The field of human tissue regeneration is expanding rapidly and applications for the technology get broader daily. Some companies already have well-established manufacturing processes and so issues relating to commercial use mostly revolve around upscaling from the lab, regulatory compliance, and overall profitability in terms of capital and operational costs.
The challenges presented by tissue regeneration – such as the need for high levels of sterility and for the elimination of all cross-contamination1 – are pushing companies toward the adoption of isolation technology in place of traditional cleanrooms, largely because the advantages of isolator technology have already been demonstrated for aseptic processing in the pharmaceutical industry.
The brief for Comecer, a specialist in isolation technology for pharmaceutical, chemical and food industry applications, was from a multinational company operating in the biotech industry that was looking to invest in a new laboratory dedicated to commercial tissue engineering. The company decided to investigate the adoption of isolation technology to guarantee complete continuity of a Grade A environment and to assure an improved quality of the final product, together with full compliance with GMP and FDA regulations. The challenge was to develop custom laboratory equipment as well as specific solutions to ensure asepsis while maintaining ease of use and operability.
To implement a Quality By Design approach, the first step was to define with the company the requisites of the process in terms of:
- the equipment needed to fulfill all cell culturing protocols
- the conditions for primary cells and consumables
- the requirements for inlets and outlets for process materials
- the expected manufacturing capacity
All the different steps in cell culturing involve skilled technicians who follow specific protocols to produce the final product. In generic terms the most important steps to reach confluency (the required number of adherent cells in a culture dish or a flask) are: supernatant removal, incubation, detaching (for monolayer culture), observation and counting.
A certain level of flexibility was also desirable for the company so that it could be ready to adapt to different techniques and products. The equipment that needed to be available throughout the process included:
- A CO2 incubator
- A refrigerator at +4°C
- A microscope
- Refrigerated centrifuge
- Other minor specific equipment
Primary cells can reach the laboratory in different conditions and packaging, while all other consumables, such as pipettes, media, flasks, bags, fetal bovine serum, trypan blue, trypsin etc., have containers already in use by the company. The need to have an inlet and an outlet for consumables, for primary cells, for the exit of the final product, and for the handling of waste and tools was given as a ‘must have’ by the company.
The initial sketch was based on the isolation technology used in general aseptic processing (e.g. in formulation, sterility testing and sterile fill finishing) and with equipment integration solutions used in active pharmaceutical ingredient processing. The idea of segregating the technical unclassified area from the operator side was taken from a concept developed in sterile vial filling – called the ‘balcony design’. The advantages given in terms of operation and maintenance of the isolators and of the integrated equipment is well recognised by the pharmaceutical industry.
Figure 2: Incubator access doors
There were no incubators, refrigerators and centrifuges on the market that could be integrated into isolators in a way that would allow the internal space of the centrifuge, incubator and refrigerator to be continuous within the Grade A (FDA – Class 100) isolator enclosure.
This Class means that everything has to be subject to vapour phase hydrogen peroxide decontamination cycles, to achieve a 6 log reduction of bacterial spores, as well as to be an integral part of the enclosure in terms of air tightness. In addition, stainless steel 316L with a mirror grade finish needed to be used for all the internal volume components where technically feasible. Two modules were defined for the purpose: the incubation module, integrating the incubator, and the separation module, integrating the centrifuge, the refrigerator and the microscope (see Figure 1). A transfer hatch connected to the separation module was to be used as an entry and exit port.
Figure 3: Refrigerator
The overall laboratory space constraint determined each module’s size, while the transfer hatch had maximum dimensions of 300 x 400 x 300mm. The isolator design was driven by GMP and the Pharmaceutical Inspection Convention and Pharmaceutical Inspection Co-operation Scheme (PIC/S) principles, and also by the reference standards and guidelines in aseptic processing within isolation technology. From this assessment, the following technical features were defined as required for the company’s application:
- Laminar airflow equipped with HEPA H14 filter: in aseptic processing, extensive manual operations through gloves can be a major source of particulate, which is why laminar airflow (providing higher volume changes in the enclosure) is preferred, as it assures a better ‘air wash’ of the working area
- Positive pressure
- Exhaust filters H14: even if not required for strictly sterile applications, the selection of this feature was mainly driven by the risk of cross-contamination between different modules and the flexibility in manipulation of hazardous materials in the isolator
- High rate of recirculation to reduce overall air consumption: each module is recirculating up to 70% of the total LAF in the chamber
Figure 4: Centrifuge
- A mock-up was created to ensure ergonomic correctness, mainly in the incubation modules where operators would be working while sitting for prolonged periods. Accessibility to all integrated equipment was also assessed
- H202 gas was state-of-the-art for the requested performance
- Different cycles for the full isolator and equipment, one rapid cycle dedicated to the transfer hatch, decontamination performance achievement to be verified, and the same for the incubator, refrigerator and the centrifuge bucket
Figure 5: Transfer hatch
- Viable and non-viable monitoring for each single module
- GAMP 5 Software to manage the whole isolator
- SCADA System for 21 CFR Part11 and supervision of all seven units.
Each isolator unit is composed of one separation module with two incubation modules on the side; a transfer hatch on the bottom of the separation module serves all three modules. The solutions shown in the pictures above include:
- Incubator access doors (Figure 2): the entire volume of 400L is accessible from the interior of the isolator through eight small doors. Each door provides access to two extractable trays.
- Refrigerator (Figure 3): 80L of capacity are accessible from the interior of the isolator chamber through a single door. The internal volume therefore is equipped with sliding trays.
- Centrifuge (Figure 4): refrigerated centrifuge with an airtight drum made of anodised aluminium.
- Transfer hatch (Figure 5): accessible from the separation module floor.
- Viable and non-viable monitoring (Figure 6) and storage area (not shown): a storage volume was defined for media and other process material. The microscope has been equipped with a digital camera connected to an external monitor; the electroporation cuvette (used to maximise electroporation efficiencies for cells) has been placed within the chamber, while the control box is outside.
Figure 6: Viable and non-viable monitoring equipment
The isolators were equipped with all the sensors and probes typically needed for this kind of application, e.g. an onboard glove integrity tester, an anemometer, a temperature and relative humidity sensor, a manometer to check for filter obstructions, an automatic leak integrity verification prior to the decontamination cycle, etc.
The final layout of the manufacturing suite has resulted in a laboratory side classified as Grade D according to GMP guidelines, while the technical side in the back of the isolators (needed for maintenance access) is unclassified.
As a result, the specific integration solutions developed within this project are leading to their implementation in other regenerative medicine fields, such as cell therapy, stem cell manipulation and tissue engineering.
1. http://www.comecer.com/isolation/isolator-glove-boxes/regenerative-medicine/sterility-cross-contamination-challenges-tissue-engineering-regenerative-medicine/CONTACT Massimiliano Cesarini, Team Leader for Regenerative Medicine Solutions COMECER SpA Via Maestri del Lavoro, 90 48014 Castel Bolognese (RA), Italy T +39 0546656375 email@example.com www.comecer.com