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Techniques to Distinguish and Separate Viral Capsids

There can be major differences in therapeutic outcomes from using empty versus full viral capsids

Andy Tay, PhD

Andy Tay, PhD is a freelance science writer based in Singapore. He can be reached at

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Viral vectors are an important tool for gene and cell therapy that are used to pack and deliver therapeutic nucleic acids to target cells. In gene therapy, viral delivery is performed in vivo, while in cell therapy, viral delivery can be done in vivo or ex vivo.

The typical manufacturing process of viral vectors uses adherent cell lines, like human embryonic kidney (HEK293) cells, that are easily genetically engineered, exhibit rapid growth rate, and have the ability to grow in a serum-free culture. During virus assembly, viral capsids—proteins that contain the genomic material of the virus—may be packaged fully or partially with nucleic acids while others remain empty. This exact biology is unclear, but it can be partly attributed to a dysfunctional viral packaging system due to under- or over-expression of the proteins orchestrating the packaging steps.

Viral vector characterization is an essential quality control step. The proportion of empty, partially filled, and full capsids must be determined as the first two types of capsids are considered impurities that may negatively impact product efficacy and safety. Fifty to 95 percent of the total viral vectors generated from the cell culture might be empty or partially filled, and the exact proportion varies by manufacturers, production workflow, and the health of the cells.

Clinical trials have found that empty capsids can induce immune responses and reduce transgene expressions, but even when there is 10-fold excess of empty or partially filled capsids over full capsids, therapeutic efficacy can still be achieved. Interestingly, empty viral capsids may enhance gene transfer by acting as decoys and reducing rapid clearance of fully packaged viral vectors from the body by pre-existing immunity. 

While there is ongoing debate around the functions of empty and partially filled capsids, from the manufacturing and product angle, these capsids represent impurities, so there is great incentive to identify and separate them from full capsids to ensure product consistency.

Techniques for identification

To purify viral vectors, first there must be methods to distinguish the desirable viruses from the contaminants before separating them. Methods for viral capsid characterization are also key to determine whether the purification process works. For instance, after purification and before product release, viral vectors must be characterized again for quality control. Here, we will discuss the more popular methods to identify the different types of viral capsids as these methods can provide rich information on what purifying techniques to subsequently use. Note that in most instances, techniques for viral capsid characterization and purification go hand-in-hand and understanding the strengths and limitations of each method will allow manufacturers to combine methods and produce the best viral vector product.

Electron microscopy is the standard method to distinguish between empty and partially filled capsids. With electron microscopy, manufacturers can use standard sample preparation protocols to visualize the “fullness” of viral capsids and estimate the proportion of full capsids. It is an expensive technique with extremely low throughput, making it unsuitable for routine viral vector characterization but very helpful as a final step for quality control. Manufacturers are encouraged to use the highly accurate electron microscopic method after viral vector purification to make sure that the viral capsids are fully filled before product release.

There is rising interest in developing cheaper methods with higher throughput. Another technique uses differential UV absorbance at different wavelengths. Empty capsids in purified vector samples lower the absorbance ratio, and can be estimated using an established model. As this measurement can be performed quickly and cheaply, it can be used routinely for viral vector characterization. The caveat for this technique is that it requires highly purified samples, as impurities from culture solution can affect the measurements. To capitalize on the strengths of differential UV absorbance measurement, manufacturers can consider using this method at different stages of purification including after each step of ultra-centrifugation and chromatography to characterize the effectiveness of each purification method and protocol. Continual measurements using this quick and cheap method can also help refine the purification protocols, such as changing time for ultra-centrifugation and the flow rate in chromatography.

Electron microscopy and UV absorbance measurements are the more popular methods to characterize viral capsids, but there are emerging methods that offer other advantages. One such technique to distinguish viral capsids is charged detection mass spectrometry (CDMS). Conventional MS has difficulties measuring mass heterogeneity of viral particles, which CDMS overcomes by concurrently measuring the charge of single particles. While CDMS is a sensitive technique, this method requires expensive MS instrumentation and may not be available in most viral production facilities. Manufacturers can consider using this method in a manner similar to electron microscopy, for characterization of the final viral vectors before product release. Nevertheless, the industry’s gold-standard for pure viral vector is still determined through electron microscopy and manufacturers need to first present data to show that CDMS provides comparable performance before this would be widely accepted as an industry alternative.

