How to Develop a Robust Viral Safety Strategy for Gene Therapies

adeno-associated virus, viral vector

Gene therapies offer a promising hope of cure against rare diseases and other diseases like cancer, with over 1,000 ongoing clinical trials. The increasing need for safe and effective gene therapies is emphasizing the importance of viral safety within the viral vector manufacturing process. This will enable meeting global regulatory timelines and providing faster access to innovative therapies for patients in need.

Gene therapies are evolving to become promising therapeutic options for patients with rare diseases. But so far, scientists have just explored the tip of the iceberg with gene therapies; comparable to where the healthcare industry was with monoclonal antibodies roughly three decades ago. 

As the fastest growing sector in biotechnology, the gene therapy market is bracing to meet the high unmet medical needs of rare disease patients. Pharma and biotech companies are realizing that to enable increased access to gene therapies, production processes need to be improved and optimized.

The science behind viral vectors and their production 

Viral vectors are used to deliver the corrective genes into patient cells to trigger the production of proteins that are missing in patients with rare diseases. A commonly used virus for this purpose is the adeno-associated virus (AAV).

Despite the complexity of viral vectors, the process template to manufacture and purify them does not vary significantly from that used for traditional molecules such as monoclonal antibodies.

The upstream process involves growing cells in a suitable medium with the intention to produce high concentrations of the desired viral vector. A typical AAV production workflow uses specialized mammalian or insect cells, known as packaging cells. These cells serve as hosts for the eventual assembly of the viral vector particles containing the gene of interest.

During the upstream process, multiple plasmids, one of which is embedded with the corrective gene, are introduced into the chosen packaging cell type to help produce the desired viral vector particles. To achieve the desired quantities of viral vector end-products, the packaging cells are multiplied inside a bioreactor.

This is followed by the downstream process of breaking open the packaging cells to release the viral vectors. Purification steps follow to isolate the virus from other residual packaging cell contaminants, such as cell debris, cell protein, cell DNA, and cell culture media components. 

The purification process usually begins with clarification, which uses a combination of filtration and centrifugation technologies. The harvested material is treated with nucleases to eliminate any extraneous DNA, such as through Benzonase® endonuclease treatment.

Chromatography is also leveraged amidst other filtration steps (like Tangential Flow Filtration and 0.2µ filtration) to obtain a cleaner, concentrated end-product.

The severe consequences of viral contamination

Ratish Krishnan, Merck, viral safety, viral vectors, gene therapy
Ratish Krishnan, Senior Strategy Consultant, Novel Modalities, Merck

It is crucial to ensure viral safety throughout the entire manufacturing process. “Virus contamination can happen anywhere in the process chain from the point of origin to the point of use,” explained Ratish Krishnan, Senior Strategy Consultant, Novel Modalities at Merck*. 

“If customers are using serum-containing media, this adds possible contamination risks via the raw materials used. Contaminants can be generated during other production steps as well. Adventitious viruses are one such type of undesired contaminant that can be unintentionally introduced during a manufacturing process.” 

Contamination can result in lost batches of viral vector products, triggering investigation incidents, subsequent plant shutdowns, and decontamination efforts, and could result in financial losses worth millions of dollars. Compromised viral safety can also result in more serious clinical consequences, such as adverse events in patients.

These grave consequences of failing to uphold viral safety in their processes have driven many biotech companies to actively study and invest in robust viral safety strategies. 

This can entail implementing viral, DNA, or bacterial contaminant clearance technologies throughout the production process. The adventitious viruses are inactivated either via a pH detergent treatment or by removal methods such as chromatography and/or filtration. 

The challenges with ensuring viral safety 

Viral safety techniques currently employed in an AAV production process are heavily borrowed from processes used for monoclonal antibodies.

“Monoclonal antibodies have been around for a couple of decades now and their production is well established, and processes have been templated. However, in the case of AAV production, the industry is still on a learning path,” cautioned Krishnan. 

