Introduction to Cell Substrates in Vaccine Production:
Cell substrates have been a never-ending source of concern about the potential for transmitting infectious diseases and/or cancer. The initial key decision in selecting cell substrates for vaccine production was to minimize the risk of cancer transmission through live viral vaccines. This led to the restriction of acceptable production cells to those obtained from primary tissues, while excluding cells derived from human tumors—such as HeLa cells.
Risks Associated with Tumorigenic Cell Lines:
Since immortal cell lines like HeLa are tumorigenic, they are avoided in vaccine development, as vaccines are administered to healthy individuals for disease prevention, making safety just as critical as effectiveness. Therefore, the risk of the oncogene being passed on in the cellular DNA, which may unavoidably be a component of the vaccine’s active ingredient, makes the risks associated with tumorigenic cells—even if they are not intact in the product—unacceptable.
Regulatory Evolution and WHO Guidance:
Numerous conferences have led to a scientific consensus that there are three main potential issues with animal-cell substrates: transforming proteins, viruses, and DNA. In 1986, WHO created a WHO Study Group on Cell Substrates to explore cell substrate concerns in greater depth. The Study Group came to the conclusion that CCLs are generally appropriate when it is shown that the production process removes potentially hazardous human infecting viruses, lowers DNA to tolerable levels, and/or stops its biological activity.
They also came to the conclusion that CCLs should not be disregarded as potential biological production substrates.
The guidelines defining acceptable levels of Residual Host Cell DNA (cDNA) in biological products by various regulatory agencies have evolved in the last decades. The WHO increased the acceptable limit to ≤10 ng per parenteral dose which was previously determined to be ≤100 pg in 1987.
Use of Tumorigenic CCLs in Vaccine Development:
During the 1990s and into the 2000s, a variety of CCLs were explored as cell substrates for biological products in development because, like the cell lines referred to above, they offered significant advantages during production (e.g., rapid growth and high expression). They include the tumorigenic cell lines –
- HeLa for adeno-associated virus vector HIV vaccines
- PER.C6 for influenza and HIV vaccines
- Madin–Darby canine kidney (MDCK) for influenza vaccines
Cell lines that appear to have an infinite capacity for population doubling are known as continuous cell lines. Previously known as “established,” it is frequently referred to as “immortal.”
Vero Cells- A Preferred Continuous Cell Line:
The most commonly used cell line for vaccine production by regulatory bodies at the moment is the Vero cell line, which was the first continuous mammalian cell line created by African green monkeys in 1962. This is because Vero cells may be cultured and infected on micro-carrier beads in both serum-free medium and large-scale fermenters without reducing productivity. The PER.C6 cell line was the first tumorigenic cell line to be evaluated for use in the creation of a live virus vaccination.
It was used for the development and production of a replication-defective adenovirus vectored HIV-1 vaccine
When developing viral vaccines, it is essential to consider and address several safety concerns. These include the possible presence of remaining live cells in the final product that could pose a tumorigenic risk to humans, traces of residual DNA from the cell substrates used, and the potential contamination by unintended agents—such as hidden viruses—that may have played a role in the tumorigenic nature of the original cells.
Key Safety Considerations in Viral Vaccine Development:
Since the 1980s, some CCLs have been employed to produce safe and efficient vaccines and biotherapeutics.
Despite advantages of CCLs based on consistency and reproducibility of the reconstituted cell populations for an indefinite period by facility of cell bank system, easier growth than DCLs, adaptation to grow in a serum-free medium, ability to be grown on microcarriers for large production in bioreactors, there are various risks associated with use of CCL in viral vaccine formulation as CCLs may express endogenous viruses, and some are tumorigenic in immunosuppressed animal models.
Human and non-human primate lymphocytes and macrophages may carry latent viruses such as herpesviruses and retroviruses. CCLs of non-hematogenous cells from human and non-human primates may contain viruses or have viral genes integrated into their DNA. Since Animal cell substrates are commonly used in the manufacture of biological products, (rcDNA), they may remain in the final manufactured product. The ability of DNA to cause a normal cell to change and maybe become tumorigenic could be the source of its oncogenic activity. The major mechanism through which this could occur would be the introduction of an active dominant oncogene since such dominant oncogenes could directly transform a normal cell. Other mechanisms would require that the rcDNA transforms through insertional mutagenesis, and have been considered less likely since the frequency of integration of DNA is generally low.
Oncogenic activity from cell substrates could be due either to the cell substrate DNA or to an oncogenic viral agent present in the cells. When appropriate, and particularly for vaccines, cell DNA and cell lysates should be examined for oncogenicity in a test approved by the NRA/NCL.
It is necessary to examine the oncogenic risk of DNA when considering the use of tumorigenic cells in biological production. This would be particularly crucial for live attenuated virus vaccines, as chemical inactivation of the DNA is not feasible and nuclease digestion and DNA quantity reduction are the only ways to lower the biological activity of DNA.
For products such as monoclonal antibodies and subunit vaccines manufactured in tumorigenic cell substrates, it is necessary to demonstrate the clearance of DNA by the manufacturing process. There may be instances where CCL DNA is considered to pose a higher level of risk because it contains specific elements such as infectious retroviral sequences. Under these circumstances, steps are taken to reduce the risks of cDNA. The small non-coding RNA molecules – microRNA (miRNA) – that are more stable and have the capacity to modulate gene expression can contain certain miRNA genes that can be oncogenic.
Growth-promoting proteins may be secreted by cells that are used to produce biologicals, but the risks from these substances are limited but in exceptional circumstances, growth factors may contribute to oncogenesis.
References:
- Technology Networks Cell Science (2024). Cell Culture Basics: Equipment, Fundamentals and Protocol
- World Health Organization (2010). Recommendations for the evaluation of animal cell cultures as substrates for the manufacture of biological medicinal products and for the characterization of cell banks