In the light of growing interest in regenerative therapies, Massimiliano Cesarini, Comecer Group, describes the redesign of a tissue regeneration lab to improve sterility and reduce production costs based on the use of isolation technology
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:
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.
Figure 1: Schematic showing the proposed activities and equipment for each module
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:
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:
Figure 4: Centrifuge
Figure 5: Transfer hatch
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:
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 firstname.lastname@example.org www.comecer.com