Viral Vectors (VV), Capsids

Viral vectors illustration

Gene therapy and vaccines use viral vectors to protect the nucleic acid administered into patients and to direct it to a target tissue. Although significant advances have been achieved for the purification of viral vectors during manufacturing by chromatography and filtration, the separation of full and empty viral particles (capsids) often remains unresolved, as well as the discrimination from capsids with undesired cargo (truncated genes, host-cell DNA, plasmid DNA). This can result in potential patient safety and drug efficacy issues, due to significant dosing of empty or undesired viral particles, raising concerns in respect to immunogenic and other adverse reactions. Therefore, a thorough analysis of the full/faulty/empty viral vector ratio is required as extended characterization to establish, transfer, scale, and validate a robust pharmaceutical manufacturing process and a safe drug product.

Analytical ultracentrifugation is the gold standard for the investigation of these important aspects. Appropriate detection systems and experience in experimental design and interpretation of results is required to resolve the different species and to identify their nature by more than just educated guesses. Nanolytics offers more then 20 years of dedicated experience in AUC and industry leading proprietary detection systems. Our expert team can support you in designing appropriate experimental strategies, reproducibly executing complex setups, and interpreting the results to provide insights into your product and process.

CASE STUDY: AAV capsids loaded with DNA.

The case study is a complex system of AAV capsids, partially empty or loaded, with various aggregates. The system is unraveled by a combination of sedimentation velocity and density gradient techniques. The components are addressed by a threefold access: by sedimentation rate, by density, and by spectroscopic properties.

The upper panel shows the sedimentation coefficient distributions measured with absorbance at two wavelengths. Whereas the left species represents empty capsids, as can be confirmed by the A280/A260 ratio, the others represent a mixture of filled capsids and dimers of empty capsids, cosedimenting at similar rates. Yet higher aggregates are found in low concentration at high sedimentation rates. In a density gradient, these species are separated by their density alone, and the species’ absorbance ratios allow to unambiguously identify the three major species as empty capsids (monomers and dimers) and partially/fully loaded capsids. Their density allows to calculate the composition orthogonally.