Part 3: Chromatography Purification for Viral Vectors

The growing number of viral vectors used in the production of therapeutics requires scalable processes for their purification. How can chromatography support the application of viral vectors and other new modalities?

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Chromatography is a fundamental technique for viral vector purification in GMP processes, especially at large scales. At smaller scales, research teams often opt to use ultra-centrifugation instead. However, when scaling up, ultracentrifugation is not viable, and chromatography techniques are typically used instead. This transfer represents a significant challenge for the developmental journey of a viral vector. The shift from ultracentrifugation to chromatography might change the viral product's critical quality attributes (CQAs), leading to issues such as regulatory hiccups. It is essential to understand the properties of your viral particle, exploit this knowledge for purification, and avoid changes in technologies as you develop your process.  

Viral vectors are very diverse, which introduces the first hurdle: there is no "one-size fits all" purification approach that will work for all viruses and vectors. Even within one virus family, such as adenoviruses, there are significant differences in the spike proteins on the surface of the viruses that affect how easily they can be purified.

Typical Impurities in Viral Vector Purification  

Many viral vectors like adenoviruses have to be produced from cell cultures and need a lysis step. This inevitably introduces a lot of cellular debris into the virus preparation, which must be removed. Careful handling is essential as the viral particles can become entangled with cellular contents (such as genomic DNA), making them much harder to purify.  

Another important consideration is possible contamination with several other types of viral particles. During the lysis steps, the virus preparation may become contaminated with other viruses. It is also inevitable that the virus preparation will contain the right viruses packaged with a different DNA sequence or containing no DNA whatsoever. These can be difficult to remove because, for most separation techniques, viral particles with and without a nucleic acid payload seem to be exactly the same.

Properties That Aid Separation  

All viral vectors have unique protein motifs on the surface, which lend themselves to purification via affinity chromatography (AC). The purpose of these protein motifs is to bind cellular ligands. If a similar ligand can be used during chromatography, it would be possible to capture the viral particles.  

However, there is a significant downside to using affinity chromatography. Currently, there is no universal ligand to capture even one family of viral particles such as AAVs. This means that every time the virus particle changes, it is possible that the capture ligand would also have to change. This can be time-consuming, expensive, and not conducive to platform production processes.

A useful solution to this problem is to use an ion exchanger instead. Using ion-exchange chromatography (IEX) for the capture step means that the same column can be used for every virus serotype. Cation exchange chromatography can remove the majority of process- and product-related impurities when properly developed, making it an attractive option for a platform process.

Regardless of the capture step, the final polishing step almost always utilizes anion exchange chromatography (AEX) for the separation of empty and full viral capsids. The subtle charge difference between empty and full capsids allows AEX to achieve sufficient resolution of the two particles, which would otherwise co-elute with different chromatography modes.

If the viral preparation is dirty, a hydrophobic interaction chromatography (HIC) step can be included before the capture step to clean up the preparation before capturing the viral particles.

Matrix of Choice  

At the time of writing, there is only one drawback to monoliths and membranes: there are limited binding ligands available. Viral vectors are incredibly diverse. Even viruses in the same family can have different spike proteins. This feature makes developing a platform process based on selected monolith and membrane matrixes challenging, if not impossible. Therefore, in some cases, it is necessary to use resins to purify viral vectors. Resins are more established and available with a wide range of ligands, while the monoliths and membranes are still catching up.

Viral Vector Production Relies on Efficient Chromatography Solutions

Most gene therapy applications rely on the use of viruses as vectors. The stringent and reliable purification of viral vectors is essential for the safety and efficacy of advanced therapies. Learn more about the application of chromatography methods to the production of viral vectors and other emerging modalities.

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