Mary Kay Bates is senior global applications scientist at Thermo Fisher Scientific.

Mary Kay Bates

The cell therapy market is growing, driven by scientific advances and the potential offered for the treatment of a wide range of medical conditions, from cancers to autoimmune and infectious diseases. Worth $7.8 billion in 2019, the global cell therapy market is set to reach $48 billion by 2027, a compound annual growth rate of 25.6% from 2020–2027 (1). This trend is mirrored in regulatory submissions; FDA anticipates more than 200 cell and gene therapy investigational new drug submissions and 10–20 approvals per year by 2025 (2).

As with any pharmaceuticals developed for human use, the production of cell therapies follows good manufacturing practices (GMP). While not prescriptive, GMP is a framework for procedures, personnel, training, and record-keeping to ensure product safety, purity, efficacy, and quality. Production equipment, including carbon dioxide (CO

) incubators used for cell therapy, must comply with these principles.

Cell therapy development is made possible through advances in cell biology understanding and a shift from working in long-established immortalized cell lines to a focus on stem cells and primary cells. These cells are highly sensitive and reactive to external cues, and require carefully controlled conditions that closely replicate those seen 

 to achieve optimal cell growth and proper gene expression.

In addition to providing carefully controlled conditions for proper cell growth, CO

 incubators used in cell therapy production should offer independently proven methods for control of cell culture contamination. For cell therapy manufacturing, the cell production process most often takes place in a cleanroom, since particulates represent a risk to cells that will be injected into a human patient. Particulate contamination needs to be carefully controlled in the cleanroom by filtration and air exchange. Different types of cleanrooms control airborne particulates to different levels. Inside the cleanroom itself, manual and automated methods are used for the disinfection of surfaces and equipment, but such practices can damage equipment materials and electronics. To minimize damage, laboratory equipment should be tested to demonstrate compatibility with chemicals and application methods.

Regulatory audits help ensure the safety, efficacy, purity, and quality of a product by reviewing documentation that accompanies every step of the process. A CO

 incubator supported with a comprehensive documentation package will help ease the path to regulatory approval.

In the production of cells for human therapy, CO

 incubators must provide a highly controlled environment to support optimal cell growth, ensuring proper gene expression, limiting stress responses, and preserving characteristics critical to the success of the therapy. Equipment design and engineering play a critical role in how cells grow; different incubator models show widely different performance (3), even if functional specifications are similar. Ideally, CO

 incubators used in cell production should offer recovery of all conditions (i.e., temperature, CO

 gas concentration, and humidity) in 10 minutes or less following a 30-second door opening. Fast recovery ensures sensitive stem and primary cells remain in their ideal growth environment (see 

) for the maximum time during production, helping to ensure high-quality cell therapy products.

Table I. Recommended control parameters for a carbon dioxide incubator. Click to enlarge.

One technical aspect involved in the recovery of parameters is the quality and location of sensors. Sensors used to measure temperature, gas concentration, and relative humidity should be located inside the chamber where the cells are incubated, rather than using a bypass sensor located in a separate electronics compartment (4). In-chamber sensors are positioned to measure and react to the same conditions experienced by the cultured cells.

Another design element critical to parameter recovery and uniformity of conditions in the incubator chamber is active air circulation. Warm air rises, and CO

 gas sinks because it is heavier than air. Without active air circulation, atmospheric conditions will stratify, meaning cultures in different areas of the incubator experience different conditions. To ensure the consistent growth of all cultures, a circulating fan is required.

Microbial contamination is a significant concern for cell therapy, with mycoplasma species discovered in 15–35% of all cell cultures in 2015 (5). Different approaches to contamination control can be used, but the best way to select such a method is to look for proof of efficacy. For example, a dry heat sterilization cycle built into a CO

 incubator should be proven according to the precepts in the pharmacopeias from the United States (6) and the European Union (7). Both require proof of elimination of one million specific, heat-resistant bacterial endospores. The US Pharmacopeia additionally requires an “overkill” approach for a total 12-log sterility assurance level (SAL). Both pharmacopeias require hot air to be continuously circulated using a fan. To ensure no cold spots where microorganism could survive, the entire incubation chamber should be mapped to confirm that all areas reach the specified temperature.

A 12-log SAL sterilization is effective at eliminating microorganisms in the chamber, but an in-chamber high-efficiency particulate air (HEPA) filtration system provides 24/7 protection from airborne microorganisms. This filtration is important because each time the incubator door is opened, surrounding air can enter the incubator interior, including circulating microbes. Recovery time of the incubator to cleanroom conditions is an important parameter to consider. For example, in a Thermo Scientific incubator design, an H13-rated HEPA filter combined with active airflow captures all microorganisms regardless of size, providing ISO Class 5 cleanroom conditions in five minutes following a 30-second door opening. This design filters the entire chamber air volume every 60 seconds, approaching zero particulates circulating over time. The filters are rated for one year of average use.

Removing the risk of non-viable particles

The dangers posed by microscopic particles are not limited to microorganisms. Non-viable particulates from personnel, equipment, and consumables also carry safety and efficacy risks: 22% of FDA recalls of sterile injectables between 2008 to 2012 were due to non-viable particulates (8), representing the second leading cause of recalls between 2009 to 2019 (9). Personnel regularly monitor particulate counts in a cleanroom to ensure compliance. The most common particulates are bits of metal, glass, plastic, hair, rubber, cell debris, and fabric (10).

