Pharmacology of Recombinant Adeno-associated Virus Production

Abstract

Recombinant adeno-associated viral (rAAV) vectors have been used in more than 150 clinical trials with a good safety profile and significant clinical benefit in many genetic diseases. In addition, due to their ability to infect non-dividing and dividing cells and to serve as efficient substrate for homologous recombination, rAAVs are being used as a tool for gene-editing approaches. However, manufacturing of these vectors at high quantities and fulfilling current good manufacturing practices (GMP) is still a challenge, and several technological platforms are competing for this niche. Herein, we will describe the most commonly used upstream methods to produce rAAVs, paying particular attention to the starting materials (input) used in each platform and which related impurities can be expected in final products (output). The most commonly found impurities in rAAV stocks include defective particles (i.e., AAV capsids that do contain the therapeutic gene or are not infectious), residual proteins from host cells and helper viruses (adenovirus, herpes simplex virus, or baculoviruses), and illegitimate DNA from plasmids, cells, or helper viruses that may be encapsidated into rAAV particles. Given the role that impurities may play in immunotoxicity, this article reviews the impurities inherently associated with each manufacturing platform.

Keywords: AAV; gene therapy; impurities; manufacturing; quality controls; viral vectors.

Figure 1

Electron Microscopy Images of rAAV Particles Original magnification ×75,000. Electron-dense particles observed by electron microscopy (EM) after negative staining with uranyl acetate correspond to empty particles (white arrow, right panel). A full rAAV particle is indicated by a black arrow.

Figure 2

Schematic Representation of Raw Materials (Input) and Product-Related Impurities (Output) in Each System Used for the Production of rAAV Vectors Asterisk (*) indicates depending on culture medium composition and on the lysis and clarification steps. The percentages of DNA contaminants were published in Lecomte et al., Ye et al., and Penaud-Budloo et al.