Cell Culture Contamination in Research and Development
It is vital that potential contamination risks are managed throughout the cell culture process.
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Cell culture underpins many aspects of research and development, from laboratory experiments to large-scale manufacturing processes in the food and pharmaceutical industries. Managing potential contamination risks is, therefore, an important concern for researchers and manufacturers alike and must be factored in from the earliest stage of selecting cells and reagents, through to purifying and quality-assuring end products.
In this article, we hear from three experts working in or alongside academic, industry and clinical settings to help ensure the integrity and safety of cells and cell-derived products.
Ensuring the integrity of cell lines
As the source of starting cell lines for experiments and production processes, cell banks must have strict processes and procedures in place to detect and prevent contamination. There are multiple types of contamination to consider: infectious agents, misidentified cells and contamination with undesired chemical contaminants.
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When it comes to infectious agents, the main challenges are mycoplasma infection, and bacterial or fungal contamination caused by poor aseptic practices, said Laura Steenpass, head of the Human and Animal Cell Lines Department at the Leibniz Institute DSMZ – German Collection of Microorganisms and Cell Cultures GmbH.
“Mycoplasma are not a new problem, but a challenge because you just can’t see this infection when you look at your cultures daily. It depends on the type of cells you’re culturing; some of them might sense mycoplasma and react with a change in their proliferation after a few weeks but then they are already well established.”
It’s therefore essential to routinely test for mycoplasma. When the DSMZ receives a new cell line, they quarantine it in a specific culture area and as soon as they have enough material, they test for mycoplasma using a PCR-based assay.
“This step is more efficient thanks to ready-mixed PCR primers that can detect different mycoplasma,” said Steenpass. “There are also several rapid panel-based assays for mycoplasma, some of them using fluorescently labeled compounds that bind to the mycoplasma to give a fluorescent readout.”
If the cells are negative for mycoplasma (and common viruses, also tested with a panel), they are expanded and frozen down to a master cell bank. The next step is to create a working cell bank, which is tested for mycoplasma again before being released. Only then, if negative, will the cells be used for experiments or for distribution to others.
The two most common sources of mycoplasma contamination are humans – we carry it, for example, in our nasal mucus – and cell culture supplements. Good aseptic cell culture techniques can mitigate the former, and the latter is usually solved by screening or treating cell culture reagents before use.
However, the use of animal-derived products also brings additional contamination risks. For example, serum containing the remnants of antibiotics is a major problem in assays where antibiotics such as tetracycline are used to induce or select for certain cell phenotypes.
“There are initiatives to replace the use of animal-derived ingredients with chemically defined media for cell culture for both ethical and practical reasons,” said Steenpass. “Chemically defined media is expensive, but it can solve some of the other challenges with animal-derived cell culture products, such as inter-batch standardization and antibiotic contamination.”
A further source of contamination is cell mixing, and testing for cell authenticity is one of the first steps that occurs before a cell line can be expanded into master and working cell banks. “For human cells, this is reasonably straightforward and requires serotyping cells using short tandem repeat (STR) analysis – akin to paternity testing for cells,” explained Steenpass.
For other cell lines such as mouse cells, which are more genetically similar, specific protein markers are required to distinguish between, say, a kidney or a liver cell. For fibroblasts which lack clear differentiating markers, this is even more of a challenge. “In the future, it’s likely this step will be carried out using single nucleotide polymorphism (SNP) arrays,” said Steenpass. “These are more accurate than STRs but are also currently more expensive.”
Rapid detection of contamination in industrial processes
Challenges with cell culture contamination are a significant consideration during the development and manufacture of many cell-derived products – especially an increasingly wide range of biopharmaceuticals. All biological drugs are made using cell culture, which starts in early development with small stable cell line cultures and is scaled up in later development and in product manufacturing to large bioreactors, reaching 10,000–20,000 liter capacity.
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“At this scale, the risk and consequences of contamination is high,” explained Dr. Chikkathur N. Madhavarao, a biologist at the US Food and Drug Administration (FDA), Center for Drug Evaluation and Research. “It takes just one bacterium to get into the culture, and within a few hours to a day, the whole batch will be contaminated, leading to high costs not only through wasted production reagents and time but also the costs of handling the contaminated product and cleaning the bioreactor. It’s crucial to detect any contamination as soon as possible to reduce production downtime.”
Most large-scale cell culture for biopharmaceuticals are also done in the absence of antibiotics, because traces of antibacterial agents could remain in the product and even small amounts could be harmful to patients who may be immunogenic to them. This makes it paramount to detect subtle changes in bioreactor cultures quickly and sensitively.
“There are some routinely used process parameters such as dissolved oxygen content and medium pH that are continuously monitored using in situ probes in bioreactors,” explained Madhavarao. “If the culture is contaminated, you’ll see a rapid consumption of oxygen and a drop in pH as the contaminant microbes consume glucose and produce lactic acid.”
However, while studying the production of an IgG1 monoclonal antibody, Madhavarao and colleagues found a new process parameter that could be used to detect contamination. An in-line conductivity/capacitance probe was being used to monitor the growth of Chinese hamster ovary (CHO) cells in batch cultures.
