Genetic stability studies: could process parameters change your virus?

By adjusting your process conditions, you may increase productivity, but how does it influence the genetic stability?
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Written by expert: Monika, Associate Director Viral Vector Development

Despite having very limited coding capacity, most of the RNA viruses are surprisingly good at escaping the hosts immune response and withstanding antiviral drugs. RNA viruses achieve this by multiplying rapidly, coupled with high mutation rates compared to DNA viruses. An important cause for the high mutation rate of RNA viruses is caused by their RNA polymerase enzyme. This viral enzyme, which is responsible for copying the genetic code of the virus, lacks proofreading ability. Therefore, mistakes made by the polymerase enzyme during replication are not corrected. This results in a pool of different genotypes in the virus population, ready for adaptation. If circumstances are changing, the genetic variability of RNA viruses allows them to quickly adapt to the new situation.

In a natural environment, this change in circumstances can be for example the immune response of the host. However, during manufacturing of a vaccine, process conditions like pH, medium and temperature could put a selection pressure on your virus population. This could have far-reaching consequences on your final product concerning immunogenicity and even vaccine safety.

Example: genetic stability of poliovirus

With a 99% reduction in polio cases worldwide since 1988, the global eradication of polio is now in sight. Since the 1950s, two vaccines are used to protect people from polio infection; an inactivated polio vaccine (IPV) and an oral polio vaccine (OPV). IPV is produced using an inactivated virus either based on wild type polio strains (cIPV) or on live attenuated strains (sIPV) and is administered by injection. OPV is a live-attenuated (weakened) vaccine, administered orally. OPV is significantly cheaper than IPV, but its nature as a live RNA virus bears the risk that it could evolve over time into more neurovirulent variants.

Mutations occurring on the surface-protein regions can change the immunogenicity of the virus. Meaning, the vaccine will generate a different immune response in the host than intended. This is a concern both in the production of IPV and OPV. It is essential to monitor the changes in the genetic content of poliovirus strains during the production process. We have to avoid that process conditions are changing the virus in a non-desired way, and ensure the final product contains the intended virus strain.

Testing the genetic stability

The genetic stability can be tested by serial passages of the virus followed by next generation sequencing (NGS) analysis after each passage. NGS is a relatively new approach. This approach allows researchers to analyze each nucleotide position of the viral genome. Thereby, we can detect all mutations present in the virus population. By analyzing various passages of the virus, the changes and possible drifts in genetic content can be traced over the consecutive viral replication cycles. The impact of certain process conditions on the mutation rate can be investigated using a DoE (design of experiment) study. This allows us to assess the impact of individual process parameters. Additionally, it allows us to see the combined effect of the parameters on certain mutations.

It’s not only titer that matters

By adjusting your process conditions, you may increase productivity, but be aware, it is not only titer that matters. Your increased yield could be a result of a major genetic drift that happened in the virus population while the virus was adapting to the new circumstances. Mutant analysis by PCR and restriction enzyme cleavage (MAPREC) is the currently preferred in vitro test for the monitoring of vaccine safety and consistency during production. NGS can be a good alternative to MAPREC. The use of NGS is increasingly supported by regulatory agencies. We strongly advise to check the genetic stability of your virus during the production process. Additionally, we advise to check the genetic content of your final product. These steps are important to assure process consistency and safety.

We are dedicated to help bring biopharmaceuticals to the market at higher speed, with reduced costs, and with a higher success rate. Batavia Biosciences has vast experience in developing and manufacturing virus-based products. Our experienced virus experts are well equipped to take on any challenge associated with production of virus-based biopharmaceuticals.


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