Cleanroom-viable CO2 hatcheries for cell treatment
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Creating improved technologies for cleanroom equipment used for cell therapy process development and manufacturing means including materials optimised for stringent cleaning protocols and reducing potential for contaminants including microorganisms and non-viable particulates
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The requirements for CO2 incubators also include creating ideal growth conditions for patient samples and optimised shelving that ensures a high throughput per footprint.
Important considerations for cleanroom compatibility and cell therapy manufacturing
1) Cleaning and disinfection compatibility
2) Very low particulate emission and technologies to limit microbial contamination
3) Complete certificate and documentation package
4) Optimised space for large vessel types to achieve a high throughput
1) Cleaning and disinfection compatibility
For a CO2 incubator used in cell therapy development and production, a brushed 304 stainless-steel exterior is recommended. It is more resistant than a traditional painted steel exterior, for use with chemical disinfectants for manual cleaning and with vaporised hydrogen peroxide (VHP) procedures. Inside the incubator chamber, interior components that are electropolished stainless steel reduce microscopic structures that could provide areas for microbial growth. Ingress protection 54 (IP54)-rated exterior casing and a silicone-sealed touchscreen display protect the electronics, further supporting compatibility with common cleanroom protocols.
2) Low particulate emission and technologies to prevent from microbial contamination
Since a final cell-based product to be injected into a human patient is inherently limited in the amount and ways that it can be purified, particulate management remains a primary concern in the cell therapy manufacturing setting. Microbial contamination remains the number one cause of recalls for biological pharmaceuticals. But these are not the only microscopic contaminants of concern. Non-viable particulates were the reason for 22% of US FDA recalls of sterile injectables from 2008-2012[1] and the second leading cause of recalls from 2009 to 2019 (3). Tiny articles from fabrics, stainless steel, glass, plastics and more represent a variety of dangers for a patient, from an unintended immune response to a pulmonary embolism. And since the production equipment itself is responsible for an estimated 15% of particle generation in a cleanroom[2], equipment that helps to control particulate emissions is extremely advantageous[3].
A CO2 incubator that offers an active particle control system to control the emission of particulates in the air is proven to be highly suitable for use in an ISO Class 5 and Grade A/B cleanroom environment.
As your partner in production, a CO2 incubator should also offer proven solutions for contamination control and maximum time at set parameters so that cells express the proper characteristics and potency. An in-chamber circulating fan not only provides fast recovery from a door opening and optimised uniformity, but also the opportunity for in-chamber HEPA filtration. With a carefully designed air circulation system, recovery of all parameters in ten minutes following a thirty second door opening can be achieved. The same system driving HEPA filtration can reach ISO Class 5 conditions in five minutes or less, capturing particulates of any size. An on-demand dry heat sterilisation cycle should be tested by an independent third party laboratory, working according to the standards in the international pharmacopeias to reach a 12-log Sterilisation Assurance Level (SAL). These standards also require continuous air circulation during the entire cycle. Forty-eight point temperature mapping demonstrates that all areas reach and maintain the specified sterilisation temperature, to ensure there are no cold areas where microbes could survive and regrow.
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The table above shows results from a proven sterilisation, showing greater than six-log reduction of Bacillus atropheus spores, the specified biological indicator for dry heat sterilisation, and of Geobacillus stearopthermophilus spores, the specified biological indicator for autoclave sterilisation.
This cycle held at 180 ˚C for forty-five minutes. Doubling of this sterilisation time at 180 ˚C represents an “overkill” approach for a total 12-log SAL.[4]
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The figure above shows a forty-eight point temperature map during a CO2 incubator dry-heat sterilisation cycle during which all areas reach and hold at 180 ˚C or greater for at least ninety minutes.[4]
3) Certificates and documentation
A certified cleanroom-compatible CO2 incubator is one that has been tested according to ISO 14644-1 requirements and is proven to adhere to air quality limits within grade A/B cleanroom standards. In addition to testing the design, each unit should undergo complete end of line testing. To facilitate the audit process for on-site qualification, an equipment manufacturer should provide certificates and documentation to cover 1) product certificates, specifications and materials of composition 2) Factory Acceptance Test documentation and 3) clear guidance for cleaning, disinfection, routine maintenance and replacement of parts and accessories, as well as information on performance.
4) Optimised space for large vessel types to achieve a high throughput
With the right culture vessel and an environment that promotes proper growth, high expression of desired receptors, and optimum critical quality attributes (CQAs), C 2O incubators designed for cell therapy production can be part of an efficient scale-out program. Recognising the importance of maximising efficiency and valuable cleanroom space, recently available optimised shelving systems further maximise CO2 incubator chamber space to provide a high throughput per footprint.
Standard shelving systems are limited in terms of weight capacity and efficient use of incubator interior space. But by re-thinking shelving with designs specific to selected vessel types that better manage heavier loads, the capacity and handling can improve tremendously.