“We found there was something strange happening with conductivity measurements when we had a contaminated culture,” he explained. “Abnormal increases in conductivity consistently corresponded to bacterial contamination.”
They verified this by growing bacteria independently outside the bioreactor setting and titrating the bacteria and found that conductivity matched the levels of bacterial growth.1 Moreover, contaminated bioreactor runs had sharp increases in conductivity rates hours before levels of dissolved oxygen dropped.
“These probes are already routinely used in bioreactors, so it’s now feasible to use this approach as an additional parameter that can detect bacterial contamination at an earlier stage than other process parameters,” said Madhavarao.
Collaborating to prevent contamination
Contamination with viruses is a particular concern in the development and manufacturing of biopharmaceuticals as they are more challenging to detect.2 The Consortium on Adventitious Agent Contamination in Biomanufacturing (CAACB) was established to share industry information about contamination issues and events, to promote learning and sharing across the sector.
“Although viral contamination is rare in biopharmaceutical production, there were silos of information within companies and unless there was a material event where a company had insufficient material to provide to patients, there’s no obligation to report instances of contamination,” said Stacy Springs, executive director, MIT Center for Biomedical Innovation (CBI).
The CAACB started with a project collecting data on virus contaminations in biomanufacturing. A pilot started out with six companies that worked with the CBI to create a comprehensive set of questions that were both valuable and feasible for industry to answer.
Today, the consortium is a living organization with about 25–30 companies involved and sharing data on an ongoing basis. The results have helped to highlight different contamination risks for different cells, likely sources of contamination and the effectiveness of various detection and viral safety methods.
“Importantly, our findings show that the three main pillars of viral safety work effectively – namely, selection of low-risk raw materials, testing of cell banks and in-process materials, and removal or inactivation of potentially undetected viruses during production purification,” said Springs. “But with more cell and gene therapies now in development, it is intrinsically more difficult to utilize all three pillars of viral safety, because you’re unable to conduct the final step of viral clearance.”
Another area that’s changing is the more predominant use of next-generation sequencing (NGS). Following a revision to the ICH Q5A(R2) viral safety guideline, companies are beginning to assess when and how they can replace in vivo viral safety tests with NGS, for example in cell bank testing or in product testing.
“We surveyed the CAACB membership to assess the value of the in vivo virus test. We found that there wasn’t a single case where the in vivo test was positive for a virus contamination that was not also detected in another parallel assay. This data, as well as data on reliability, questions the value of the in vivo assay and highlights the need for alternatives such as NGS,” said Springs.3 “I think the implementation of NGS will become very important in the future and it’s something the regulatory community is also exploring and embracing.”
The CBI is now launching a new effort to survey which bacteria and fungi have been affecting production of cell and gene therapy products. This initiative will extend beyond the core CAACB membership because considerable data on these products exists in academic hospital settings or other GMP environments that are not necessarily the company manufacturing them.
“In the 30+ years of using recombinant DNA technology for making these kinds of drugs, there has never been a pathogen safety issue – I think that says a lot to the fact that current safety measures work really well, but we have to be ever vigilant, especially as so many more of these biologic drugs are in development.”
About the interviewees:
Prof. Laura Steenpaß is Head of the Department of Human and Animal Cell Cultures at the Leibniz Institute DSMZ and Professor of Cell Biology at the Zoological Institute of the Technische Universität Braunschweig. The focus of her work at the DSMZ is on the use of human cancer cell lines and pluripotent stem cells for research into genetically caused diseases.
Dr. Chikkathur N. Madhavarao is a biologist in the Office of Pharmaceutical Quality Research in CDER, US FDA, that investigates the process and product quality relationships. Dr. Madhavarao has been investigating for over 12 years at FDA the impact of variations in manufacturing process variables on the critical quality attributes (CQAs) of therapeutic proteins and the novel application of process analytical technology (PAT) tools to improve process understanding and control.
Dr. Stacy Springs is the executive director at the MIT Center for Biomedical Innovation (CBI).
The Center integrates the Institute’s technical, scientific, and management expertise to solve
complex biopharmaceutical challenges. CBI leads multi-stakeholder, multidisciplinary research and educational initiatives with real world impact, including MIT’s Biomanufacturing Consortium, (BioMAN), and its Consortium on Adventitious Agent Contamination in Biomanufacturing, (CAACB).
References:
1. Morris C, Madhavarao CN, Yoon S, Ashraf M. Single in-line biomass probe detects CHO cell growth by capacitance and bacterial contamination by conductivity in bioreactor. Biotechnol J. 2021;16(12):e2100126. doi: 10.1002/biot.202100126
2. Barone PW, Wiebe ME, Leung JC, et al. Viral contamination in biologic manufacture and implications for emerging therapies. Nat Biotechnol. 2020;38(5):563-572. doi: 10.1038/s41587-020-0507-2
3. Barone PW, Keumurian FJ, Neufeld C, et al. Historical evaluation of the in vivo adventitious virus test and its potential for replacement with next generation sequencing (NGS). Biologicals. 2023;81:101661. doi: 10.1016/j.biologicals.2022.11.003
