When we consider the various structural aspects of the VP proteins that need to be investigated – disulfide bridges and the associated higher order structure, N and C-termini, glycosylation and post translational modifications (PTMs) – it is clear that such an assessment falls within the ICH Q6B guidelines, which is the accepted guidance document for characterization of protein biotherapeutics. The use of mass spectrometric techniques in conjunction with various on- and off-line liquid chromatographic techniques produces a detailed structural understanding of the VP coat proteins and their PTMs and is applicable to different AAV serotypes. Furthermore, the application of these techniques, or at least a subset of them, during production of the gene therapeutic can highlight if any deleterious events have occurred to the proteins resulting in, for example, increased deamidation which could give cause for concern with regards to efficacy of the product. Of course, since therapies derived from adeno associated viral vectors require that the protein and nucleic come together to produce correctly packaged and active particles, it is necessary to analyze the final, packaged product to assess for the degree of capsid filling. This can be performed by analytical ultracentrifugation, which can also be used as a mechanism of identifying fragments and aggregates in the sample alongside the technique of differential light scattering.
It is worth bearing in mind that ICH Q6B also covers process impurity analysis such as process residuals and Host Cell Proteins (HCPs). The gene therapy guidelines from the FDA recommends that these types of components are screened for in an impurity analysis (1).
1. Chemistry, manufacturing and control (CMC) information for human gene therapy investigational new drug applications (INDs) Guidance for industry. FDA 2020.
2.Giles, A.R., et al. (2018). Deamidation of amino acids on the surface of Adeno-Associated Virus capsids leads to charge heterogeneity and altered vector function. Molecular Therapy, 26, 2848-2862.