Monoclonal antibodies tend to be smaller molecules compared to the viral vectors, and considerations for purity, concentration, and scalability of the vectors are still evolving.

Scientists still need to understand how the traditional approaches established for monoclonal antibodies can be optimized for viral vectors and evaluate where new products or processes need to be developed.

Krishnan added: “The end-product in gene therapy is a virus particle, which brings with it the obvious challenge of differentiating your product of interest from other undesired viruses. This means having a clear strategy is important even in early stages of process development.”

The emerging regulatory expectations around gene therapies also pose a major challenge. The open guidance available on the market does not specify the level of viral clearance or end-product purity that companies need to aim for, which makes it tricky to devise production and viral safety approaches for these therapies.

Established contract development and manufacturing organizations (CDMOs), such as Merck’s BioReliance® viral and gene therapy CDMO business, can leverage their experience to address these challenges.

By working in close collaboration with a CDMO, biotech companies can ensure that viral safety is maintained throughout the viral vector manufacturing process.

An integrated viral safety assurance approach

Providing an insight on the proven viral safety assurance approaches Merck has optimized, Krishnan said, “We recommend a multifaceted approach towards viral risk mitigation. The three pillars we have found to be essential in keeping adventitious viruses out of your adeno-associated viruses are prevent, detect and remove.”

Merck, viral safety, viral vectors, gene therapy manufacturing
The three pillars that are essential in keeping adventitious viruses out of adeno-associated viruses

The first pillar, prevent, ensures that the creation of an environment suitable for undesired viruses is excluded in the product stream. This can be done by careful selection and pretreatment of raw materials, through pre-treatment such as high-temperature short-time processing and nanofiltration, as well as including stringent checks throughout the viral vector production process as early as raw material sourcing and selection.

The next pillar, detect, incorporates different types of testing throughout the process, from cell bank biosafety tests to assays that reliably detect potential contamination in the upstream or downstream steps. 

And the last pillar, remove, uses state-of-the-art nanofiltration and chromatographic separation technologies alongside optimized, sterilized reagents to remove adventitious viruses. 

“We focus on providing a comprehensive and integrated approach ensuring we systematically cover the three important pillars. Our offerings include key bioprocessing products used in AAV production coupled with our manufacturing expertise, various testing services, access to our viral clearance labs, and more,” summarized Krishnan.

“We choose optimization strategies that are suited to specific customer requirements. Our holistic approach builds on our experience in the viral vector space, the latest best practices in the industry, enabling us to suggest additions that provide added insurance throughout the customer’s production process.”

The future of viral safety in gene therapies

Today, the growing number of clinical trials using AAV provides a sense of optimism for the treatment of rare diseases. 

With a promising product in the pipeline, it is ideal to devise a comprehensive biosafety approach from early development stages, explained Krishnan: “During process development, biosafety tends to get overlooked. However, at later stages, redesigning the process is a difficult challenge that could have been avoided with proactive thinking.”

With increasing gene therapy market approvals, regulatory guidelines will get clearer, and planning ahead will become even more crucial. Having a robust viral clearance strategy is going to become an expectation, rather than a nice-to-have, predicted Krishnan. 

As regulatory authorities are working closely with manufacturers to realize the commercialization of novel therapies, technology providers like Merck, with access to the required expertise and services, are optimizing their approach based on evolving regulatory landscapes and customer needs. 

Merck will continue to be a key partner for our customers in the viral safety assurance space. We will continue to generate data on our existing bioprocessing portfolio, and introduce purpose-built products and services with the end goal of solving our customers’ toughest challenges in gene therapies,” concluded Krishnan.

To hear more from Ratish Krishnan on how to implement techniques for adventitious virus removal in your viral vector process, check out this recent webinar or visit the company website to speak to the expert team!

*The life science business of Merck operates as MilliporeSigma in the U.S. and Canada.

Images via and Merck

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