As one example, the standard shelving system for the Thermo Scientific™ Heracell™ Vios™ 250i CR CO2 Incubator holds four of ScaleReady’s G-Rex® 500M-CS bioreactors. With an optimised shelving system, the capacity can be increased to ten of the G-Rex® 500M-CS units, an increase of 150 %.
Since in a cleanroom production environment, most CO2 incubators are stacked, the new shelving provides a potential capacity of 20 G-Rex® 500M-CS per incubator footprint (as per example 25.1 in x 34.6 in). Each of ScaleReady’s G-Rex® 500M-CS can produce up to twenty billion cells in ten days. Regardless of whether the production goal is autologous or allogeneic immune cell therapy, this is an impressive output per footprint. The production volume combined with the cost-effectiveness of using CO2 incubators compared to standalone bioreactors such as rockers supports the attractiveness of CO2 incubators for cell therapy development and manufacturing. For these critical applications, it is important to carefully evaluate all features, ensuring proven performance.
1. Tawde, SA. (2015) Particulate matter in injectables: Main cause for recalls. Journal of Pharmacovigilance 03.
2. Clarke D, Stanton J, Powers D, et al. (2016) Managing particulates in cell therapy: Guidance for best practice. Cytotherapy 18(9):1063-1076.
3. Thermo Scientific Smart Note: What is a certified compatible CO2 incubator design, and why is this an important consideration for any cell therapy or gene therapy process? (2021) Thermo Fisher Scientific COL34101 0321.
4. Thermo Scientific Steri-Run sterilization cycle proves total sterilization. (2015) Thermo Fisher Scientific ANCO2STERIRUN 0215.
New, Detailed Genetic Roadmap of Glaucoma | Technology Networks
A new, detailed genetic roadmap of glaucoma – the world’s leading cause of irreversible blindness – will help researchers develop new drugs to combat the disease, by identifying potential target areas to stall or reverse vision loss.
The research, one of the largest and most detailed stem cell modelling studies reported for any disease, is published today in Cell Genomics.
By comparing stem cell models of the retinal ganglion cells of people with Primary Open Angle Glaucoma to those without the disease, more than 300 novel genetic features of these cells were uncovered.
The findings are the result of a national collaboration led by Professor Alex Hewitt (Centre for Eye Research Australia, University of Melbourne and University of Tasmania), Professor Alice Pébay and Dr Maciej Daniszewski (University of Melbourne) and Ms Anne Senabouth and Professor Joseph Powell (Garvan Institute of Medical Research).
Professor Hewitt, who is Head of Clinical Genetics at CERA, says the study will lead to a better understanding of the mechanisms that damage retinal ganglion cells and lead to the onset of glaucoma.
This will help researchers develop new drugs to combat glaucoma, by identifying potential new areas to target to stall or reverse vision loss caused by the disease.
Creating retinal ganglion cells
Healthy retinal ganglion cells – which transmit visual information from the eye to the brain via the optic nerve – are essential for vision. In glaucoma, the gradual damage and death of these cells leads to a progressive, irreversible decline in sight.
“Glaucoma is often an inherited condition and comparing diseased retinal ganglion cells with healthy one is an effective way to increase our understanding of the mechanisms that contribute to vision loss,’’ says Professor Hewitt.
Professor Pébay, whose team lead the stem cell aspects of this work, says: “Until recently that’s been impossible because you cannot obtain or profile retinal ganglion cells from living donors without an invasive procedure.”
To overcome this challenge, the scientists used Nobel Prized-winning induced pluripotent stem cell (iPSC) technology to ‘reprogram’ skin cells provided by donors into a stem cells that were then turned into a retinal ganglion cell in the lab.
They then mapped the individual genetic expression of almost a quarter of a million cells to identify features that could impact on the way genes are expressed in the cell, impacting its function, and potentially contributing to vision loss.
Identifying genetic features
The researchers identified 312 unique genetic features in the retinal ganglion cell models that warrant further investigation.
“The sequencing identifies which genes are turned on in a cell, their level of activation and where they are turned on and off – like a road network with traffic lights,’’ says Professor Powell, whose team led the analysis of world leading dataset.
“This research gives us a genetic roadmap of glaucoma and identifies 312 sites in the genome where these lights are blinking.
“Understanding which of these traffic lights should be turned off or on will be the next step in developing new therapies to prevent glaucoma.’’
Professor Hewitt, an ophthalmologist, says the research provides hundreds of new targets for researchers developing new drugs to treat glaucoma which is predicted to affect more than 80 million people globally by 2040.
“Current therapies are limited to slowing vision loss by reducing pressure in the eye – but they do not work for all patients and some people continue to lose many retinal ganglion cells and vision despite having normal eye pressure.
“The rich source of genetic information generated by this research is an important first step towards developing new treatments that go beyond lowering eye pressure and can reverse damage and vision loss.’’