor must meet the donation standards for hematopoietic products as determined by the blood bank/apheresis unit. Eligibility is also determined depending on the presence of T-cell immunity against the virus of interest. This assessment is conducted by the hematology diagnostic unit by means of in-vitro assays and cytometry.

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  Once a donor has been determined eligible, the attending physician submits an application under the exceptional drug access program, attaching the minutes of the committee meeting where approval was granted. Subsequently, a consultation is made with the hospital's pharmacy department to discuss the possibility to administer the drug under the compassionate use rule if the treatment is not part of an active clinical trial. All of these documents are submitted to the hospital's medical director, together with an application under the exceptional drug access program prepared by the pharmacy department.

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  The pharmacy department must upload all the required documents (application by the hematology unit, approval by the committee, application of the pharmacy department with the hospital medical director's, and the ATU technical manager's authorization to produce the ATMP) to the AEMPS exceptional drug access program website to obtain an authorization to use the drug in individual patients. In addition, the product's PEI (investigational new drug) authorization number, as assigned by AEMPS, must be stated and any clarifications requested by AEMPS about the product must be addressed.

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  Once AEMPS’ authorization has been secured, both the hospital's apheresis unit and the ATU can set about planning the production process. The former must call in donors and schedule the lymphocyte apheresis procedure, while the latter must secure the reagents needed and prepare the compounding room, the batch documentation and the required materials.

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  The ATMP can now be produced. This procedure, which will be explained in detail below, basically includes obtaining a donor sample, compounding the ATMP in GMP conditions, dosing and packaging the drug, the release of the end product by the technical manager and, last but not least, administration of the ATMP.

Fig. 2.

Applying for an authorization to compound advanced therapy medicinal products.

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The HUP's hematology department contacted the ATU for the production of several ATMPs in GMP conditions. All of such ATMPs were to be based on T-cells specifically targeted at viruses related to publicly-funded projects within a multicenter clinical trial (Nr EudraCT 2018-000911-25).

The ATU has since 2019 been accredited by AEMPS for the production of 4 different kinds of allogeneic T-cells specifically targeted to cytomegalovirus (CMV), Epstein–Barr virus (EBV), adenovirus (AdV), and BK polyomavirus (BKV), obtained from peripheral blood. Moreover, the ATU has recently been authorized to produce T-cells targeted at JC polyomavirus (JCV) for the treatment of progressive multifocal leukoencefalopathy.

These ATMPs, known as virus-specific T cells (VSTs), are indicated for the treatment of viral reactivations either refractory to standard pharmacological treatment or unable to receive it because of toxicity issues. Infusion of VSTs endows the receptor with virus-specific immunity without increasing the risk of them developing graft-versus-host disease (GvHD). The selection of donor T-cells takes place by antigenic stimulation with a viral peptide and immunomagnetic capture of the specific IFNɣ-producing cells, using the automated CliniMACS Prodigy® platform.

The starting material is obtained through leukapheresis of a donor who is seropositive for the virus of interest. It must contain between 1.0 and 1.5×109 nucleated cells and, following the performance of a series of tests to verify its integrity and eligibility for the process it will undergo, it is adjusted to a final volume of ≤100 ml. A sterile connector is used to transfer the starting material to the tubes in the CliniMACS Prodigy® system, keeping the circuit closed for the duration of the process to avoid any potential contamination.

The T-cells specific for the virus of interest are selected from the total pool of donor lymphocytes by antigen stimulation with a viral peptide and immunomagnetic capture through IFNɣ production, using the CliniMACS Prodigy® platform. This process takes place over a period ranging between 4 and 6 h at 37 °C.

Once analyzed by flow cytometry, the total volume of the final product is dosed taking into consideration the weight of the recipient and the specifications of the final product (viability, purity, maximum dose, and maximum content of impurities). The processing and packaging of the final product takes place in a grade A environment using a horizontal laminar flow cabinet located in a grade B cleanroom.

Production of the 5 types of VST results in a single dose, which is prepared by wet compounding and administered at once. For that reason, the production process does not involve cryopreservation of the final product.

The release of the final product takes place in 2 stages: an early phase, based on partial results, and a final phase, once all the results are available. The critical stages of the process are controlled by analyzing both the intermediate products and the final product.

Before early release of an ATMP a quality control procedure is implemented, which consists of a microbiologic assay (Gram-positive cultures) and a flow cytometry test (post-stimulation and final product), where counts and viability analyses of lymphocyte and IFNɣ-marked T-cell populations are performed. Such analyses make it possible to ensure that only T-cells specifically targeted against the virus of interest have been selected and that they comply with pre-determined purity criteria. The quality examination required before the ATMP can be released is completed with a series of sterility, mycoplasma and endotoxin assays.

The product is then transported at a temperature ranging between 2 and 8 °C. A maximum of 24 h may elapse between early release of the ATMP and its infusion into the patient's bloodstream. The production process spans a maximum period of 36 h (range: 22–36) and takes place in an almost entirely closed system, except for the reconstitution and reagent addition phases. In our case, the process was performed in a grade A environment in a grade C cleanroom. Production was automated (CliniMACS Prodigy®) and all reagents were filtered before use.