Figure 1.

Distribution of adeno-associated virus (AAV)8 particles in cell lysates and culture media changes with time. Four groups (n = 3/group) of HEK 293T/17 cells on 15 cm plates were transfected to produce AAV-FVIII. Cells and culture media were harvested on days 3, 5, 6, and 7 and analyzed by AAV8 capsid enzyme-linked immunosorbent assay to determine the distribution of viral genomes. AAV-FVIII showed an increase in production when culture was extended from day 3 (1.1 × 10¹³ ± 9.2 × 10¹¹ and 3.6 × 10¹³ ± 2.5 × 10¹² total capsids in the lysate and media, respectively) to day 5 (6.7 × 10¹² ± 6.7 × 10¹¹ and 4.5 × 10¹³ ± 2.6 × 10¹² total capsids in the lysate and media, respectively), day 6 (5.0 × 10¹² ± 1.9 × 10¹¹ and 5.3 × 10¹³ ± 3.3 × 10¹² total capsids in the lysate and media, respectively), and day 7 (3.0 × 10¹² ± 1.3 × 10¹⁰ and 5.0 × 10¹³ ± 1.9 × 10¹² total capsids in the lysate and media, respectively) post-transfection, with day 5 showing a 9% increase over day 3 and day 6 showing a 24% increase over day 3 (* denotes significant difference versus day 3; Ɨ denotes significant difference versus day 5; and ǂ denotes significant difference versus day 6 as measured by analysis of variance, P = 0.004). Viral titer decreased from day 6 to 7, but was still 14% higher than on day 3. 76% of AAV-FVIII particles were in the culture media at day 3, and this proportion increased to 87% by day 5, 91% by day 6, and 94% by day 7.