Finally, there is an innovative use of microfluidic electrophoresis to quantify the presence of genomic and protein content from digested viral capsids. The applied voltages break down viral capsids into their components—genetic cargo and proteins—and by comparing these to a standard reference, the proportion of full viral capsids can be determined. The advantage of this technique is that it is scalable with fast turnaround time. The use of microfluidic channels also means that low sample volume is needed for characterization, making up for an inability to recover viral capsids. The high throughput and small sample volumes are making microfluidic techniques increasingly popular with the biotech industry. Although the use of microfluidics for viral capsid characterization is still new, it is likely we will see similar platforms being deployed in the future. Many microfluidic systems have been developed for cell characterization, purification, and recovery, and while the size scale of cells is different from viral vectors, similar concepts can be applied in the form of nanofluidics for integrated viral capsid characterization and purification. 

Additional capability to separate

Once fully filled viral capsids can be distinguished from empty and partially filled ones, the information can be used to integrate= other methods for viral vector purification. Here, we will describe methods that have integrated characterization (some of which is explained in the earlier section), purification, and recovery.

Viral vectors with different ratios of empty/partially filled and full capsids show different sedimentation profiles, and an analytical ultracentrifuge can be used to monitor their ratios in real time with optical detection and provide a means to purify the samples. Researchers have also combined cryogenic electron microscopy (cryo-EM) with analytical ultracentrifugation, followed   by advanced image processing, to provide a more accurate quantification of full viral capsids. This is motivated by the ability of cryo-EM to visualize viral vectors in their native or near-native states with more accurate characterization of their capsid states. 

Ultracentrifugation can process and purify large volume samples, but it is not as sensitive as chromatography, which can purify samples based on parameters like size and charge. Chromatography is likely the most widely used tool for viral vector characterization and purification in the industry as they can be optimized to isolate vectors with different properties and for small- to large-scale production. 

Size exclusion chromatography is widely used as a polishing purification step for viral vectors as it can distinguish and separate monomeric capsids from bigger aggregates and other impurities. McIntosh and colleagues combined size exclusion chromatography with multiangle light scattering, ultraviolet light absorbance, and refractive index detectors to measure capsid size, aggregation, integrity, and content, followed by recovering of viral capsids based on size exclusion principles. This integrated system is powerful as it enables manufacturers to assess and refine the performances of their chromatography purification protocols using three methods, which can all be performed cheaply and quickly. This can be particularly important when the purification protocol is new or when manufacturers are purifying a new type of viral vector product and protocols have to be tweaked continually to produce high-quality products.

It is useful to note that chromatography columns can also make use of additional exclusion criteria like surface charges for enrichment because fully filled viral capsids are generally less positively charged than empty capsids in a salt solution or buffer. The advantage of using both size and surface charges for viral vector purification is that the product will be of higher purity because more contaminants can be removed. One such method is anion exchange chromatography. The choice of buffer, additives like metal ions and detergents, and elution speed can further enrich the isolation of full viral capsids, depending on the serotypes. For instance, it has been shown that high magnesium chloride concentration better eluted empty capsids and removing sodium chloride from the low pH elution buffer, significantly improved the recovery for recombinant adeno-associated virus type 5. The mechanism in which buffer and metal ions affect chromatography outcomes is poorly understood but it could be due to how a buffer differently affects the availability and binding of metal ions to full and empty viral capsids. Therefore, researchers are encouraged to work with service providers who have optimized purification protocols for different viruses and are able to share these protocols. Although the mechanism is unclear, experimental data is still a powerful tool to know that the choice of chromatography material and process works.

Key takeaways

The structure, purity, and content of viral capsids play an important role in affecting their functions in gene delivery and subsequent clinical outcomes. While there are numerous methods for viral vector characterization and recovery, techniques vary by throughput, sample volume requirement, need for specialized instruments, and ability to resolve intermediate, partially filled viral capsids, making integration of different methods the best option for many. 

Quality checks of viral capsids during manufacturing are crucial to ensure that patients are getting the treatments they need. Methods for viral vector characterization and purification should be considered together as they impact how purification protocols are optimized and determine whether a product is pure enough for release. Quality control technology using methods like chromatography will contribute to formulating the best viral vector products.