At the completion of each large-scale production run, the rAAV-containing fractions undergo analysis for a range of characteristics considered important for rAAV preclinical development. In a 100-liter preparation of rAAV6-GFP vector, the vector eluted from the immunoaffinity column in a 2-liter fraction () was concentrated to 40
ml. Analysis by CsCl isopycnic gradient centrifugation showed that approximately 50% of the capsid proteins corresponded in density to full, or DNA-containing, capsids (, top). Both quantitative polymerase chain reaction (qPCR) and fluorescent dye binding (SYBR Gold; Molecular Probes Invitrogen, Carlsbad, CA) of extracted vector DNA were used to quantify the nucleic acid content in the vector fractions. With highly purified vector preparations, the values generated by the two assays were similar (e.g., Fraction 19, qPCR, 4.59
vg/ml; direct DNA measurement, 5.83
vg/ml) (, bottom). The extracted vector DNA, analyzed by agarose gel electrophoresis, was of the predicted size (Urabe et al.
). Determining the percentage of empty and filled capsids by electron microscopy (EM) in the vector preparation (, top) is more convincing when used with CsCl isopycnic gradient-processed materials corresponding to a high-density fraction (for filled particles) (, middle) and a low-density fraction (for empty particles) (, bottom). A second orthogonal assay, such as CsCl isopycnic gradients and subsequent analyses, strengthens the filled particle content estimation. Determining particle dispersion in solution, that is, aggregation state, using dynamic light scattering, is important especially when establishing the rAAV formulation, for example, concentration and excipient composition, as well as storage conditions. The appearance of a single peak at approximately 20
nm indicates that the particles are monodisperse (). Electron microscopy provides useful information about the capsid morphology and corroborates other results regarding aggregation, concentration, and empty versus filled capsids. In vitro
bioassays provide convenient methods for determining the relative potency and for defining the in vivo
dosage base. However, there are shortcomings because not all vectors express readily assayable products, for example, small interfering RNA (siRNA), and in vitro
transduction efficiencies in commonly used cell lines, for example, HEK-293 cells, vary widely depending on vector serotype.
FIG. 5. (A) Elution profile of rAAV6-GFP from AVB Sepharose HP. The clarified insect cell lysate (100 liters) was applied (0.3 liter/min) to the 20-cm column and washed with 10 liters of phosphate-buffered saline until the ultraviolet (UV) absorbance stabilized (more ...)
To date, we have successfully produced rAAV with essentially wild-type capsids for serotypes 1, 2, 4, 6, and 8. The methods introduced by Urabe and colleagues for serotype 2 served as the template for generating these other AAV serotypes. So far, AAV5 remains the exception to this process, in that VP1 was not expressed at the desired level. Rather than evaluating the codon usage for the type 5 cap gene, substituting the type 5 VP1 unique region with the AAV2 homolog abrogated the problem. Also, AAV4 capsids are poorly expressed, resulting in low vector yields. However, not much effort was expended for producing AAV types 4 and 5. It is possible that the difficulties with these two serotypes result from codon usage differences between invertebrates and mammalian cells.
If the protein and nucleic acid components comprising rAAV particles produced in invertebrate and mammalian systems are indistinguishable, then the activities of the vectors are also expected to be indistinguishable. An independent laboratory tested insect cell-produced rAAV2-GFP, using C12 cells infected with adenovirus, and reported 2.17
GCU/ml and 4
VG/ml (GCU, green cell units; VG, vector genomes determined by qPCR). The GCU:VG ratio was 18.4, which is considered a low value for rAAV2 produced in either insect or mammalian cells (R.O. Snyder, personal communication, January 2002). In other cases, the lower infectious activities of rAAV preparations were attributable primarily to the particle composition as well as to the presence of process impurities. As described previously, the particle composition is readily assessable by polyacrylamide gel electrophoresis with silver staining and Western blotting. Expression of relatively low levels of VP1 resulted in low-infectious particles. However, as discussed, the stoichiometry of the capsid proteins is adjustable by altering the initiation codon, thereby producing fully active vector particles.