While approximately 70% of non-viable particulates come from personnel in the cleanroom, about 15% come from the equipment (10) used to grow and process the cultures. With this in mind, equipment manufacturers are starting to consider how their designs can better complement a cleanroom setting and limit the number of particulates released. A cleanroom-certified design is carefully tested by a qualified industry institute with clearly documented procedures, such as those outlined in ISO 14644-14 (11).

In a design used in cleanroom-certified incubators from Thermo Fisher Scientific, for example, a HEPA filtration system limits particulate release to the cleanroom. The entire external casing is sealed, and a vacuum system captures particulates, passing them to the HEPA filter at the rear (see 

). Incoming air to cool the electronics is also filtered.

Figure 1. Active particulate control in an incubator (Thermo Scientific). Click to enlarge. Figure is courtesy of the author.

It is impossible to test all chemical disinfectants available globally, but some common usage exists. For example, hydrogen peroxide is a broad-spectrum disinfectant and, in low concentrations of 1–6%, it is generally compatible with paint, stainless steel, glass, and plastics. For any chemical disinfectant, it is important to follow the manufacturer’s recommendations for dwell time, recommended dilution, and personnel protection. It is best practice to follow any aggressive disinfectant, including hydrogen peroxide, with a 70% ethanol (EtOH) or 70% isopropanol (IPA) solution to remove any residues and protect from chemical buildup that, over time, could degrade equipment materials. It is particularly important to remove strong chemical residues from a cell culture incubator to limit fumes that could harm cultured cells and elicit stress responses (12,13).

Another approach common to cleanroom sterilization is fumigation using vaporized hydrogen peroxide (VHP). Depending on the provider, VHP can include a range of hydrogen peroxide concentrations and added chemicals such as peracetic acid. Condensation of high concentration VHP chemicals can damage incubator materials over time and can cause peeling of painted steel surfaces. For these reasons, any VHP process should carefully control condensation—commonly referred to as a ‘dry process’—and provide proof of sterilization and neutralization of the chemicals to prevent potential harm to equipment, cultured cells, and personnel.

For increased compatibility with chemical disinfectants and VHP, a brushed 304 stainless-steel exterior is recommended. Ingress protection 54 (IP54)-rated electronics and a silicone-sealed touchscreen display increase compatibility with such processes, protecting from dirt and splashed liquids. An electropolished stainless-steel incubator chamber and components mean reduced microscopic structures for easier cleaning and limited areas for microorganism attachment.

To ease equipment qualification and help meet audit requirements, full factory acceptance testing (FAT) and applicable certificates and sourcing documentation are essential. Such documentation should be provided by the CO

Preventive maintenance checklists and evidence

Replaceable parts list and dates of last replacement

EC, UL, REACH, and ISO 13485 certificates

Supporting the growing cell therapy market

 incubators are critical for the creation and maintenance of optimal cell growth conditions, encouraging the cellular responses seen 

 that are necessary for cell therapy success. A cleanroom-compliant CO

 incubator is certified to control particulate emission, withstand stringent cleaning protocols, and provide conditions for sensitive cells to promote their consistent growth and expression. 

, 2020-2027, Sept. 2020.
2. FDA, “Statement from FDA Commissioner Scott Gottlieb, MD and Peter Marks, MD, PhD, Director of the Center for Biologics Evaluation and Research on New Policies to Advance Development of Safe And Effective Cell and Gene Therapies,” Press Release, Jan. 15, 2019.
3. Thermo Fisher Scientific, “Which Incubation Parameters Are Most Important for Proper Cell Growth and Expression?” Thermo Scientific Smart Note (2015).
4. Thermo Fisher Scientific, “Why Does the Location of Sensors in my CO

 Incubator Affect Responses from my Cultured Cells?” Thermo Scientific Smart Note (2015).
5. C.N. Wilder and Y. Reid, “Mycoplasma Quality Control of Cell Substrates and Biopharmaceuticals,” 

, USP <Chapter 1229>, “Sterilization of Compendial Articles,” 

, Sections 5.1.1-5.1.2 10th ed. (EDQM, Strasbourg, France, 2020).
8. S.A. Tawde, “Particulate matter in injectables: Main cause for recalls,” 

 online 03 (1) (2015).
9. J.S. Eglovitch, “FDA: Despite Improvement, Particulate-Related Injectables Recalls Remain a Concern,” 

 (April 25, 2019).
10. D. Clarke et al., 

 18 (9) 1063–1076 (2016).
11. K. Wronski, M.K. Bates, and L. Low, “Compliance Testing Demonstrates a New CO₂ Incubator Merits Certification for Use in Grade A/B Environments,” Thermo Fisher Scientific, pending publication (2021).
12. S. Shen, L. Yuan, and S. Zeng, 

, 21 (12) 973–978 (2009).
13. C. McDermott, et al., 

Vol. 45, No. 8
August  2021
Pages: 36–38

When referring to this article, please cite it as M. Bates, “Designing Incubators for Cell Therapy Manufacturing,” 

Articles

Controlling conditions and preventing contamination are crucial for cell growth.